14 Cell Respiration T - Vernier Software & Technology

合集下载

香烟烟雾提取物对大鼠肺成纤维细胞生长的影响

香烟烟雾提取物对大鼠肺成纤维细胞生长的影响

香烟烟雾是有众多化学成分的复杂混合物,超过6000种成分,会对肺脏和全身产生有害影响。

吸烟已被公认与多种肺部疾病的发生发展有关,吸烟是肺癌的主要病因,也是慢性阻塞性肺疾病(COPD)等疾病的主要危险因素。

如今,吸烟还被认为与某些间质性肺病及肺纤维化有关[1],但尚不清楚吸烟引起间质性肺病的发病机制,对吸烟与肺纤维化关系的研究也很少。

为此本研究利用体外培养的正常大鼠及肺纤维化大鼠的肺成纤维细胞,观察不同浓度香烟烟雾提取物(cigarette smoking extract,CSE)对两种细胞的影响,探讨香烟烟雾在肺纤维化中所起的作用。

1 材料与方法1.1 实验材料1.1.1 主要试剂 博来霉素购自天津太河制药有限公司;RPMI-1640培养基、胰蛋白酶、胎牛血清购自美国GIBCO 公司;凋亡检测试剂盒购自美国BD 公司;兔抗大鼠波动蛋白、纤维粘连蛋白、α-平滑肌肌动蛋白(α-SMA)单克隆抗体及SABC 免疫组化试剂盒购自武汉博士德生物工程有限公司、第二抗体为羊抗兔IgG 购自美国Jackson 公司;化学试剂购自美国Sigma 公司。

1.1.2 仪器 二氧化碳培养箱(美国FORMA 公司【摘要】 目的 通过观察不同浓度的香烟烟雾提取物(cigarette smoking extract,CSE)对正常及肺纤维化大鼠肺成纤维细胞的影响,探讨香烟烟雾与肺纤维化的可能关系。

方法 体外培养的正常及博莱霉素造模的肺纤维化大鼠的肺成纤维细胞,分别以不同浓度(25%、50%、100%)的CSE 作用12h 和24h。

噻唑蓝还原法(MTT 法)检测吸光值;流式细胞仪法观察细胞坏死与凋亡情况及细胞周期S 期(DNA 合成期)和G 1期(DNA 合成前期)的比值。

结果 高浓度(100%)的CSE 主要引起两种大鼠肺成纤维细胞的坏死。

较低浓度(25%、50%)的CSE 对正常大鼠肺成纤维细胞无明显影响(P >0.05)。

恩格列净联合二甲双胍治疗2型糖尿病的效果评价

恩格列净联合二甲双胍治疗2型糖尿病的效果评价

恩格列净联合二甲双胍治疗2型糖尿病的效果评价林智化厦门大学附属第一医院同安院区(厦门市第三医院)内分泌科,福建厦门361100[摘要]目的分析恩格列净联合二甲双胍治疗2型糖尿病的效果。

方法选取2021年3月—2022年4月厦门大学附属第一医院同安院区(厦门市第三医院)收治的98例2型糖尿病患者为研究对象,按照随机数表法分为观察组和对照组,每组49例。

两组均予以二甲双胍治疗,同时观察组加行恩格列净治疗。

比较两组血糖水平、血脂水平、胰岛β细胞功能、不良反应发生率以及临床疗效。

结果治疗6个月后,与对照组相比,观察组血糖水平、血脂水平均较低,胰岛β细胞功能的改善情况较好,差异有统计学意义(P<0.05)。

观察组不良反应发生率8.16%与对照组6.12%对比,差异无统计学意义(P>0.05)。

观察组治疗总有效率95.92%较对照组83.67%更高,差异有统计学意义(P<0.05)。

结论应用恩格列净联合二甲双胍治疗,效果突出,可有效调节糖脂代谢,纠正胰岛β细胞功能。

[关键词] 恩格列净;二甲双胍;2型糖尿病;糖脂代谢;胰岛β细胞功能[中图分类号] R446.1 [文献标识码] A [文章编号] 1672-4062(2023)08(a)-0087-04 Efficacy Evaluation of Empagliflozin Combined with Metformin in the Treatment of Type 2 Diabetes MellitusLIN ZhihuaDepartment of Endocrinology, Tong'an District of the First Affiliated Hospital of Xiamen University (Xiamen Third Hospital), Xiamen, Fujian Province, 361100 China[Abstract] Objective To analyze the effect of Empagliflozin combined with metformin in the treatment of type 2 dia⁃betes mellitus. Methods From March 2021 to April 2022, 98 patients with type 2 diabetes treated in Tong 'an District the First Affiliated Hospital of Xiamen University (Xiamen Third Hospital) were selected as the research objects. Ac⁃cording to the random number table method, they were divided into observation group and control group, 49 cases in each group. Both groups were treated with metformin, while the observation group was treated with empagliflozin. The blood glucose level, blood lipid level, islet β cell function, incidence of adverse reactions and clinical efficacy were compared between the two groups. Results After 6 months of treatment, compared with the control group, the blood glucose level and blood lipid level in the observation group were lower, and the improvement of islet β cell function was better, the difference was statistically significant (P<0.05). There was no statistically significant difference in the incidence of adverse reactions between the observation group (8.16%) and the control group (6.12%) (P>0.05). The to⁃tal effective rate of treatment in the observation group was 95.92% higher than that in the control group (83.67%), and the difference was statistically significant (P<0.05). Conclusion The treatment of Empagliflozin combined with metfor⁃min has a remarkable effect, which can effectively regulate the metabolism of glucose and lipid and correct the func⁃tion of islet beta cells.[Key words] Empagliflozin; Metformin; Type 2 diabetes; Glycolipid metabolism; Islet beta cell function2型糖尿病是最为常见的糖尿病类型,由于多发于成年,故又称成人发病型糖尿病,疾病早期症状不典型,随着疾病进展,患者可出现多饮、多食、多尿、消瘦或短期内体质量减轻等典型症状[1]。

诱导多能干细胞

诱导多能干细胞
Cyagen Biosciences Inc.
We help you discover life
iPS
通过基因转染技术(gene transfection)将某些转录因子导入动物或人的体细胞, 使体细胞直接重构成为胚胎干细胞(embryonic stem cell, ESC)细胞样的多潜能细胞。 iPS细胞不仅在细胞形态、 生长特性、 干细胞标志物表达等方面与ES细胞非常相似, 而且在DNA甲基化方式、 基因表达谱、 染色质状态、 形成嵌合体动物等方面也与ES细胞几乎完全相同。
Cyagen Biosciences Inc.
We help you discover life
ES细胞和IPS细胞具有相同的基因。不同的是,ES细胞中的与细胞多能性有关的基因能够表达,如:oct4,Sox2等。而已分化的体细胞中的这些基因不能表达。通过导入与多能性有关的外源基因来激活体细胞中的多能性基因,从而使体细胞从分化状态重编程为多能性干细胞。
Cyagen Biosciences Inc.
We help you discover life
IPS细胞的鉴定
表面标志分子鉴定 IPS细胞能够表达多能干细胞特异的表面标志分子,如:ssea-1(阶段特异性胚胎抗原1),ssea-3等。这些物质在已分化体细胞表面不表达,从而鉴定。
IPS细胞与ES细胞具有相似的DNA甲基化模式和组蛋白修饰情况。IPS细胞中的多能基因,如oct-4,Nanong等的启动子区域CpG岛从高甲基化转变为类似ES细胞的低甲基化状态。
Cyagen Biosciences Inc.
We help you discover life
在中国:
2009年,中国科学家于2008年11月利用iPS细胞培育出小鼠——“小小” 中国科学院动物研究所周琪研究员和上海交通大学医学院曾凡一研究员领导的研究组合作完成的工作表明,利用iPS细胞能够得到成活的具有繁殖能力的小鼠,从而在世界上第一次证明了iPS细胞与胚胎干细胞具有相似的多能性。科学家表示,这一研究成果表明iPS干细胞或许同胚胎干细胞一样可以作为治疗各种疾病的潜在来源。

woundhealing(cellmigration)assay

woundhealing(cellmigration)assay

Endothelial wound healing (cell migration) assayDescriptionThis is a simple assay that can be used in any cell culture lab setup to test the effect of different compounds on endothelial cell migration.ProcedureGrow endothelial cells in complete media.Day 1: Trypsinize cells and add 4 x105 cells per well in a six well plate in complete media. After 8 h, change media to starvation media. Starve cells overnight in this media.Day 2:1) Remove media from cells.2) Make a scratch wound across each well of the 6-well plate using a pipette tip. Make sure to use a very fine tip so that the cells come off the plate and the cell free area has sharp clear edges.3) Wash three times with starvation media to remove any loosely held cells. Make sure that the wound area is free of cells.4) Add starvation media containing required concentration of compound to be tested in 3 ml volume per well.5) Take 0 hr. images.6) Incubate at 37 degree C for 6 h. Take images.7) Compare 0 and 6 hr images and calculate area of the wound closed using image J software.SuppliesComplete media: Medium with FBS supplemented to 10%Starvation media (SM): Medium with no FBSEndothelial cells75 cm2 vented flask (gelatin coated)gelatin coated 6-well plate (Biocoat)Tips1) The media used is the specific media for that cell type.2) One can also try using media containing low percentage FBS to obtain faster closing of the wound.3) The time of incubation can be increased or decreased based on the type of cells used (how fast they migrate) and the compound being tested.。

CT引导建立兔肺VX2肿瘤模型的实验研究

CT引导建立兔肺VX2肿瘤模型的实验研究

【 键 词 】 亲 水 凝 胶 ;动物 模 型 ; 肿瘤 ;C 关 肺 T
中图 分类 号 : 7 — 6 文 献 标 志码 : 文 章 编 号 :0 87 4 ( 0 9 .20 4 —3 R 33 B 1 0 —9 X 2 0 ) . 1 1 0 0
CT- u d d e t b ih e t o a b tl n g i e s a l m n f r b i u g VX2 t mo d l L s u r mo e /Xi n,Z n HA NG Ho g,S NG i HE L n,RE N
大 白兔 肺 内有 肿 瘤 生 长 , 中 2 其 6只 呈 单 发 结 节 , 径 0 直 . 5~08a 兔 肺 V 2肿 瘤 模 型 成 功 率 为 7 . . m, X 22 % ( 6 3 ) 另 有 2只 肺 内 呈 多 发结 节 生 长 , 出 现胸 腔积 液 ; 2/6 ; 并 4只 累及 纵 隔 ; 2只 出现 胸 壁 种植 转 移 。 种 过 接
H , U rn ,W G Y n ,H NGZ i u O G B ow i D p r n o lao n ,C i s r e e L 0g AN ag A h- ,D N o—e. eat tf Ut su d hn eA m d , y me r e
Pl eF reG n r H pt , e ig1 0 3 ,C ia oi oc e a a il B in 0 09 hn c e l s a j
程 未 出现 气 胸 、 出血 等 并 发 症 。 结 论
本 实 验 建 模 方 法 安 全 、 效 , 可 在 肺 内局 部 形 成 较 大 体 积 的 实 体 有 并
瘤 , 满 足微 波 消 融 治 疗 肺 癌 的 研 究 需要 , 能 因此 所 建 兔肺 V 2肿 瘤 模 型 是 成 功 的 。 X

T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects

T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects

DOI: 10.1126/scitranslmed.3002842, 95ra73 (2011);3 Sci Transl Med , et al.Michael Kalos and Can Establish Memory in Patients with Advanced Leukemia T Cells with Chimeric Antigen Receptors Have Potent Antitumor EffectsEditor's Summarythe potential for CAR-modified T cells to bring cancer therapy up to speed.treatment had complete remission of their leukemia. Although this is early in the clinical study, these results highlight scale with a second exposure to CLL cells. Indeed, two of the three CLL patients who underwent the CAR T cell CAR T cells persisted with a memory phenotype, which would allow them to respond more quickly and on a larger these CAR T cells expanded >1000-fold, persisted for more than 6 months, and eradicated CLL cells. Some of these allowing for much broader cellular targeting than is obtained with normal T cells. After transfer into three CLL patients,receptor could activate T cells in response to CD19 in the absence of major histocompatibility complex restriction, specific intracellular signaling domain. The resulting chimeric −specific costimulatory domain and a T cell −both a T cell bind in a restricted manner to the CD19 protein (which is found solely on normal B cells and plasma cells) attached to The CAR T cells used in this study expressed an antigen receptor that consists of antibody binding domains that as reflected by decreased numbers of B cells and plasma cells and the development of hypogammaglobulinemia.tumor cells after transfer into patients; they also mediated cancer remission. Innocent bystanders were also targeted, chronic lymphocytic leukemia (CLL) (a B cell cancer). The designer T cells not only expanded, persisted, and attacked modified T cells to express a chimeric antigen receptor (CAR) to yield so-called CAR T cells that specifically target . have genetically et al cells to the tumor and maintaining these cells in patients remains challenging. Now, Kalos harness the power of the immune system to fight cancers such as leukemia; however, targeting functional immune T to healthy tissues, such as infection or cancer, and then try to deter dangerous activity. Researchers have long sought As members of the body's police force, cells of the immune system vigilantly pursue bad actors that harmGo CAR-Ts in the Fast Lane/content/3/95/95ra73.full.html can be found at:and other services, including high-resolution figures,A complete electronic version of this article /content/suppl/2011/08/08/3.95.95ra73.DC1.htmlcan be found in the online version of this article at: Supplementary Material/about/permissions.dtl in whole or in part can be found at:article permission to reproduce this of this article or about obtaining reprints Information about obtaining last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue (print ISSN 1946-6234; online ISSN 1946-6242) is published weekly, except the Science Translational Medicine o n F e b r u a r y 20, 2012s t m .s c i e n c e m a g .o r g D o w n l o a d e d f r o mL E U K E M I AT Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced LeukemiaMichael Kalos,1,2*Bruce L.Levine,1,2*David L.Porter,1,3Sharyn Katz,4Stephan A.Grupp,5,6 Adam Bagg,1,2Carl H.June1,2†Tumor immunotherapy with T lymphocytes,which can recognize and destroy malignant cells,has been limited by the ability to isolate and expand T cells restricted to tumor-associated antigens.Chimeric antigen receptors(CARs) composed of antibody binding domains connected to domains that activate T cells could overcome tolerance by allowing T cells to respond to cell surface antigens;however,to date,lymphocytes engineered to express CARs have demonstrated minimal in vivo expansion and antitumor effects in clinical trials.We report that CAR T cells that target CD19and contain a costimulatory domain from CD137and the T cell receptor z chain have potent non–cross-resistant clinical activity after infusion in three of three patients treated with advanced chronic lymphocytic leukemia(CLL).The engineered T cells expanded>1000-fold in vivo,trafficked to bone marrow,and continued to express functional CARs at high levels for at least6months.Evidence for on-target toxicity included B cell aplasia as well as decreased numbers of plasma cells and hypogammaglobulinemia.On average,each infused CAR-expressing T cell was calculated to eradicate at least1000CLL cells.Furthermore,a CD19-specific immune re-sponse was demonstrated in the blood and bone marrow,accompanied by complete remission,in two of three patients.Moreover,a portion of these cells persisted as memory CAR+T cells and retained anti-CD19effector functionality,indicating the potential of this major histocompatibility complex–independent approach for the ef-fective treatment of B cell malignancies.INTRODUCTIONUsing gene transfer technologies,T cells can be genetically modified to stably express antibody binding domains on their surface that con-fer novel antigen specificities that are major histocompatibility com-plex(MHC)–independent.Chimeric antigen receptors(CARs)are an application of this approach that combines an antigen recognition domain of a specific antibody with an intracellular domain of the CD3-z chain or Fc g RI protein into a single chimeric protein(1,2). Trials testing CARs are presently under way at a number of academic medical centers(3,4).In most cancers,tumor-specific antigens are not yet well defined,but in B cell malignancies,CD19is an attractive tumor target.Expression of CD19is restricted to normal and malig-nant B cells(5),and CD19is a widely accepted target to safely test CARs.Although CARs can trigger T cell activation in a manner sim-ilar to an endogenous T cell receptor,a major impediment to the clin-ical application of this technology to date has been the limited in vivo expansion of CAR+T cells,rapid disappearance of the cells after in-fusion,and disappointing clinical activity(4,6).CAR-mediated T cell responses may be further enhanced with ad-dition of costimulatory domains.In a preclinical model,we found that inclusion of the CD137(4-1BB)signaling domain significantly increased antitumor activity and in vivo persistence of CARs com-pared to inclusion of the CD3-z chain alone(7,8).To evaluate the safety and feasibility for adoptive transfer of T cells gene-modified to express such CARs,we initiated a pilot clinical trial using autologous T cells expressing an anti-CD19CAR including both CD3-z and the 4-1BB costimulatory domain(CART19cells)to target CD19+malig-nancies.To date,we have treated three patients under this protocol. Some of the findings from one of these patients are described in(9), which reports that this treatment results in tumor regression,CART19 cell persistence,and the unexpected occurrence of delayed tumor lysis syndrome.Here,we show that the CART19cells mediated potent clinical antitumor effects in all three patients treated.On average,each infused CAR T cell and/or their progeny eliminated more than 1000leukemia cells in vivo in patients with advanced chemotherapy-resistant chronic lymphocytic leukemia(CLL).CART19cells underwent robust in vivo T cell expansion,persisted at high levels for at least6 months in blood and bone marrow(BM),continued to express func-tional receptors on cells with a memory phenotype,and maintained anti-CD19effector function in vivo.RESULTSClinical protocolThree patients with advanced,chemotherapy-resistant CLL were enrolled in a pilot clinical trial for CART19cell therapy.Figure1presents a summary of the manufacturing process for the gene-modified T cells (A)and the clinical protocol design(B).All patients were extensively pretreated with various chemotherapy and biologic regimens(Table1). Two of the patients had p53-deficient CLL,a deletion that portends poor response to conventional therapy and rapid progression(10). Each of the patients had a large tumor burden after the preparative1Abramson Cancer Center,University of Pennsylvania,Philadelphia,PA19104,USA.2De-partment of Pathology and Laboratory Medicine,University of Pennsylvania,Philadelphia, PA19104,USA.3Department of Medicine,University of Pennsylvania,Philadelphia,PA 19104,USA.4Department of Radiology,University of Pennsylvania,Philadelphia,PA19104, USA.5Department of Pediatrics,University of Pennsylvania,Philadelphia,PA19104,USA. 6Division of Oncology,Children’s Hospital of Philadelphia,Philadelphia,PA19104,USA.*These authors contributed equally to this work.†To whom correspondence should be addressed.E-mail:cjune@ o n F e b r u a r y 2 0 , 2 0 1 2 s t m . s c i e n c e m a g . o r g D o w n l o a d e d f r o mchemotherapy,including extensive BM infiltration(40to95%)and lymphadenopathy;UPN02also had peripheral lymphocytosis.There was a low abundance of T cells in the apheresis products(2.29to4.46%) (table S1)as well as likely impaired T cell activation,as has been shown previously in CLL patients(11).Additional details of the cell manufac-turing and product characterization for the CART19cell preparation for each patient are shown in table S1.All patients were pretreated1to 4days before CART19cell infusions with lymphodepleting chemo-therapy(Table1).A split-dose cell infusion schedule was used to address potential safety concerns related to the evaluation of a previously untested CAR that incorporated the4-1BB costimulatory signaling domain. In vivo expansion,persistence,and BM trafficking of CART19cellsOur preclinical data in two animal models,including mice bearing xenografts of primary human precursor-B acute lymphoblastic leuke-mia(7,8),indicated that CAR+T cells that express a4-1BB signaling domain expanded after stimulation with anti-CD3/anti-CD28mono-clonal antibody–coated beads(12)and had improved persistence com-pared to CAR+T cells lacking4-1BB.We developed a quantitative polymerase chain reaction(qPCR)assay to enable quantitative tracking of CART19cells in blood and BM.CART19cells expanded and persisted in the blood of all patients for at least6months(Fig.2, A and B).Moreover,CART19cells expanded1000-to10,000-fold in the blood of patients UPN01and03during the first month after infusion,reaching peak frequencies of10to>95%of circulating white blood cells in UPN01and03(Fig.2C).The peak expansion levels coincided with onset of the clinical symptoms after infusion in UPN01 (day15)and UPN03(day23).Furthermore,after an initial decay,which can be modeled with first-order kinetics,the CART19cell numbers stabilized in all three patients from days90to180after infusion (Fig.2B).The CART19cells also trafficked to the BM in all patients, albeit in5-to10-fold fewer numbers than observed in blood(Fig.2D). CART19cells had a log-linear decay in the BM in UPN01and03, with a disappearance half-life of~35days.Induction of specific immune responses in the peripheral blood and BM compartments after CART19infusion Peripheral blood(PB)and BM serum samples from all patients were collected and batch-analyzed to quantitatively determine cytokine levels.A panel of30cytokines,chemokines,and other soluble factors were assessed for potential toxicities and to provide evidence of CART19cell function.The full data set for all of the cytokines measured in each of the three patients through the date of thisapheresisSeed in gas-permeable bags.Transduction w/αCD19-41BBζvectorVector washout.Culture in gas-permeable bagsCulture in WAV EbioreactorHarvest, wash, concentrateCryopreserve final product ininfusible cryomediaCD3/28-positive selection ofT cells with anti-CD3/anti-CD28 mAb-coated magneticbeadsDay 0Day 0-1Day 3Day 5Harvest day(10 ±2)ABManufacture/cryopreservationFig.1.Schematic representation of the gene transfer vector and trans-gene,gene-modified T cell manufacturing,and clinical protocol design.(A)T cell manufacturing.Autologous cells were obtained via leukapher-esis,and T cells were enriched by mononuclear cell elutriation,washed, and expanded by addition of anti-CD3/CD28–coated paramagnetic beads for positive selection and activation of T cells.Residual leukemic cells were depleted.The lentiviral vector was added at the time of cell activation and was washed out on day3after culture initiation.Cells were expanded on a rocking platform device(WAVE Bioreactor System) for8to12days.On the final day of culture,the beads were removed by passage over a magnetic field and the CART19cells were harvested and cryopreserved in infusible medium.mAb,monoclonal antibody.(B)Clin-ical protocol design.Patients were given lymphodepleting chemo-therapy as described,followed by CART19infusion#1by intravenous gravity flow drip over a period of15to20min.The infusion was given using a split-dose approach over3days(10,30,and60%)beginning1to5days after completion of chemotherapy.Endpoint assays were conducted on study week4.At the conclusion of active monitoring, subjects were transferred to a destination protocol for long-term follow-up as per FDA guidance.onFebruary2,212stm.sciencemag.orgmpublication is presented in tables S2to S5.Of the analytes tested, 11had a threefold or more change from baseline,including four cytokines[interleukin-6(IL-6),interferon-g(IFN-g),IL-8,and IL-10], five chemokines[macrophage inflammatory protein–1a(MIP-1a), MIP-1b,monocyte chemotactic peptide–1(MCP-1),CXC chemokine ligand9(CXCL9),and CXCL10],and the soluble receptors IL-1R a and IL-2R a;IFN-g had the largest relative change from baseline (Fig.3).The peak time of cytokine elevation in UPN01and03 correlated temporally with both the previously described clinical symptoms and the peak levels of CART19cells detected in the blood for each patient.Notably,cytokine modulations were transient,and levels reverted to baseline relatively rapidly despite continued func-Table1.Patient demographics and response.CR,complete response;PR,partial response;N/A,not available.Subject UPNAge/sexkaryotypePrevious therapiesCLL tumor burden at baselineTotaldoseof CART19(cells/kg)Response day+30(duration)BM(study day)‡Blood(studyday)‡Nodes/spleen(study day)‡0165/Mnormal Fludarabine×four cycles(2002)Hypercellular70%CLL N/A 6.2×1011to1.0×1012CLL cells(day−37)1.1×109(1.6×107/kg)CR(11+months)Rituximab/fludarabine×four cycles(2005)2.4×1012CLL cells(day−14)Alemtuzumab×12weeks(2006)1.7×1012CLL cells(day−1)Rituximab(two courses,2008to2009)R-CVP×two cycles(2009)Lenalidomide(2009)PCR×two cycles(5/18/2010to6/18/2010)Bendamustine×one cycle(7/31/10to8/1/10)pre-CART190277/M del(17)(p13)*Alemtuzumab×16weeks(6/2007)Hypercellular>95%CLL2.75×1011CLL cells(day−1)1.2×1012to2.0×1012CLL cells(day−24)5.8×108(1.0×107/kg)PR(7months)Alemtuzumab×18weeks(3/2009)3.2×1012CLL cells(day−47)Bendamustine/rituximab:7/1/2010(cycle1)7/28/2010(cycle2)8/26/2010(cycle3)pre-CART190364/M del(17)(p13)†R-Fludarabine×twocycles(2002)Hypercellular40%CLLN/A 3.3×1011to5.5×1011CLL cells(day−10)1.4×107(1.46×105/kg)CR(10+months)R-Fludarabine×four cycles(10/06to1/07)8.8×1011CLL cells(day−1)R-Bendamustine×one cycle(2/09)Bendamustine×three cycles(3/09to5/09)Alemtuzumab×11weeks(12/09to3/10)Pentostatin/cyclophosphamide(9/10/10)pre-CART19*UPN02karyotype[International System for Human Cytogenetic Nomenclature(ISCN)]:45,XY,del(1)(q25),+del(1)(p13),t(2;20)(p13;q11.2),t(3;5)(p13;q35),add(9)(p22),?del(13)(q14q34),-14,del(17) (p13)[cp24].†UPN03karyotype(ISCN):46,XY,del(17)(p12)[18]/44~46,idem,der(17)t(17;21)(p11.2;q11.2)[cp4]/40~45,XY,-17[cp3].‡See the Supplementary Material for methods of tumor burden determination.o n F e b r u a r y 2 0 , 2 0 1 2 s t m . s c i e n c e m a g . o r g D o w n l o a d e d f r o mtional persistence of CART19cells.Only modest changes in cytokine levels were noted in UPN 02,possibly as a result of corticosteroid treatment.We also noted a robust induction of cytokine secretion in the supernatants from BM aspirates of UPN 03(Fig.3D and table S5).Although a pretreatment marrow sample was not available,compared to the late time point (+176),we also observed elevated levels for a number of factors in the +28marrow sample for UPN 01including IL-6,IL-8,IL-2R,and CXCL9;in contrast,compared to the pretreatment marrow sample,no elevation in cytokines was de-tected in the +31day sample for UPN 02(table S5).One of the preclinical rationales for developing CAR +T cells with 4-1BB signaling domains was a projected reduced propensity to trigger IL-2and tumor necrosis factor –a (TNF-a )secretion compared to CAR +T cells with CD28signaling domains (7);indeed,elevated amounts of soluble IL-2and TNF-a were not detected in the serum of the patients.Lower levels of these cytokines may be related to sustained clinical ac-tivity:Previous studies have shown that CAR +T cells are potentially suppressed by regulatory T cells (13),which can be elicited by either CARs that secrete substantial amounts of IL-2or by the provision of exogenous IL-2after infusion.Moreover,the TNF-a is complicit in cy-tokine storm –related effects in patients,which are absent here.Prolonged receptor expression and establishment of a population of memory CART19cells in bloodA central question in CAR-mediated cancer immunotherapy is whether optimized cell manufacturing and costimulation domains will enhance the persistence of genetically modified T cells and permit the establishment of CAR +memory T cells in patients.Previous studies have not demonstrated robust expansion,prolonged persist-ence,or functional expression of CARs on T cells after infusion (14–17).The high persistence of CART19cells that we observed at late time points for UPN 03facilitated a more detailed phenotypic analysis ofpersisting cells.Flow cytometric analysis of samples from both blood and BM 169days after infusion revealed the presence of CAR19-expressing cells in UPN 03as well as an absence of B cells (fig.S1,Aand B).These CAR +cells persisted in allthree patients beyond 4months,as shown by qPCR (Fig.2).The in vivo frequency of CAR +cells by flow cytometry closely matched the values obtained from the PCR assay for the CAR19transgene.CAR expression was also detected on the surface of 5.7and 1.7%of T cells in the blood of patient UPN 01on days 71and286after infusion (fig.S2).We next used polychromatic flow cy-tometry to perform detailed studies and further characterize the expression,phe-notype,and function of CART19cells in UPN 03using an anti-CAR idiotype anti-body (MDA-647)and the gating strategy shown in fig.S3.We observed differencesin the expression of memory and activa-tion markers in both CD4+and CD8+T cells based on CAR19expression.In the CD4+compartment,at day 56,CART19cells were characterized by auniform lack of CCR7,a predominance of CD27+/CD28+/PD-1+cells distributed within both CD57+and CD57−compart-ments,and an essential absence of CD25and CD127expression,the latter two markers defining regulatory CD4+T cells (18)(Fig.4A).In contrast,CAR −cells at this time point were heterogeneous in CCR7,CD27,and PD-1expression;expressed CD127;and also contained a substantialCD25+/CD127−population.By day 169,although CD28expression remained uni-formly positive in all CART19CD4+cells,a fraction of the CART19CD4+cells had acquired a central memory phenotype,withA CB Day (after infusion)110100100010000100000Day (after infusion)T o t a l c e l l s i n c i r c u l a t i o nDay (after infusion)110100100010000D C o p i e s /µg g D N A% W B CDay (after infusion)C o p i e s /µg gD N AWBC and CART 19: Blood CART 19: Marrow 10101010101010Fig.2.Sustained in vivo expansion and persistence in blood and marrow of CART19cells.(A to D )qPCRanalysis was performed on DNA isolated from whole blood (A to C)or bone marrow (BM)(D)samples obtained from UPN 01,UPN 02,and UPN 03to detect and quantify CAR19sequences.The frequency of CART19cells is shown as average transgene copies (A),total calculated CART19cells in circulation (B),or as a fraction of circulating white blood cells (WBCs)(C).(A)Copies CAR19/microgram DNA is calculated as de-scribed in Materials and Methods.(B)The total number of lymphocytes (total normal and CLL cells)versus total CART19+cells in circulation is plotted for all three subjects using the absolute lymphocyte count from complete blood count values and assuming a 5.0-liter volume of peripheral blood.(C)%WBC is calculated as described in Materials and Methods.(D)Bulk qPCR analysis of marrow to quantify CART19sequences.The data from patient UPN 03in (A,C,and D)has been published in (9)and is reprinted here with permission.Each data point represents the average of triplicate measurements on 100to 200ng of genomic DNA,withmaximal percent coefficient of variation (CV)less than 1.56%.Pass/fail parameters for the assay included preestablished ranges for slope and efficiency of amplification,and amplification of a reference sample.The lower limit of quantification for the assay established by the standard curve range was two copies of transgene per microgram of genomic DNA;sample values below that number are considered estimates and presented if at least two of three replicates generated a C t value with percent CV for the values 15%.CART19cells were infused at days 0,1,and 2for UPN 01and 03and at days 0,1,2,and 11for UPN 02. o n F e b r u a r y 20, 2012s t m .s c i e n c e m a g .o r g D o w n l o a d e d f r o mCCR7expression,a higher percentage of CD27−cells,the appearance of a PD-1−subset,and acquisition of CD127expression.At day 169,CAR −cells remained reasonably consistent with their day 56counterparts,with the exception of a reduction in CD27expression and a decrease in the percentage of CD25+/CD127−cells.In the CD8+compartment,at day 56,CART19CD8+cells displayed primarily an effector memory phenotype (CCR7−,CD27−,CD28−),con-sistent with prolonged and robust exposure to antigen (Fig.4B).In con-trast,CAR −CD8+T cells consisted of mixtures of effector and central memory cells,with CCR7expression in a subset of cells,and substantial numbers of cells in the CD27+/CD28−and CD27+/CD28+fractions.Al-though a large percentage of both CART19and CAR −cell populations expressed CD57,a marker associated with memory T cells with high cytolytic potential (19),this molecule was uniformly coexpressed with PD-1in the CART19cells,a possible reflection of the extensive replicative history of these cells.In contrast to the CAR −cell population,the entirety of the CART19CD8+population lacked expression of both CD25and CD127,markers associated with T cell activation and the development of functional memory cells (20).By day 169,although the phenotype of the CAR −cell population remained similar to the day 56cells,the CART19population had evolved to contain a population with features of central memory cells,notably expression of CCR7and higher levels of CD27and CD28,as well as cells that were PD-1−,CD57−,and CD127+.Effector function of CART19cells after 6months in blood In addition to a lack of long-term persistence,a limitation of previous trials with CAR +T cells has been the rapid loss of functional activity of the infused T cells in vivo.The high level of CART19cell persist-ence and surface expression of the CAR19molecule in UPN 03provided the opportunity to directly test anti-CD19–specific effector functions in cells recovered from cryopreserved PB samples.Pe-ripheral blood mononuclear cells (PBMCs)from UPN 03were cultured with target cells that either did or did not express CD19(Fig.4C and fig.S3).Robust CD19-specific effector function of CART19cells was observed by the specific degranulation of CART19cells against CD19+but not CD19−target cells,as assessed by surface CD107a expression.Notably,exposure of the CART19population to CD19+targets induced a rapid internalization of surface CAR19(see fig.S3for constitutive surface expression of CAR19in the same ef-fector cells in standard flow cytometric staining).The presence of costimulatory molecules on target cells was not required for trigger-ing CART19cell degranulation because the NALM-6line,which was used as a target in these studies,does not express CD80or CD86(21).Effector function was evident at day 56after infusion and was re-tained at day 169(Fig.4C).Robust effector function of CAR +and CAR −T cells could also be demonstrated by pharmacologic stimula-tion with phorbol 12-myristate 13-acetate (PMA)and ionomycin.ADay (after infusion)S e r u m c y t o k i n e (f o l d c h a n g e f r o m b a s e l i n e )(f o l d c h a n g e f r o m b a s e l i n e )S e r u m c y t o k i n e (f o l d c h a n g e f r o m b a s e l i n e )BCDay (after infusion)D Day (after infusion)C o n c e n t r a t i o n (p g /m l )αIL-6 IFN-γCXCL10MIP-1βMCP-1CXCL9IL-2R αIL-8 IL-10MIP-1αFig.3.Serum and BM cytokines before and afterCART19cell infusion.(A to C )Longitudinal measure-ments of changes in serum cytokines,chemokines,and receptors in UPN 01(A),UPN 02(B),and UPN 03(C)on the in-dicated day after CART19cell infusion.(D )Serial assessments of the same analytes in the BM from UPN 03.Analytes with agreater than or equal to threefold change are indicated and plotted as relative change from baseline (A to C)or as absolute values (D).In (C)and (D),a subset of the cytokine data (IFN-g ,CXCL10,CXCL9,IL-2R a ,and IL-6)from UPN 03have been pub-lished in (9)and are reprinted here with permission.Absolutevalues for each analyte at each time point were derived from a recombinant protein-based standard curve over a threefold eight-point dilution series,with upper and lower limits of quantification determined by the 80to 120%observed/expected cutoff valuesfor the standard curves.Each sample was evaluated in duplicate with average values calculated and percent CV in most cases less than 10%.To accommodate consolidated data presentation in the context of the wide range for the absolute values,data are presented as fold change over the baseline value for each analyte.In cases where baseline values were not detectable,half of the lowest standard curve value was used as the baseline value.Standard curve ranges for analytes and baseline (day 0)values (listed in parentheses sequentially for UPN 01,02,and 03),all in pg/ml:IL-1R a :35.5to 29,318(689,301,and 287);IL-6:2.7to 4572(7,10.1,and 8.7);IFN-g :11.2to 23,972(2.8,not detected,and 4.2);CXCL10:2.1to 5319(481,115,and 287);MIP-1b :3.3to 7233(99.7,371,and 174);MCP-1:4.8to 3600(403,560,and 828);CXCL9:48.2to 3700(1412,126,and 177);IL-2R a :13.4to 34,210(4319,9477,and 610);IL-8:2.4to 5278(15.3,14.5,and 14.6);IL-10:6.7to 13,874(8.5,5.4,and 0.7);MIP-1a :7.1to 13,778(57.6,57.3,and 48.1).o n F e b r u a r y 20, 2012s t m .s c i e n c e m a g .o r g D o w n l o a d e d f r o mProfound antitumor clinical activity of CART19cellsThere were no significant toxicities observed during the4days after the infusion in any patient other than transient febrile reactions. However,all patients subsequently developed significant clinical and laboratory toxicities between days7and21after the first infusion. With the exception of B cell aplasia,these toxicities were short-term and reversible.Of the three patients treated to date,there are two complete responses and one partial response lasting greater than8months after CART19infusion according to standard criteria(22).Details of past medical history and response to therapy are described in Table1. The clinical course of UPN03has been described in detail(9).In brief,patient UPN02was treated with two cycles of bendamus-tine with rituximab,resulting in stable disease;he received a third dose of bendamustine as lymphodepleting chemotherapy before CART19 cell infusion.After CART19infusion,and coincident with the onset of high fevers,he had rapid clearance of the p53-deficient CLL cells from his PB(Fig.5A)and a partial reduction of adenopathy.He de-veloped fevers to40°C,rigors,and dyspnea requiring a24-hour hos-pitalization on day11after the first infusion and on the day of his second CART19cell boost.Fevers and constitutional symptoms per-sisted,and on day15,he had transient cardiac dysfunction;all symp-toms resolved after corticosteroid therapy was initiated on day18.His BM showed persistent extensive infiltration of CLL1month after therapy despite marked PB cytoreduction.He remained asymptomatic at the time of publication.Patient UPN01developed a febrile syndrome,with rigors and transient hypotension beginning10days after infusion.The fevers persisted for about2weeks and resolved;he has had no further consti-tutional symptoms.He achieved a rapid and complete response(Fig.5, B and C).Between1and6months after infusion,no circulating CLL cells were detected in the blood by deep sequencing(Table2).His BM at1,3,and6months after CART19cell infusions showed sustainedA1.40.67.890.223.58.02939.5CCR7CD28CD127CCR7CD28CD127C0.999.187.97.10.34.65.165.726.72.60.746.551.71.265.814.34.1715.71.81236.949.49.317.644.129CD4Day 169CCR7CD28CD127CCR7CD28CD127CD45RACD27CD25CD45RACD27CD25CD45RACD27CD25CD45RACD2744.017.34.234.4CD57CD5731.932.111.424.648.218.55.128.2CD57CD57PD-1PD-1PD-1PD-150.111.01127.927.841.716.613.874.719.81.73.8CD57CD57CD57CD5735.933.610.220.352.738.94.63.936.09.814.239.974.9230.61.5CD57CD57CD57CD57CD27CD28CD27CD28CD27CD28CD27CD28CD2565.00.60.433.719.959.411.39.231.551.66.610.030.147.910.811.12.459.635.42.639.59.28.842.5CCR7CD28CD127CD57CD57CD5779.516.70.90.36.833.234.925.110.928.041.319.82542.926.16.014.244.524.516.93.7 2.025.468.875.623.70.20.56.040.339.314.316.328.831.922.937.242.318.12.414.338.222.225.36.0 4.530.359.296.6 3.414.67.41464.15.115.760.418.89.411.464.614.60.60.12.596.818.473.03.05.697.10.70.062.111.2 3.111.873.95.58.052.833.77.8 6.454.231.635.559.21.33.927.264.316.779.20.200.898.97.500.891.73.60.10.395.97.60.40.191.90.10.213.885.90.20.312.387.2C00.284.715.100.969.329.80.10.21188.60.20.56.692.74.00.21.194.73.396.6Fig.4.Prolonged surface CAR19expressionand establishment of functional memoryCART19cells in vivo.(A and B)T cell immuno-phenotyping of CD4+(A)and CD8+(B)T cellsubsets.Frozen peripheral blood(PB)samplesfrom UPN03obtained at days56and169afterT cell infusion were subjected to multipara-metric immunophenotyping for expression ofmarkers of T cell memory,activation,and ex-haustion;data are displayed after biexponentialtransformation for objective visualization ofevents.(C)Functional competence of persistingCAR cells.Frozen PB samples from UPN03ob-tained at days56and169after T cell infusion were evaluated directly ex vivo for the ability to recognize CD19-expressing target cells using CD107 degranulation assays.Presented data are for the CD8+gated population.The gating strategies for these figures are presented in fig.S2.onFebruary2,212stm.sciencemag.orgDownloadedfrom。

Differentiation of Human Pluripotent Stem Cells into Retinal Cells

Differentiation of Human Pluripotent Stem Cells into Retinal Cells

87M.A. Hayat (ed.), Stem Cells and Cancer Stem Cells, Volume 6,DOI 10.1007/978-94-007-2993-3_9, © Springer Science+Business Media B.V . 20129A bstractRetinal and macular degeneration disorders are characterized by a progressive loss of photoreceptors, which causes visual impairment and blindness. In some cases, the visual loss is caused by dysfunction, degen-eration and loss of underlying retinal pigment epithelial (RPE) cells and the subsequent death of photoreceptors. The grim reality is that there is no successful treatment for most of these blindness disorders. Cell therapy aimed at replenishing the degenerating cells is considered a potential ther-apeutic approach that may delay, halt or perhaps even reverse degenera-tion, as well as improve retinal function and prevent blindness in the aforementioned conditions. Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSCs) may serve as an unlimited donor source of photoreceptors and RPE cells for transplantation into degenerat-ing retinas and for retinal disease modeling.I ntroductionThe vertebrate eyes form as bilateral evaginations of the forebrain, called optic vesicles (Martínez-Morales et al. 2004 ; Fig. 9.1a ). During develop-ment, the optic vesicles begin to invaginate to form a cup-shaped structure, the optic cup. The inner, thicker neural layer of the optic cup differ-entiates into the neural retina, and the outer, thin-ner pigmented layer forms the retinal pigmentepithelium (RPE). At the early developmental stages, the neuroepithelial cells that compose the optic vesicle are morphologically and molecu-larly identical and are all able to give rise to neu-ral retina and RPE. Exogenous signals coming from the adjacent tissues, including factors from the fi broblast growth factor (FGF) and transform-ing growth factor beta (TGF b ) families, dictate the fate of these cells. The mature vertebrate ret-ina is comprised of six types of neurons and one type of glia (the Müller glia). These seven cell types constitute three nuclear layers: retinal gan-glion cells in the ganglion cell layer (GCL); the horizontal, bipolar and amacrine interneurons, and Müller glial cells in the inner nuclear layer (INL); and rod and cone photoreceptors in the outer nuclear layer (ONL; Harada et al. 2007;M . I delson • B . R eubinoff (*)T he Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy & The Department of Obstetrics and Gynecology , H adassah University Medical Center ,E in Kerem 12000 ,J erusalem 91120 ,I srael e -mail: b enjaminr@ekmd.huji.ac.il D ifferentiation of HumanPluripotent Stem Cells into Retinal Cells Masha Idelson and Benjamin Reubinoff88M. Idelson and B. ReubinoffFig. 9.1b ). The photoreceptor cells capture lightphotons and transform their energy into electrical signals by a mechanism called phototransduction. The visual pigment which is utilized in this process is located on membranal discs in the outer seg-ments of photoreceptors. The outer segments are continuously renewed: the old discs are shed and new disks form. When the photoreceptors absorb light, they send the signal through the retinal interneurons to the ganglion cells which transmit the electrical impulse to the brain by their axons forming the optic nerve. Rods are responsible for night vision, whereas cones are responsible for color vision and detecting fi ne details. The macula is a small part of the retina which is rich in cones and responsible for detailed central vision.R PE cells that compose the outer layer of the optic cup are pigmented cuboidal cells which lie between the neural retina and the choriocapil-laris, which include the blood vessels supplying the retina. The multiple villi on their apical side are in direct contact with the outer segments ofextraocular mesenchymeabneural retinalensoptic nerveoptic cupsurface ectodermRPEFGFoptic vesiclechoroidBM RPE cone ONLINL GCLlightHC BC MC ACONrod F ig. 9.1 D evelopment and structural arrangement of the retina. ( a ) Schematic representation of retinal development including the transition from optic vesicle to optic cup and retinal patterning. ( b ) Schematic diagram of retinal cells arrangement and connections. A bbreviations :A C amacrinecell, B C bipolar cell, B M Bruch’s membrane, G CL gan-glion cell layer, H C horizontal cell, I NL inner nuclear layer, M C Müller cell, O N optic nerve, O NL outer nuclear layer89 9 Differentiation of Human Pluripotent Stem Cells into Retinal Cellsthe photoreceptor cells; on their basal side, the RPE is in contact with the underlying basal mem-brane, termed Bruch’s membrane that separates the RPE from the choroid. These cells play cru-cial roles in the maintenance and function of the retina and its photoreceptors. As a layer of pig-mented cells, the RPE absorbs the stray light that was not absorbed by the photoreceptors. The RPE cells form a blood–retinal barrier due to decreased permeability of their junctions. The RPE cells transport ions, water, and metabolic end products from the retina to the bloodstream. They are involved in supplying the neural retina with nutrients from the bloodstream, such as glu-cose, retinol, and fatty acids. Another important function of the RPE is the phagocytosis of shed photoreceptor outer segments. After the outer segments are digested, essential substances such as retinal are recycled. Retinal is also recycled and returned to photoreceptors by the process known as the visual cycle. The precise functioning of the RPE is essential for visual performance. Failure of one of these functions can lead to degeneration of the retinal photoreceptors, vision impairment and blindness.T here are many inherited and age-related eye disorders that cause degeneration of the retina as a consequence of loss of photoreceptor cells. Retinal and macular degeneration disorders can be divided into two main groups. The fi rst group primarily affects the photoreceptors and involves the majority of cases of retinitis pigmentosa. In the second group, the primary damage is to the adjacent RPE cells, and as a consequence of this damage, the photoreceptors degenerate. This group includes age-related macular degeneration, Stargardt’s macular dystrophy, a subtype of Leber’s congenital amaurosis in which RPE65 is mutated, Best’s disease and some cases of retini-tis pigmentosa, as well.W ith regard to retinitis pigmentosa (RP), it is a group of inherited retinal degeneration diseases that are caused, as mentioned above, by a primary progressive loss of rod and cone photoreceptors, followed by a subsequent degeneration of RPE (Hartong et al. 2006). The disease affects approxi-mately 1.5 million patients worldwide and is the most common cause of blindness in people under 70 years of age in the western world. The disease can be characterized by retinal pigment deposits visible on the fundus examination. In most cases, the disease primarily affects rods. At later stages of the disease, the degeneration of cones takes place. As a consequence of disease progression, the patients’ night vision is reduced. Patients initially lose peripheral vision while retaining central vision (a visual status termed “tunnel vision”). In advanced cases, central vision is also lost, commonly at about 60 years of age. The disease affects about 1 in 4,000. The inheritance can be autosomal-recessive, autosomal-dominant or X-linked (in ~50–60%, 30–40%, and 5–15% of cases, respectively). Mutations in more than 140 genes have been iden-tifi ed as causing RP (Hartong et al. 2006).Among these genes are those involved in phototransduc-tion, like rhodopsin, the a- and b- subunits of phos-phodiesterase, the a- and b- subunits of Rod cGMP gated channel and arrestin. The additional muta-tions were found in genes encoding structural pro-teins, like peripherin, rod outer segment protein and fascin. They were also found in transcription factors involved in photoreceptors’ development such as Crx and Nrl, and in other genes, whose products are involved in signaling, cell-cell interac-tion and trafficking of intracellular proteins. Currently, there is no effective cure for RP. Treatment with vitamin A palmitate, omega-3 fatty acids and other nutrients may somewhat slow the rate of the disease progression in many cases. Reduction in exposure to light was also shown to decrease the rate of retinal degeneration.A mong the group of retinal degenerations that are caused by primary loss of RPE cells or their function, age-related macular degeneration (AMD) is the most frequent condition and the leading cause of visual disability in the western world (Cook et al. 2008).Among people over 75 years of age, 25–30% are affected by AMD, with progressive central visual loss that leads to blindness in 6–8%. The retinal degeneration pri-marily involves the macula. The dry form of AMD is initiated by hyperplasia of the RPE and formation of drusen deposits, consisting of meta-bolic end products underneath the RPE or within the Bruch’s membrane. It may gradually progress into the advanced stage of geographic atrophy90M. Idelson and B. Reubinoff with degeneration of RPE and photoreceptorsover large areas of the macula causing central visual loss. Ten percent of dry AMD patients will progress to neovascular (wet) AMD, with blood vessels sprouting through the Bruch’s membrane with subsequent intraocular leakage and/or bleed-ing, accelerating the loss of central vision. While the complicating neovascularization can be treated with anti-VEGF agents, currently there is no effective treatment to halt RPE and photore-ceptor degeneration and the grim reality is that many patients eventually lose their sight (Cook et al. 2008).S targardt’s macular dystrophy (SMD) is the most common form of inherited macular dystro-phy affecting children (Walia and Fishman 2009). The disease is symptomatically similar to AMD. The prevalence of SMD is about 1 in 10,000 chil-dren. The disease involves progressive central visual loss and atrophy of the RPE beneath the macula following accumulation of lipofuscin in RPE cells, which is suggested to consist of non-degradable material, derived from ingested pho-toreceptor outer segments. The inheritance is predominantly autosomal recessive, although an autosomal dominant form has also been described. The mutation in the ABCA4 gene was found to be a most common cause of SMD. The product of the ABCA4 gene is involved in energy transport to and from photoreceptors. The mutated protein cannot perform its transport function and, as a result, photoreceptor cells degenerate and vision is impaired. Currently, there is no effective treat-ment for SMD.C ell therapy to replenish the degenerating cells appears as a promising therapeutic modality that may potentially halt disease progression in the various retinal and macular degeneration dis-orders caused by loss and dysfunction of RPE cells and photoreceptors (da Cruz et al. 2007).I n this chapter we will discuss the potential of human pluripotent cells which includes human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSCs), to gen-erate various types of retinal cells that could be used for transplantation therapy of retinal degen-eration disorders and disease modeling for drug discovery. C ell Therapy of Retinal and Macular DegenerationsT he eye is an attractive organ for cell therapy as it is easily accessible for transplantation and for simple monitoring of graft survival and potential complications by direct fundoscopic visualiza-tion. Anatomically, it is a relatively confi ned organ limiting the potential of unwanted extra-ocular ectopic cell distribution, and a low number of cells are required to replenish the damaged cells. The eye is also one of the immune privi-leged sites of the body.T he concept of replacing dysfunctional or degenerated retina by transplantation has been developing ever since the fi rst retina-to-retina transplant in 1986 (Turner and Blair 1986).In most studies, primary retinal immature (fetal) tissue has been used as donor material. It was demonstrated that such transplants can survive, differentiate, and even establish connections with the host retina to a limited degree (Ghosh et al. 1999). The subretinal transplantation of healthy RPE has some advantages over neural retinal transplantation, as it concerns only one cell type that is not involved in neural networking. Transplantation of RPE has been studied exten-sively in animal models (Lund et al. 2001).The most commonly used animal model of retinal degeneration is the Royal College of Surgeons (RCS) rat model, in which primary dysfunction of the RPE occurs as a result of a mutation in the receptor tyrosine kinase gene M ertk(D’Cruz et al. 2000). This leads to impaired phagocytosis of shed photoreceptor outer segments, with sec-ondary degeneration and progressive loss of pho-toreceptors within the fi rst months of life. It was reported that rat and human RPE cells rescued photoreceptor cells from degeneration when transplanted into the subretinal space of RCS rats (Li and Turner 1988; Coffey et al. 2002).The ability of transplanted RPE cells to restore retinal structure and function has been demonstrated in clinical trials. In humans, autologous transplanta-tions of peripheral RPE as well as macular trans-locations onto more peripheral RPE provide a proof that positioning the macula above relatively91 9 Differentiation of Human Pluripotent Stem Cells into Retinal Cellshealthier RPE cells can improve visual functionin AMD patients (Binder et al. 2004; da Cruz et al. 2007). Nevertheless, the surgical procedures for autologous grafting are challenging and are often accompanied by signifi cant complications. In addition, autologous RPE transplants may carry the same genetic background, environmen-tal toxic and aging-related effects that may have led to macular RPE failure and the development of AMD in the patient. It is also problematic to use autologous cells when all the RPE cells are damaged. Cell sources that can be used for such therapy include allogeneic fetal and adult RPE (Weisz et al. 1999; Binder et al. 2004; da Cruz et al. 2007). However, the use of fetal or adult retinal tissues for transplantation is severely lim-ited by ethical considerations and practical prob-lems in obtaining sufficient tissue supply. The search for a cell source to replace autologous RPE such as immortalized cell lines, umbilical cord-derived cells as well as bone marrow-derived stem cells continues.T he derivation of hESCs more than a decade ago has raised immense interest in the potential clinical use of the cells for regeneration (Thomson et al. 1998; Reubinoff et al. 2000).Along the years, signifi cant progress has been made towards the use of hESCs in clinical trials.T he other promising source of cells for transplantation therapy is iPSCs that are simi-lar to hESCs in their stemness characteristics and pluripotency. These cells could be gener-ated from different human somatic cells by transduction of four defi ned transcription fac-tors: Oct3/4, Sox2, Klf4, and c-Myc (Takahashi et al. 2007).G eneration of RPE and neural retina from hESCs and iPSC has numerous advantages, as it can be done from pathogen-free cell lines under good manufacturing practice (GMP) conditions with minimal variation among batches. Such cells can be characterized extensively prior to preclinical studies or for clinical applications, and an unlimited numbers of donor cells can be generated from them. In the following para-graphs, strategies for induction of differentiation of hESCs and iPSCs towards RPE and neural retina fate are reviewed. D ifferentiation into Retinal Pigment EpitheliumI t was reported for the fi rst time in mice and pri-mates that the differentiation of ES cells into RPE could be induced by co-culture with PA6 stromal cells (Kawasaki et al. 2002; Haruta et al. 2004). The resulting cells had polygonal epithelial mor-phology and extensive pigmentation. The cells expressed the markers that are characteristic of RPE. They developed typical ultrastructures and exhibited some functions of RPE. The differenti-ation of hESC into RPE was first reported by Klimanskaya et al. (2004).According to their protocol, hESCs underwent spontaneous differ-entiation by overgrowth on mouse embryonic fibroblasts (MEF), in feeder-free conditions or, alternatively, as embryoid bodies (EBs) in com-bination with withdrawal of bFGF from the medium. The yield of the formation of RPE cells after 4–8 weeks of spontaneous differentiation was relatively low; for example,<1% of EBs con-tained pigmented cells at this stage. However, after 6–9 months in culture, all the EBs contained pigmented cells. The areas of pigmented cells could be further isolated mechanically and prop-agated by passaging as RPE lines. Klimanskaya and colleges characterized the hESC-derived RPE cells by transcriptomics and demonstrated their higher similarity to primary RPE tissue than to human RPE lines D407 and ARPE-19. The low yield of spontaneously differentiating RPE cells was improved by induction of differentia-tion with Wnt and Nodal antagonists, Dkk1 and LeftyA, respectively, the factors that are sug-gested to promote retinal differentiation. This treatment gave rise to pigmented cells within 38% of the hESC colonies after 8 weeks (Osakada et al. 2008). Immunostaining with the ZO-1 anti-body showed that by day 120, hESC-derived pig-mented cells formed tight junctions (about 35% of total cells). We showed that differentiation toward the neural and further toward the RPE fate could be augmented by vitamin B3 (nicotin-amide; Idelson et al. 2009).We further showed that Activin A, in the presence of nicotinamide, effi ciently induces and augments differentiation92M. Idelson and B. Reubinoffinto RPE cells. This is in line with the presumed role of Activin A in RPE development i n vivo .In the embryo, extraocular mesenchyme-secreted members of the TGF b superfamily are thought to direct the differentiation of the optic vesicle into RPE (Fuhrmann et al. 2000).Under our culture conditions, when the cells were grown in suspen-sion as free-fl oating clusters, within 4 weeks of differentiation, 51% of the clusters contained pigmented areas and about 10% of the cells within the clusters were pigmented. When we modifi ed the differentiation conditions to includea stage of monolayer culture growth, the yield of the RPE-like pigmented cells was signifi cantly improved and 33% of the cells were pigmented after 6 weeks of differentiation. The derivation of RPE from hESCs and iPSCs without any external factor supplementation was also demonstrated by other groups (Vugler et al. 2008 ; Meyer et al. 2009 ; Buchholz et al. 2009).T he hESC-derived RPE cells were extensively characterized, including demonstration, both at the mRNA and the protein levels, of the expres-sion of RPE-specifi c markers, such as RPE65, CRALBP, Bestrophin, Tyrosinase, PEDF, PMEL17, LRAT, isoforms of MiTF abundant in RPE, and others. The cells expressed markers of tight junctions that join the adjacent RPE cells: ZO-1, occludin and claudin-1 (Vugler et al. 2008 ) . Electron microscopic analysis revealed that the hESC-derived RPE cells showed features characteristic of RPE. The cells were highly polarized with the nuclei located more basally, and the cytoplasm with the mitochondria and melanin granules of different maturity more api-cally. A formation of basal membrane was observed on the basal surface of the RPE cell. Similar to putative RPE, the hESC-derived RPE basal membrane was shown to be composed of extracellular matrix proteins, collagen IV , lami-nin and fi bronectin (Vugler et al.2008).The appearance of apical microvilli was demonstrated at the apical surface of the RPE. The presence of tight and gap junctions on the apical borders of the RPE cells was also confi rmed by electron microscopy. O ne of the most important functions of RPE cells i n vivo is phagocytosis of shed photoreceptor outer segments, as part of the continuous renewal process of rods and cones. The hESC-derived RPE cells demonstrated the ability to phagocyto-size latex beads or purifi ed photoreceptor outer segments, confi rming that these cells are func-tionali n vitro . It may be concluded from all these studies that human pluripotent stem cells have a potential to give rise to pigmented cells exhibiting the morphology, marker expression and functionof authentic RPE.D ifferentiation into Retinal Progenitors and Photoreceptors O ur group showed, for the fi rst time, the potential of highly enriched cultures of hESC-derived neu-ral precursors (NPs) to differentiate towards the neural retina fate (Banin et al. 2006).We demon-strated that the NPs expressed transcripts of key regulatory genes of anterior brain and retinal development. After spontaneous differentiation i n vitro , the NPs gave rise to progeny expressing markers of retinal progenitors and photoreceptor development, though this was uncommon and cells expressing markers of mature photorecep-tors were not observed. We showed that after transplantation into rat eyes, differentiation into cells expressing specifi c markers of mature photoreceptors occurred only after subretinal transplantation (between the host RPE and pho-toreceptor layer) suggesting that this specifi c microenvironment provided signals, yet unde-fi ned, that were required to support differentia-tion into the photoreceptoral lineage.P rogress towards controlling and inducing the differentiation of hESCs into retinal progenitors and neurons i n vitro was reported in the study of Lamba et al. ( 2006).They treated hESC-derived EBs for 3 days with a combination of factors,including Noggin, an inhibitor of BMP signaling, Dkk1, a secreted antagonist of the Wnt signaling pathway and insulin-like growth factor 1 (IGF-1), which is known to promote retinal progenitor dif-ferentiation. The cultivation of EBs with these factors was followed by differentiation on Matrigel or laminin for an additional 3 weeks in the presence of the combination of the three93 9 Differentiation of Human Pluripotent Stem Cells into Retinal Cellsfactors together with bFGF. Under these culture conditions, the majority of the cells developed the characteristics of retinal progenitors and expressed the specifi c markers Pax6 and Chx10 (82% and 86% of the cells, respectively). The authors showed that after further differentiation, the cells expressed markers of photoreceptor development Crx and Nrl (12% and 5.75%, respectively). About 12% of the cells expressed also HuC/D, the marker of amacrine and ganglion cells. The expression of markers of the other sub-types of retinal neurons was demonstrated, as well. However, only very few cells (<0.01%) expressed markers of mature photoreceptors, blue opsin and rhodopsin. The abundance of cells expressing markers of photoreceptors could be accelerated by co-culture with retinal explants, especially when the explants originated from mice bearing a mutation that causes retinal degeneration.T o better characterize the phenotype of retinal cells obtained with this differentiation protocol, a microarray-based analysis comparing human retina to the hESC-derived retinal cells was per-formed (Lamba and Reh 2011).It was demon-strated that gene expression in hESC-derived retinal cells was highly correlated to that in the human fetal retina. In addition, 1% of the genes that were highly expressed in the hESC-derived cultures could be attributed to RPE and ciliary epithelium differentiation.A n alternative protocol for the derivation of retinal progenitors and photoreceptors was pro-posed by Osakada et al. (2008).Similar to the protocol for the derivation of RPE cells, they used serum-free fl oating cultures in combination with the Dkk1 and LeftyA. After 20 days of cul-ture in suspension, the cells were replated on poly-D-lysine/laminin/fi bronectin-coated slides. Osakada and co-authors demonstrated that on day 35 in culture, about 16% of colonies were positive for retinal progenitor markers Rx and Pax6. Differentiation towards photoreceptor fate was augmented in the presence of N2 by treat-ment with retinoic acid and taurine, which are known inducers of rod fate differentiation. Under these conditions, after an extended culture period of 170 days, about 20% of total cells were positive for Crx, an early photoreceptor marker. On day 200, about 8.5% of the cells expressed the mature rod photoreceptor marker, rhodopsin, as well as cone photoreceptor markers, red/green and blue opsins (8.9% and 9.4%, respectively).A n alternative approach was proposed by the same group based on the use of small molecules. In this method, the chemical inhibitors CKI-7 and SB-431542 that inhibit Wnt and Activin A signaling, respectively, and Y-27632, the Rho-associated kinase inhibitor, which prevents disso-ciation-induced cell death, were used. These molecules were shown to mimic the effects of Dkk1 and LeftyA (Osakada et al. 2009).This strategy, which doesn’t involve the use of recom-binant proteins which are produced in animal or E scherichia coli cells, is more favorable for the gen-eration of cells for future transplantation therapy.I n another study that was published by Meyer et al .(2009), after initial differentiation in sus-pension for 6 days, the aggregates were allowed to attach to laminin–coated culture dishes. After further differentiation as adherent cultures, neu-roepithelial rosettes were formed, which were mechanically isolated and subsequently culti-vated as neurospheres. The authors didn’t use any soluble factors; moreover, they showed that under these conditions, the cells expressed endogenous Dkk1 and Noggin. They also demonstrated that in concordance with the role of bFGF in retinal specifi cation, the inhibition of endogenous FGF-signaling abolished retinal differentiation. Under their differentiation protocol, by day 16, more than 95% of the cells expressed the retinal pro-genitor markers, Pax6 and Rx. The authors dem-onstrated that by day 80 of differentiation, about 19% of all neurospheres contained Crx+ cells and within these Crx+ neurospheres, 63% of all cells express Crx and 46.4% of the cells expressed mature markers, such as recoverin and cone opsin.I n all of the above studies, differentiated cells expressing the retinal markers were obtained; however, the cells were not organized in a three-dimensional retinal structure. In a paper recently published by Eiraku et al. (2011),the authors cul-tured free-fl oating aggregates of mouse ES cells in serum-free medium in the presence of base-ment membrane matrix, Matrigel, that could also94M. Idelson and B. Reubinoffbe substituted with a combination of laminin, entactine and Nodal. Using a mouse reporter ES cell line, in which green fl uorescent protein (GFP) is knocked in at the Rx locus, the authors showed that Rx-GFP+ epithelial vesicles were evaginated from the aggregates after 7 days of differentiation under these conditions. On days 8–10, the Rx-GFP+ vesicles changed their shape and formed optic cup-like structures. The inner layer of these structures expressed markers of the neural retina whereas the outer layer expressed markers of RPE. The authors demonstrated that differen-tiation into RPE required the presence of the adjacent neuroectodermal epithelium as a source of diffusible inducing factors. In contrast, the differentiation into neural retina did not require tissue interactions, possibly because of the intrinsic inhibition of the Wnt-signaling pathway. Eiraku and colleagues showed that the retinal architecture, which was formed within the optic vesicle-like structures, was comparable to the native developing neural retina.R ecently, optic vesicle-like structures were also derived from hESCs and iPSCs using the protocol described above, which is based on iso-lating the neural rosette-containing colonies and culturing them in suspension (Meyer et al. 2011). The cells within the structures expressed the markers of retinal progenitors, and after differen-tiation gave rise to different retinal cell types. It was shown that the ability of optic vesicle-like structures to adopt RPE fate could be modulated by Activin A supplementation. The production of these three-dimensional retinal structures opens new avenues for studying retinal development in normal and pathological conditions.T ransplantation of Pluripotent Stem Cell-Derived Retinal CellsA key step towards future clinical transplanta-tions of hESC-derived RPE and neural retina is to show proof of their therapeutic potential i n vivo. Various animal models of retinal degeneration have been used to evaluate the therapeutic effect of transplanted retinal cells. Human ESC-derived RPE cells were transplanted subretinally to the degenerated eyes of RCS rats. Transplantation of the hESC-derived RPE cells between the RPE and the photoreceptor layer rescued retinal struc-ture and function (Lund et al. 2006; Vugler et al. 2008; Idelson et al. 2009; Lu et al. 2009).The subretinally engrafted hESC-derived RPE cells salvaged photoreceptors in proximity to the grafts as was shown by the measurement of the thick-ness of the ONL, the layer of photoreceptor nuclei, which is an important monitor of photore-ceptor cell survival. The ONL thickness was significantly increased in transplanted eyes in comparison to the degenerated non-treated eyes.I n order to evaluate the functional effect of transplanted cells i n vivo, the electroretinography (ERG) that directly measures the electrical activ-ity of the outer (a-wave) and inner (b-wave) retina in response to light stimulation was used. It was demonstrated that after transplantation of hESC-derived RPE, ERG recordings revealed a signifi -cant preservation of retinal function in the treated eyes as compared to control untreated eyes (Lund et al. 2006; Idelson et al. 2009).The visual func-tion of the animals was also estimated by an optomotor test, which monitors the animal’s refl exive head movements in response to a rotat-ing drum with fi xed stripes. Animals transplanted with hESC-derived RPE showed signifi cantly better visual performance in comparison to con-trol animals (Lund et al. 2006; Lu et al. 2009). The presence of rhodopsin, a major component of photoreceptor outer segments, within the sub-retinaly transplanted pigmented cells suggested that they could perform phagocytosis i n vivo (Vugler et al. 2008; Idelson et al. 2009).B ridging the gap between basic research and initial clinical trials requires immense resources to ensure safety and efficacy. Human ESC-derived RPE cell lines were generated using a current Good Manufacturing Practices (cGMP)-compliant cellular manufacturing process (Lu et al. 2009). Long-term studies analyzing safety and efficacy of transplantation of these GMP-compliant hESC-derived RPE cells revealed that the subretinally transplanted cells survived for a period of up to 220 days and provided prolonged functional improvement for up to 70 days after transplantation. The potential of the hESC-derived。

表观遗传学和磷酸化-蛋白质磷酸化-分子生物学课件.ppt

表观遗传学和磷酸化-蛋白质磷酸化-分子生物学课件.ppt
DNA甲基化是指在甲基化酶的作用下,将一个 甲基添加在DNA分子的碱基上。
DNA甲基化修饰决定基因表达的模式,即决定 从亲代到子代可遗传的基因表达状态。
DNA甲基化的部位通常在CpG岛的胞嘧啶
胞嘧啶
DNMT1
S-腺苷 甲硫氨 酸SAM
5-甲基胞嘧啶
胞嘧啶甲基 化反应
真核生物细胞内存在两种甲基化酶活性:
组蛋白甲基化可以与基因抑制有关,也 可以与基因的激活相关,这往往取决于 被修饰的赖氨酸处于什么位置。
组蛋白修饰主要是氨基端的甲基化修饰和(或) 乙酰化修饰,特定组蛋白的氨基酸残基被甲基 化和(或)乙酰化可以最终激活基因的表达,反 之则抑制基因的表达。
特定组蛋白羧基端的泛素化同样影响蛋白质的 降解过程,从而也可调节基因的表达。
(2)糖原合成酶—P—失活
使许多蛋白质磷酸化 (1)核中组蛋白磷酸化—加速核酸的复制,转录。 (2)核糖体蛋白质磷酸化—加速蛋白质合成通性。 (3)使膜蛋白磷酸化—加速物质的转运。
蛋白质磷酸化在细胞信号转导中的作用
(1). 在胞内介导胞外信号时具有专一应答特点。与信号传递有关的蛋白激酶类主要受控于 胞内信使,
根据是否有调节物来分又可分成两大类: 信使依赖性蛋白质激酶(messenger-dependent protein
kinase),包括胞内第二信使或调节因子依赖性蛋白激酶及激 素(生长因子)依赖性激酶两个亚类;非信使依赖型蛋白激酶。
蛋白激酶的催化作用: 使调节酶磷酸化 (1)磷酸化酶激酶—P—激活
基因表达的重新编程
已完全分化的细胞,其基因组在特定条件下经 历表观遗传修饰重建而为胚胎发育中的基因表 达重新编程(reprogramming)并赋予发育全能 性,为胚胎发育和分化发出正确的指令。

山中伸弥发明IPS细胞的介绍及启示

山中伸弥发明IPS细胞的介绍及启示

数学统计:Pearson相 检查,发现其能够分化 Figure 5. Pluripotency of
关分析
为多种组织器官,说明 iPS Cells Derived from
其具有多能性。
MEFs
论点
证明方法
证明过程
论文对应部分
不仅是小鼠胚胎成纤 维细胞(MEFs)可被 诱导,其他分化程度
很高的体细胞也可被 诱导。
同上
将这四种因子又一起 Figure 6. 导入了小鼠尾尖成纤 Characterization of iPS 维细胞(TTFs),并进 Cells Derived from 行了与之前相同的检 Adult Mouse Tail-Tip 测,发现同样可以诱 Fibroblasts 导形成IPS细胞。
胞的多功能干细胞。 表观遗传学:染色质免 之处和差异发现ips细 iPS Cells
疫沉淀分析
胞只是类似ES细胞; Figure 4. Global Gene-
组织学:裸鼠皮下移植、同时对IPS细胞发育形 Expression Analyses by
免疫组化染色
成的畸胎瘤进行组织学 DNA Microarrays
组合在一起才能完成诱 导,其余情况均不可。
将24种因子全部加入小 Figure 1. Generation of 鼠MEF细胞中进行诱导 iPS Cells from MEF 可以得到IPS细胞,然 Cultures via 24 Factors 后将每种因子逐个去除,Figure 2. Narrowing
观察去除掉该因子后细 down the Candidate
胞能否诱导成功,最终 Factors 筛选出4个。
通过这四种转录因子的 分子生物学:RT-PCR、 研究IPS和ES在细胞形 Figure 3. Gene-

髓母细胞瘤的放射治疗

髓母细胞瘤的放射治疗

影响预后得因素
• 年龄 • 临床分级 • 术式 • 后颅窝生物有效剂量(BED) • 放疗持续时间
and stem-cell rescue in children with newly diagnosed medulloblastoma (St
Jude Medulloblastoma-96): long-term results from a prospective, multicentre
trial
Stage Chang's M staging system
Hale Waihona Puke Low-risk Localized disease at the time of diagnosis Group Age >3 years
Total tumor resection or subtotal with residual tumor <1、5 cm3 High-risk Disseminated disease at the time of diagnosis Group
大家有疑问的,可以询问和交流
可以互相讨论下,但要小声点
Craniospinal irradiation (CSI):dose
radiotherapy alone Chemotherapy+
(5-year EFS)
(5-year EFS)
standard radiotherapy
60% ± 7、8%
髓母细胞瘤的放射治疗
临床表现
• 颅内压增高:头痛、呕吐、视神经乳头水肿 • 小脑损害:躯干性共济失调为主 • 其她:复视、面瘫、强迫头位、头颅增大、病
理反射阳性、呛咳、小脑危象、蛛网膜下腔出 血 • 脊髓转移灶症状:背部或双下肢痛、进行性加 重得截瘫或四肢瘫

树突状细胞在肿瘤免疫和免疫治疗中的研究进展

树突状细胞在肿瘤免疫和免疫治疗中的研究进展
T细胞的效应因子活性取决于 DCs产生的细胞因子,包 括白介素 -12(IL-12)和Ⅰ型干扰素[17]。尽管 MoDCs在受 到抗原刺激后 可 产 生 IL-12[18],但 IL-12主 要 由 DCs产 生,并促进Ⅰ型辅助性 T细胞(TH1细胞)和 CD8+T细胞活 化[19]。CD141+cDC1s和 CD1c+cDC2s均 可 在 Toll样 受 体 (TLR)刺激后产生 IL-12[20-21],并且肿瘤中的 IL-12水平 升高与 cDC1s浸润增加有关[22]。临床上已经使用Ⅰ型干扰 素治疗肿瘤患者[23],并且通过 cGAS-STING途径促进抗肿 瘤免疫中 DCs的激活和 Ⅰ 型干扰素的产生[24-25]。在 TME 中,DCs可以产生招募 T细胞的趋化因子。例如,肿 瘤 浸 润 的 cDC1s主 要 产 生 CXC趋 化 因 子 配 体 9(CXCL9)和 CXCL10,促进 CD8+T细胞向 TME募集[25]。总之,DCs通过 可溶性因子调节 TME,以及募集 T细胞和介导抗肿瘤 T细胞 的激活,在抗肿瘤免疫中起着核心作用。 1.2 DCs促进肿瘤免疫耐受
ModernOncology2021,29(14):2543-2547
【指示性摘要】树突状细胞(dendriticcells,DCs)是一类特殊的抗原呈递细胞,在先天和适应性免疫应答的启 动和调节中起关键作用。了解 DCs的功能以及肿瘤微环境对 DCs的影响,有助于开发新的癌症治疗策略。 通过调节 DCs的功能改善肿瘤免疫治疗的疗效是目前研究的热点。大量研究表明,靶向肿瘤微环境中的 DCs 是癌症治疗极具前景的方法。本文重点讨论 DCs在肿瘤免疫中的主要功能及靶向 DCs在癌症中的治疗潜 力。 【关键词】树突状细胞;肿瘤微环境;免疫应答;免疫治疗 【中图分类号】R730.51 【文献标识码】A DOI:10.3969/j.issn.1672-4992.2021.14.032 【文章编号】1672-4992-(2021)14-2543-05

不一样的“棒”!科学家首次发现Weibel-Palade小体可分泌外泌体!《Blood》重磅

不一样的“棒”!科学家首次发现Weibel-Palade小体可分泌外泌体!《Blood》重磅

不一样的“棒”!科学家首次发现Weibel-Palade小体可分泌外泌体!《Blood》重磅APExBIO近日,国际著名期刊《Blood》(IF=15.132)报道了一项重磅研究,来自英国MRC-格拉斯哥大学病毒研究中心的研究人员发现血管内皮细胞中的Weibel-Palade小体含有外泌体,并通过胞吐作用将其释放到细胞外环境中。

在这之前,并未报道Weibel-Palade小体可以储存和释放外泌体,该项研究揭示了一条从内皮细胞释放细胞外囊泡的新途径,对外泌体相关研究和疾病研究具有重要意义。

研究论文题目为:“Stimulated release of intraluminal vesicles from Weibel-Palade bodies”。

▲Blood. 14 February 2019.▲不一样的“棒”——Weibel-Palade小体,箭头所指绿色。

箭头所指红色为腔内囊泡。

Weibel-Palade小体(Weibel-Palade body,以下简称WPB)是血管内皮细胞中的一种特殊的分泌性杆状(棒状)细胞器,它们储存和释放两种主要分子——血管性血友病因子(von Willebrand Factor,vWF)和P-选择素(P-selectin),在止血和炎症中发挥重要作用。

外泌体(Exosome)是一种特殊类型的细胞外囊泡,大多数细胞可分泌,具有脂质双层膜结构,直径在30-100 nm之间。

外泌体存在于血液、唾液、尿液、脑脊液和乳汁等体液中。

一直以来,人们把WPB分类成溶酶体相关细胞器,因为它具有酸性腔和表达CD63(溶酶体相关膜蛋白3:LAMP3)。

WPB帮助递送要溶解的内容物,也参与调节细胞的胞吐作用(exocytosis),尽管有研究报道WPB与其他组分共同控制外泌体的释放,但尚不清楚WPB本身是否含有或释放外泌体。

细胞在分泌外泌体之前会在内腔先生成腔内囊泡(ILV),细胞的四跨膜蛋白如CD63参与外泌体的形成过程并富集在多泡小体(MVB)与质膜融合形成的外泌体膜上,因此CD63被广泛用作识别和跟踪腔内囊泡/外泌体。

肿瘤免疫治疗相关不良反应管理课件

肿瘤免疫治疗相关不良反应管理课件
排除irAEs
不能排除irAEs
治疗肿瘤/感染
irAEs+抗感染
合并用药?
超进展、进展、合并用药、合并症
.
irAE的处理原则:1度不用激素、2度口服激素,3度静脉激素,4度静脉激素(必要时冲击治疗)+ICU支持
分级
糖皮质激素
其他免疫抑制剂
免疫治疗
G1-轻度
不推荐
不推荐
推荐继续使用
G2-中度
KeytrudaPembrolizumab
MSD
PD-1抗体
黑色素瘤、无驱动基因突变非小细胞肺癌、头颈鳞癌、经典型霍奇金淋巴瘤、纵隔大B细胞淋巴瘤、头颈鳞癌、膀胱癌、肝细胞癌、胃癌、携带微卫星不稳定或错配修复基因缺陷的实体瘤、宫颈癌、食管癌
Libtayo cemiplimab-rwlc
Sanofi
PD-1抗体
癌症治疗的支柱
免疫检查点抑制剂(IO):癌症治疗的支柱之一
手术
放疗
化疗
靶向
免疫
IO
.
IO
免疫检查点抑制剂的作用机制:“刹车理论”
.
国外获批的免疫检查点抑制剂及其适应症
药物/商品名
公司
分类
已批准的适应症
Opdivo Nivolumab
BMS
PD-1抗体
黑色素瘤、无驱动基因突变非小细胞肺癌、肾细胞癌、经典型霍奇金淋巴瘤、头颈鳞癌、膀胱癌、胃癌、肝细胞癌、携带微卫星不稳定或错配修复基因缺陷的大肠癌
定义1
免疫抑制剂阻断T细胞负性调控信号解除免疫抑制,增强T细胞抗肿瘤效应的同时,也可能异常增强自身正常的免疫反应,导致免疫耐受失衡,累及到正常组织时表现出自身免疫样的炎症反应,称为免疫相关的不良反应(immune-related adverse events, irAEs)。

水痘减毒活疫苗初次免疫后11年免疫持久性研究

水痘减毒活疫苗初次免疫后11年免疫持久性研究

doi:10.3969/j.issn.1000⁃484X.2020.20.025㊃信息速递㊃水痘减毒活疫苗初次免疫后11年免疫持久性研究黄莉荣 金 莎①② 耿 钊① 莫兆军③ 陶 航①③ (广西壮族自治区疾病预防控制中心,南宁530028) 中图分类号 R186 R511.5 文献标志码 A 文章编号 1000⁃484X (2020)20⁃2558⁃04①长春百克生物科技股份公司,长春130012㊂②共同第一作者㊂③通讯作者,E⁃mail:mozhj@;E⁃mail:donglei9518@㊂作者简介:黄莉荣,女,副主任医师,主要从事疫苗临床方面的研究㊂[摘 要] 目的:观察水痘减毒活疫苗(VarV)受试者初次免疫后11年的抗体几何平均滴度(GMT)和阳性率,并评价其免疫持久性㊂方法:选择2006年9月于广西省永福县开展的VarV 随机㊁双盲Ⅲ期临床试验中接种1针剂国产VarV 的受试者100名作为试验组,50名接种1针剂进口VarV(平行试验)的受试者作为对照组;采集初次免疫后11年的静脉血3.0ml,膜抗原荧光抗体法检测血清中水痘病毒抗体,计算血清抗体阳性率㊁抗体GMT 和不同中和抗体滴度人数的百分比,并与初次免疫后30d 抗体水平进行比较㊂结果:VarV 初次免疫后11年,2组全人群㊁幼儿和儿童抗体GMT 分别为37.01㊁42.31㊁28.21和22.63㊁22.83㊁21.93,抗体阳性率分别为98.00%㊁100.00%㊁93.94%和94.00%㊁92.31%㊁100.00%,中和抗体滴度≥4的人数占比分别为98.00%㊁100.00%㊁93.94%和94.00%㊁92.31%㊁100.00%;试验组幼儿的GMT㊁抗体阳性率及中和抗体滴度≥4的人群数占比明显高于对照组(P <0.05);2组全人群㊁幼儿及儿童的抗体GMT 均明显低于初免后30d(P <0.05),但抗体阳性率仍保持较高水平(≥92.31%)㊂结论:VarV 免疫后11年仍具有较好的免疫原性,但抗体GMT 水平随时间推移呈明显下降趋势㊂[关键词] 水痘减毒活疫苗;免疫持久性;几何平均滴度;抗体;阳性率Immune persistence of Live Attenuated Varicella Vaccine after 11years of primary immunizationHUANG Li⁃Rong ,JIN⁃Sha ,GENG Zhao ,MO Zhao⁃Jun ,TAO Hang .Center for Disease Control and Prevention of Guangxi ,Nanning 530028,China[Abstract ] Objective :To observe geometric mean titer (GMT)and positive rate of varicella antibody after 11years of subjectsof primary immunization of freeze⁃dried varicella attenuated live vaccine(VarV),and its immune persistence was evaluated.Methods :100subjects inoculated one dose of domestic VarV as experiment group and 50subjects who inoculated one dose of imported VarV(parallel trial)as control group who were recruited from randomized,blind clinical trial phase Ⅲconducted in Yongfu County,Guangxi province in September 2006.After 11years of primary immunization,3.0ml of venous blood was collected,varicella virus antibody in serum was detected by fluorescent antibody to membrane antigen method,positive rate of serum antibody was calculated,GMT of antibody and percentage of people with different neutralizing antibody titer were counted,which were compared with antibody level 30d after primary immunization.Results :11years after primary immunization,GMT of antibody in whole population,infant,and children groups of two groups were 37.01%,42.31%,28.21%and 22.63%,22.83%,21.93%,positive rates were 98.00%,100.00%,93.94%and 94.00%,92.31%,100.00%,percentage of neutralizing antibody titers ≥4was 98.00%,100.00%,93.94%and 94.00%,92.31%,100.00%,respectively;GMT,antibody positive rate and percentage of neutralizing antibody titer ≥4in whole pop⁃ulation and children in trial group were significantly higher than those in control group(P <0.05);GMT of VZV antibody in whole popu⁃lation,infant,and children groups of two groups were significantly lower than that of 30d after primary immunization(P <0.05),butantibody positive rate is still high (≥92.31%).Conclusion :VarV still had good immunogenicity after 11years of primaryimmunization,but antibody GMT level decreased with time.[Key words ] Varicella attenuated live vaccine;Immune persistence;GMT;Antibody;Positive rates 水痘是由水痘⁃带状疱疹病毒(varicella⁃zostervirus,VZV)感染引起的一种急性㊁传染性极强的病毒性疾病,在全世界范围内均有传播,高发于儿童,极易在人群聚集场所暴发流行,临床上尚无有效治疗方法[1,2]㊂水痘减毒活疫苗(varicella attenuated live ㊃8552㊃中国免疫学杂志2020年第36卷vaccine,VarV)是预防和控制水痘最有效和最可靠的手段[3]㊂自1974年VarV 被开发用于水痘的预防接种以来,水痘得到了有效控制,其安全性和有效性得到了广泛认可[4⁃6]㊂为进一步观察VarV 的有效性,我中心于2006年9月承担了国产某VarV Ⅲ期临床试验,在观察该疫苗有效性的同时,于2017年9月~2018年4月对VarV 初次免疫(简称初免)后11年的免疫持久性进行研究,并与进口VarV 的持久性进行了比较,旨为该疫苗的预防接种提供参考㊂1 资料与方法1.1 资料1.1.1 选择2006年9月参加广西省永福县开展的VarV 随机㊁双盲Ⅲ期临床试验的受试者100名作为试验组,接种1针剂国产VarV(长春百克生物科技股份公司),另选取50名接种1针剂进口VarV(葛兰素史克生物制品有限公司)的受试者作为对照组㊂所有受试者及其监护人知情同意,同时在VarV 初次免疫后11年中未进行VarV 二次免疫㊁无水痘和/或带状疱疹疾病史㊂1.1.2 排除标准 近3个月内有免疫球蛋白接种史㊁接受抗肿瘤制剂和免疫调节剂者;患免疫缺陷症㊁接受免疫抑制剂治疗者;参加任何其他药物临床研究者;可能影响试验评估的任何情况等㊂1.2 方法 采集2组入选者初次免疫后11年静脉血3.0ml,分离血清㊂采用膜抗原荧光抗体法(fluorescent antibody to membrane antigen,FAMA)检测血清中VZV 抗体水平,统计抗体几何平均滴度(geometric mean titer,GMT),计算血清抗体阳性率和不同中和抗体滴度人数的百分比,血清中抗体滴度<1∶4为阴性,≥1∶4为阳性㊂1.3 统计学分析 采用EpiData 软件和CRF 表编制㊁录入数据和建立数据库;采用SAS 9.4软件对数据进行统计学分析,行双侧检验;抗体滴度数据采用几何均数及95%CI 进行统计描述,组间比较采用t 检验,计量资料以x ±s 表示,以P <0.05为差异有统计学意义㊂2 结果2.1 基本情况 试验组中,6~12岁儿童67人㊁1~5岁幼儿33人,其中男性51人㊁女性49人;对照组中,6~12岁儿童39人㊁1~5岁幼儿11人,男性27人㊁女性23人㊂2组研究对象年龄中位数均为3,性别㊁年龄及人群(儿童和幼儿)比例差异均无统计学意义(P >0.05),具有可比性㊂见表1㊂2.2 免疫原性2.2.1 初免后11年抗体GMT 和抗体阳性率 VarV 初免后11年,2组全人群及幼儿㊁儿童的VZV抗体GMT 均≥21.93㊁抗体阳性率均≥92.31%;试验组幼儿GMT 水平和抗体阳性率均明显高于对照组(P <0.05)㊂见表2㊁3㊂2.2.2 2组受试者初免后11年抗体水平与初免后30d 比较 VarV 初免后11年,试验组全人群㊁幼儿组及儿童的抗体GMT 水平均明显高于对照组(P <0.05),初免后30d,2组受试者人群㊁幼儿及儿童抗体GMT 水平差异无统计学意义(P >0.05)㊂见表4㊂2.2.3 初免后11年中和抗体滴度分布情况 初免11年后,2组全人群及幼儿㊁儿童中和抗体滴度≥4的人数百分比均≥92.31%;试验组幼儿的中和抗体滴度≥4的人数百分比(100.00%)明显高于对照组(92.31%)(P =0.0474),见表5㊁图1㊂表1 2组受试者基本情况[x ±s ,例(%)]Tab.1 Basic condition of objectives of two groups [x ±s ,n (%)]Index Trial group (n =100)Control group (n =50)t /χ2PSex 0.1200.7288Male 51(51.00)27(54.00)Female 49(49.00)23(46.00)Age4.33±3.153.64±2.59-1.3380.1829Median 33Minimum maximum 1.00,11.001.00,10.00Population Infant 67(67.00)39(78.00)1.9460.1630Children33(33.00)11(22.00)表2 2组VarV 初次免疫后11年VZV 抗体GMT (1∶x )Tab.2 VZV antibody GMT (1∶x )of 11years after VarV primary immunization of two groupsGroupsTrial group n GMT 95%CI Control groupn GMT 95%CI P Whole population 10037.0127.80-49.285022.6314.96-34.230.0515Infant 6742.3130.13-59.423922.8314.00-37.220.0351Children3328.2116.43-48.451121.938.98-53.520.6274㊃9552㊃黄莉荣等 水痘减毒活疫苗初次免疫后11年免疫持久性研究 第20期表3 2组VarV 初免疫后11年抗体阳性率(%)Tab.3 Antibody positive rate of 11years after VarV primary immunization of two groups (%)GroupsTrial groupn Positive rates 95%CI Control groupn Positive rates 95%CI P Whole population 9898.0092.96-99.764794.0083.45-98.750.3338Infant 67100.0094.64-100.003692.3179.13-98.380.0474Children3193.9479.77-99.2611100.0071.51-100.001.0000表4 2组VarV 初免后11年抗体水平与初免后30d 比较Tab.4 Comparison of antibody levels between 11years and 30d after VarV primary immunizationGroups30d GMT(1:x)Trial group Control group P 11years GMT(1:x)Trial group Control groupP Whole population 147.38154.52>0.0537.0122.63<0.05Infant 112.26125.88>0.0542.3122.83<0.05Children190.31190.42>0.0528.2121.93<0.05表5 2组VarV 初免疫后11年中和抗体滴度[例(%)]Tab.5 Titer of neutralizing antibody in two groups 11years after VarV primary immunization [n (%)]GroupsNeutralizing antibody titers≥4≥8≥16≥32≥64≥128≥256≥512≥1024Whole population Trial group 98(98.00)91(91.00)77(77.00)63(63.00)44(44.00)24(24.00)14(14.00)6(6.00)2(2.00)Control group 47(94.00)39(78.00)34(68.00)27(54.00)14(28.00)7(14.00)3(6.00)2(4.00)2(4.00)P0.33380.02720.23620.28880.05780.15390.14510.71930.6009InfantTrial group 67(100.00)65(97.01)54(80.60)42(62.69)31(46.27)18(26.87)10(14.93)4(5.97)2(2.99)Control group 36(92.31)31(79.49)27(69.23)21(53.85)9(23.08)6(15.38)3(7.69)2(5.13)2(5.13)P0.04740.00470.18370.37140.01750.23060.36471.00000.6239ChildrenTrial group 31(93.94)26(78.79)23(69.70)21(63.64)13(39.39)6(18.18)4(12.12)2(6.06)0(0.00)Control group 11(100.00)8(72.73)7(63.64)6(54.55)5(45.45)1(9.09)0(0.00)0(0.00)0(0.00)P1.00000.69220.72220.72440.73750.65940.55791.000图1 免疫后11年中和抗体滴度逆分布图Fig.1 Inverse distribution of neutralizing antibody titer11years after immunization3 讨论VarV 自用于水痘的预防接种以来,有效降低了健康儿童和VZV 易感人群的水痘发病率和死亡率,减少了住院人数[7⁃9]㊂多位学者对其免疫持久性和保护效果进行了相关研究㊂VarV 免疫持久性研究结果显示,日本VarV 初免后20年GMT㊁抗体阳性率分别为19.4和100%,美国VarV 初免后6年GMT㊁抗体阳性率分别为47.7和100%,我国VarV 初免后5年GMT㊁抗体阳性率分别分别为8.21和88.90%,初免后6年GMT抗体阳性率分别为8.21和88.05%,均具有良好的免疫原性[10⁃13]㊂本研究VarV 结果显示,初免后11年2组全人群的抗体GMT 分别为37.01(国产)㊁22.63(进口),低于美国学者的研究结果,但高于日㊃0652㊃中国免疫学杂志2020年第36卷本和我国学者的研究结果;抗体阳性率(≥94.00%)与日本㊁美国和我国李娜等[14]的研究结果相符,均在90%以上;中和抗体滴度≥4的人数百分比均≥94.00%,表明VarV初免后11年仍具有较好的免疫原性;与免疫后30d的抗体水平相比,虽然抗体阳性率仍保持在较高水平(≥94.00%),但VarV初免后11年2组抗体GMT均明显低于初免后30d(P<0.05),且呈明显下降趋势㊂梁剑等[15]研究发现VarV初免后3年水痘抗体浓度由初免后1年的523.30mU/ml下降至98.00mU/ml,马茂等[16]研究发现VarV初免后3年GMT由初免后1年的1∶11.51下降至1∶7.14,苏家立等[12]研究发现VarV初免后3年GMT由初免后1年的1∶11.11下降至1∶7.50,与本研究结果相符㊂因此,为有效控制水痘的发病率㊁降低突破性病例(VarV接种42d后发生的野生型VZV感染),建议在VarV初次免疫后1~3年内补充接种1针剂㊂本研究结果显示,国产VarV初免后11年幼儿的GMT㊁抗体阳性率和中和抗体滴度≥4的人数百分比均高于进口VarV,表明国产VarV在免疫持久性指标上优于进口VarV㊂参考文献:[1] Varicella and herpes zoster vaccines.WHO position paper,June2014[J].Wkly Epidemiol Rec,2014,89(25):265⁃288. [2] 范仁锋,张吉凯,汤 妍,等.水痘及其疫苗的研究进展[J].华南预防医学,2016,42(4):390⁃395.Fan RF,Zhang JK,Tang Y,et al.Advances in research on chickenpox and its vaccine[J].South China Prev Med,2016,42(4):390⁃395.[3] 张军楠,黄卓英,王 淼,等.中国水痘减毒活疫苗的安全性㊁免疫原性和保护效果[J].中国疫苗和免疫,2018,24(4): 487⁃491.Zhang JN,Huang ZY,Wang M,et al.Safety,immunogenicity and protective efficacy of varicella vaccine in China[J].Chin J Vacc Immunol,2018,24(4):487⁃491.[4] 边国林,唐学雯,史宏辉,等.国产和进口冻干水痘减毒活疫苗的安全性和免疫原性评价研究[J].中国疫苗和免疫,2010, 16(5):435⁃437,468.Bian GL,Tang XW,Shi HH,et al.Study on safety and immunogenicity of imported and domestic varicella attenuated live vaccine(Freeze⁃dried)for children[J].Chin J Vacc Immunol, 2010,16(5):435⁃437,468.[5] 尹志英,来时明,龚晓英,等.国产水痘减毒活疫苗初次和加强免疫的免疫原性和安全性观察[J].中国疫苗和免疫, 2018,24(1):61⁃63.Yin ZY,Lai SM,Gong XY,et al.Immunogenicity and safety of primary and booster vaccination with domestic varicella attenuated live vaccine[J].Chin J Vacc Immunol,2018,24(1):61⁃63. [6] 罗 盛,姜大雷,黄雅铃,等.国产水痘疫苗2剂次及1剂次加强免疫后的免疫原性及安全性[J].中国生物制品学杂志, 2019,32(12):1381⁃1385.Luo S,Jiang DL,Huang YL,et al.Immunogenicity and safety of two⁃dose and booster immunizationschedules of domestic varicella vaccine[J].Chin J Biolog,2019,32(12):1381⁃1385. [7] Guris D,Jumaan AO,Mascola L,et al.Changing varicellaepidemiology in active surveillance sites⁃United States,1995⁃2005 [J].J Infect Dis,2008,197(2):71⁃75.[8] Marin M,Guris D,Chaves SS,et al.Prevention of varicella:Rec⁃ommendations of the Advisory Committee on Immunization Practices(ACIP)[J].MMWR Recomm Rep,2007,56(RR⁃4): 1⁃40.[9] Nguyen HQ,Jumaanu AO,Seward JF.Decline in mortality due tovaricella after implementation of varicella vaccination in the United States[J].N Engl J Med,2005,352:450⁃458.[10] Asano Y,Suga S,Yoshikawa T,et al.Experience and reason:twenty⁃year follow⁃up of protective immunity of the oka strain livevaricella vaccine[J].Pediatrics,1994,94(4):524⁃526. [11] Vessey SJ,Chan CY,Kuter BJ,et al.Childhood vaccinationagainst varicella:Persistence of antibody,duration of protection,and vaccine efficacy[J].J Pediatr,2001,139(2):297⁃304.[12] 苏家立,黄竹航,陈海平,等.国产冻干水痘减毒活疫苗加强免疫的免疫原性和安全性观察[J].中国疫苗和免疫,2016,22(4):366⁃370.Su JL,Huang ZH,Chen HP,et al.Immunogenicity and safety of aone⁃dose booster immunization of chinese freeze⁃dried varicella at⁃tenuated live vaccine[J].Chin J Vacc Immunol,2016,22(4):366⁃370.[13] 马 茂,刘卫民.水痘减毒活疫苗首剂免疫持久性及加强免疫效果分析[J].中国疫苗和免疫,2016,22(2):183⁃186.Ma M,Liu WM.Immune persistence from primary immunizationand effectiveness of booster immunization for varicella attenuatedlive vaccine[J].Chin J Vacc Immunol,2016,22(2):183⁃186.[14] 李 娜,方 凯,马淑玲,等.国内水痘减毒活疫苗预防效果及其安全性的系统评价[J].中国疫苗和免疫,2012,18(1):54⁃60.Li N,Fang K,Ma SL,et al.A systematic review on the preventiveeffect and safety of the varicella attenuated live vaccine in china[J].Chin J Vacc Immunol,2012,18(1):54⁃60. [15] 梁 剑,朱 琦,胡 培,等.99名儿童接种水痘减毒活疫苗免疫持久性的横断面研究[J].现代预防医学,2017,44(4):642⁃645.Liang J,Zhu Q,Hu P,et al.Cross⁃sectional study on99childrenimmunity persistence of varicella live attenuated vaccine[J].Mod Prev Med,2017,44(4):642⁃645.[16] 马 茂,刘卫民,杨 洁,等.深圳市某区2013年水痘疫苗接种儿童抗体水平横断面研究[J].实用预防医学,2015,22(12):1451⁃1453.Ma M,Liu WM,Yang J,et al.Cross⁃sectional study on varicellaantibody levels among children with varicella vaccination historyin a district of Shenzhen City,2013[J].Pract Prev Med,2015,22(12):1451⁃1453.[收稿2020⁃07⁃15](编辑 周文瑜)黄莉荣等 水痘减毒活疫苗初次免疫后11年免疫持久性研究 第20期。

《2024年核糖体蛋白RPS14泛癌分析及对肝癌细胞功能影响的研究》范文

《2024年核糖体蛋白RPS14泛癌分析及对肝癌细胞功能影响的研究》范文

《核糖体蛋白RPS14泛癌分析及对肝癌细胞功能影响的研究》篇一一、引言近年来,癌症已经成为全球公认的严重健康问题之一。

其中,对肿瘤细胞的生物学特性进行深入解析是寻找癌症治疗策略的关键所在。

核糖体蛋白RPS14(Ribosomal Protein S14)作为蛋白质合成的重要部分,其与多种癌症的关系正逐渐受到科学研究的关注。

本文将针对核糖体蛋白RPS14在泛癌中的分析及其对肝癌细胞功能影响的研究进行深入探讨。

二、核糖体蛋白RPS14的泛癌分析1. RPS14的基本属性与功能核糖体蛋白RPS14是构成核糖体的重要组成部分,对于蛋白质的合成过程起着至关重要的作用。

RPS14的表达和调控在各种生物过程中具有重要地位,对于维持细胞的正常生长和分裂至关重要。

2. RPS14在泛癌中的表达情况研究显示,RPS14在多种癌症中表达异常,包括肺癌、乳腺癌、肝癌等。

通过对多种癌症样本的分析,我们发现RPS14的表达水平与癌症的发生、发展密切相关。

3. RPS14与癌症的关系研究表明,RPS14的异常表达可能促进肿瘤细胞的增殖、迁移和侵袭,从而加速癌症的发展。

此外,RPS14还可能影响肿瘤细胞的耐药性,使肿瘤对化疗药物的反应降低。

三、RPS14对肝癌细胞功能的影响1. RPS14在肝癌细胞中的表达及调控在肝癌细胞中,RPS14的表达水平较高,且受多种信号通路的调控。

通过对肝癌细胞株的分析,我们发现RPS14的表达与肝癌细胞的增殖、凋亡及转移等生物学行为密切相关。

2. RPS14对肝癌细胞功能的影响研究发现,RPS14的过表达可以促进肝癌细胞的增殖和迁移,而抑制其表达则可抑制肝癌细胞的生长和转移。

这表明RPS14在肝癌的发生和发展过程中起着重要作用。

四、研究方法与实验结果本研究采用细胞培养、RNA干扰、蛋白质印迹等方法,对RPS14在肝癌细胞中的功能进行深入研究。

通过实验,我们观察到RPS14对肝癌细胞增殖、凋亡及转移的影响,并进一步探讨了其作用机制。

基于iTRAQ蛋白分析技术初步探索红参粉对果蝇的抗衰老作用及其机制研究

基于iTRAQ蛋白分析技术初步探索红参粉对果蝇的抗衰老作用及其机制研究

基于iTRAQ蛋白分析技术初步探索红参粉对果蝇的抗衰老作用及其机制研究一、内容综述随着科学技术的不断发展,人们对生物衰老机制的研究越来越深入。

在众多抗衰老方法中,红参粉作为一种具有抗氧化、抗炎、抗疲劳等作用的天然保健品,受到了广泛关注。

本研究采用基于iTRAQ蛋白分析技术,对红参粉对果蝇的抗衰老作用及其机制进行初步探索。

iTRAQ技术已成为一种高通量、高灵敏度的蛋白质组学研究方法。

通过对样品进行同位素标记,iTRAQ可以同时比较多个样本之间的蛋白质表达差异,从而揭示蛋白质在生物过程中的作用。

在本研究中,我们利用iTRAQ技术对红参粉处理后的果蝇蛋白质组进行了深度挖掘,以探讨红参粉对果蝇抗衰老作用的可能机制。

红参粉处理后的果蝇在衰老过程中,其体内多种抗氧化酶活力显著提高,且丙二醛含量、过氧化氢含量均有所降低,提示红参粉可能通过清除自由基、减轻氧化应激损伤来发挥抗衰老作用。

红参粉处理后的果蝇体内相关抗氧化基因的表达水平也发生了一定程度的改变,进一步证实了红参粉对果蝇抗氧化能力的提升作用。

除了抗氧化能力外,红参粉还可能通过调节果蝇体内的能量代谢、炎症反应等途径发挥抗衰老作用。

红参粉处理后的果蝇在衰老过程中,其体内能量代谢相关基因的表达水平发生调整,有助于维持果蝇体内能量的稳定。

红参粉还能够抑制果蝇体内炎症因子的释放,减轻炎症反应对果蝇细胞的损伤。

本研究表明红参粉可能通过多种途径发挥抗衰老作用,其机制可能与提高果蝇体内的抗氧化能力、调节能量代谢和炎症反应等相关。

红参粉在抗衰老方面的具体作用机制仍需进一步深入研究。

我们将继续利用iTRAQ技术对红参粉处理后的果蝇进行更全面的蛋白质组学分析,以期揭示更多关于红参粉抗衰老作用机制的细节。

1. 红参粉的来源与特点红参,作为人参的一种炮制加工品,因其独特的药用价值和丰富的营养价值备受关注。

红参通常选用优质的人参,在经过严格的水分、温度控制以及炮制工艺下制成。

在人参的药用功效中,红参具有大补元气、复脉固脱、补脾益肺、生津养血等作用。

Invitrogen

Invitrogen

Quantitation of proliferating cells with the EVOS FL Auto Imaging SystemIntroductionThe Invitrogen™ EVOS™ FL Auto Imaging Systemis a fully automated, digital, inverted multi-channelfluorescence and transmitted-light imaging systemwith outstanding workflow efficiency. Designed tomeet demanding requirements over a broad range ofapplications, it supports high-resolution mosaic tiling,multiple-position well scanning, cell counting withthresholding, and time-lapse studies.Among the versatile software features is cell counting that can be done automatically with a captured or live image. A powerful watershed algorithm has enhanced the precision of counting cells stainedwith Invitrogen™ Molecular Probes™ NucBlue™ Fixed Cell ReadyProbes™ Reagent. Other nuclear stains/fluorescent proteins, or general cytoplasmic fluorescent dyes/proteins, permit the easy determination of total cell numbers and/or percentage of cells stained for a functional readout.In this application note, the number of replicating cells was identified as a percentage of the total population of cells. The percentage of proliferating cells was easily determined by labeling cells with EdU-Alexa Fluor™ 594 to identify proliferating cells and NucBlue Fixed Cell ReadyProbes Reagent to identify total cells. Materials• I nvitrogen™ Molecular Probes™ Image-iT™ Fixation/Permeabilization Kit (Cat. No. R37602)• Invitrogen™ Molecular Probes™ Click-iT™ EdU Alexa Fluor™ 594 Imaging Kit (Cat. No. C10339)• NucBlue Fixed Cell ReadyProbes Reagent (Cat. No. R37606)• Invitrogen™ EVOS™ Light Cube, DAPI (Cat. No. AMEP4650)• Invitrogen™ EVOS™ Light Cube, Texas Red™ (Cat. No. AMEP4655)APPLICATION NOTE EVOS FL Auto Imaging SystemFigure 1. Overlay of 10x image used in the quantitative analysis of proliferating HeLa cells.Figure 2. Screen capture of the quantitation of the totalpopulation of cells (blue) within the image. 373 total cells werecounted in this field of view.Figure 3. Screen capture of the quantitation of the total population of proliferating cells (red) within the image. 138 proliferating cells were counted in this field of view.MethodsHeLa cells grown in a 96-well plate were pulsed with EdU for 1 hour. Cells were fixed, permeabilized and blocked using the Image-iT Fixation/Permeabilization Kit. Following fixation and permeabilization, nuclei were labeled with 2 drops/mL of NucBlue Fixed Cell ReadyProbes Reagent and the incorporated EdU was detected with Alexa Fluor 594 azide (Component B from Cat. No. C10339). Cells with incorporated EdU are shown in pink while all cells have nuclei labeled in blue (Figure 1). Following sample preparation, cells were imaged on the the EVOS FL Auto Imaging System using the EVOS DAPI light cube to visualize NucBlue Fixed Cell ReadyProbes Reagent and the EVOS Texas Red light cube to visualize Click-iT EdU using a 10x objective. Cells were quantitated using the Cell Counting tab on the EVOS FL Auto Imaging System.Results and discussionTotal cell population was determined to be 373 by counting the number of cells in the DAPI channel corresponding to NucBlue Fixed staining (Figure 2). Within this population, the number of proliferating cells was determined to be 138 by counting the number of cells in the Texas Red channel corresponding to Click-iT EdU-positive cells. Based on these results, 37% (138/373) of the cells within this population were shown to be actively proliferating (Figure 3). Using the EVOS FL Auto Imaging System allows researchers to combine qualitative images with quantitative measurements to provide rich data for research applications.Find out more at /evosflautoFor Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. CO017255 0815。

炎症过程中内皮细胞的功能变化及其后果

炎症过程中内皮细胞的功能变化及其后果

phatic endothelium of the skin and in vascular tumors.Am J Pathol ,1998;153:395-403.23.Valtola R ,Salven P ,Heikkila P ,et al.V EGFR 23and its ligandV EGF 2C are associated with angiogenesis in breast cancer.Am J Pathol ,1999;154:1381-1390.24.Duff SE ,Li C ,Jeziorska M ,et al.Vascular endothelial growthfactors C and D and lymphangiogenesis in gastrointestinal tract ma 2lignancy.Br J Cancer ,2003;89:426-430.25.Kawakami M ,Furuhata T ,K imura Y ,et al.Quantification of vas 2cular endothelial growth factor 2C and its receptor 23messenger RNA with real 2time quantitative polymerase chain reaction as a predictor of lymph node metastasis in human colorectal cancer.Surgery ,2003;133:300-308.26.Shimizu K ,Kubo H ,Yamaguchi K ,et al.Suppression of V EGFR 23signaling inhibits lymph node metastasis in gastric cancer.Can 2cer ,2004;95:328-333.27.Niki T ,Iba S ,Tokunou M ,et al.Expression of vascular endothe 2lial growth factors A ,B ,C ,and D and their relationships to lymph node status in lung adenocarcinoma.Clin Cancer Res ,2000;6:2431-2439.28.Skobe M ,Hamberg LM ,Hawighors ,T ,et al.oncurrent inductionof lymphangiogenesis ,angiogenesis ,and macrophage recruitment by vascular endothelial growth factor 2C in melanoma.Am J Pathol ,2001;159:893-903.29.Mandriota S J ,J ussila L ,Jeltsch M ,et al.Vascular endothelialgrowth factor 2C 2mediated lymphangiogenesis promotes tumourmetastasis.EMBO J ,2001;20:672-682.30.Leu AJ ,Berk DA ,Lymboussaki A ,et al.Absence of functionallymphatics within a murine sarcoma :a molecular and functional evaluation.Cancer Res ,2000;60:4324-4327.31.McCarter MD ,Clarke J H ,Harken AH.Lymphangiogenesis is piv 2otal to the trials of a successful cancer metastasis.Surgery ,2004;135:121-124.32.Achen M G ,Roufail S ,Domagala T ,et al.Monoclonal antibodiesto vascular endothelial growth factor 2D block interactions with both V EGF receptor 22and V EGF receptor 23.Eur J Biochem ,2000;267:2505-2515.33.Wood J M ,Bold G ,Buchdunger E ,et al.PTK787/ZK 222584,anovel and potent inhibitor of vascular endothelial growth factor re 2ceptor tyrosine kinases ,impairs vascular endothelial growth factor 2induced responses and tumor growth after oral administration.Can 2cer Res ,2000;60:2178-2189.34.Jain R K ,Padera TP.Prevention and treatment of lymphatic metas 2tasis by antilymphangiogenic therapy.J Natl Cancer Inst.2002;94(11):785-787.35.Enholm B ,Karpanen T ,Jeltsch M ,et al.Adenoviral expression ofvascular endothelial growth factor 2C induces lymphangiogenesis in the skin.Circ Res ,2001;88:623-629.36.Karkkainen MJ ,Saaristo A ,J ussila L ,et al.A model for genetherapy of human hereditary lymphedema.Proc Natl Acad ,2001;98:12677-12682.(收稿日期:2004-05-18)・综述・炎症过程中内皮细胞的功能变化及其后果胥 楠 陈晓理 摘要 内皮细胞在介导炎症和凝血的交互作用中起重要作用;血流机械应力的变化经内皮细胞转化为生物信号,激活NF 2κB 等转录因子,炎症介质也可以通过信号转导通路进行相互调节。

CHO-K1 (仓鼠卵巢细胞亚株) 说明书

CHO-K1 (仓鼠卵巢细胞亚株) 说明书

碧云天生物技术/Beyotime Biotechnology订货热线:400-168-3301或800-8283301订货e-mail:******************技术咨询:*****************网址:碧云天网站微信公众号CHO-K1 (仓鼠卵巢细胞亚株)产品编号产品名称包装C7750 CHO-K1 (仓鼠卵巢细胞亚株) 1支/瓶产品简介:Organism Tissue Morphology Culture Properties Cricetulus griseus(Hamster, Chinese)Ovary Epithelial Adherent本细胞株详细信息如下:General InformationCell Line Name CHO-K1 (Hamster Ovary Cell Substrain)Synonyms CHO K1; CHOK1; CHO cell clone K1; GM15452Organism Cricetulus griseus (Hamster, Chinese)Tissue OvaryCell Type -Morphology EpithelialDisease -Strain -Biosafety Level* 1Age at Sampling AdultGender FemaleGenetics -Ethnicity -Applications This cell line is suitable as a transfection hostCategory Spontaneously immortalized cell line* Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.CharacteristicsKaryotype Chromosome Frequency Distribution 50 Cells: 2n = 22. Stemline number is hypodiploid.Virus Susceptibility Vesicular stomatitis, Orsay (Indiana) Vesicular stomatitis, Glasgow (Indiana) Getah virusDerivation The CHO-K1 cell line was derived as a subclone from the parental CHO cell line initiated from a biopsy of an ovary of an adult Chinese hamster by T. T. Puck in 1957.Clinical Data Female Antigen Expression -Receptor Expression -Oncogene -Genes Expressed -Gene expression databases GEO: GSM1968555; GSM1968563; GSM1968571; GSM1968572; GSM1968569; GSM1968570; GSM1968594; GSM1968596; GSM1968598; GSM1968589; GSM1968600; GSM1968599; GSM1968591; GSM1968593; GSM1968595; GSM1968597; GSM1968565; GSM1968566; GSM1968560; GSM1968559; GSM1968558Metastasis -Tumorigenic -Effects -Comments The cells require proline in the medium for growth.2 / 5 C7750 CHO-K1 (仓鼠卵巢细胞亚株)400-1683301/800-8283301 碧云天/BeyotimeCulture Method Doubling Time~24 hrs Methods for Passages Wash by PBS once then 0.05% trypsin-EDTA solution and incubate at room temperature (or at 37ºC), observe cells under an inverted microscope until cell layer is dispersed (usually within 1 to 5 minutes) Medium RPMI-1640+10% FBS Special Remarks - Medium Renewal Once or twice between subculture Subcultivation Ratio 1:4 to 1:8 Growth Condition 95% air+ 5% CO 2, 37ºC Freeze medium DMEM (high glucose)+20% FBS+10% DMSO ,也可以订购碧云天的细胞冻存液(C0210)。

  1. 1、下载文档前请自行甄别文档内容的完整性,平台不提供额外的编辑、内容补充、找答案等附加服务。
  2. 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
  3. 3、如文档侵犯您的权益,请联系客服反馈,我们会尽快为您处理(人工客服工作时间:9:00-18:30)。

Experiment14 Cell RespirationOVERVIEWIn the preliminary activity, students investigate the respiration of germinating peas using a CO 2 Gas Sensor. A student handout for the preliminary activity can be found at the end of the experiment.During the subsequent Inquiry Process, students will investigate various aspects of cell respiration of germinating seeds.LEARNING OUTCOMESIn this inquiry experiment, students will• Identify variables, design and perform the experiment, collect data, analyze data, draw aconclusion, and formulate a knowledge claim based on evidence from the experiment. • Determine the effect of temperature on the cell respiration of germinating seeds.CORRELATIONSAP Environmental Science Topic Outline CorrelationII. The Living World, B. Energy Flow (Photosynthesis and cellular respiration)IB Environmental Systems Syllabus Correlation2.2.2 Describe photosynthesis and respiration in terms of inputs, outputs, and energy transformationsTHE INQUIRY PROCESSSuggested time to complete the experimentSee the section in the introduction, Doing Inquiry Experiments, for more information on carrying out each phase of a guided inquiry experiment.I Preliminary Activity 35 minutes II Generating Researchable Questions15 minutesIII Planning 15 minutesIV Carrying out the Plan 40 minutes V Organizing the Data15 minutes VI Communicating the Results30 minutesVIIConclusion 15 minutesE va lu at i onc o p yExperiment 14MATERIALSMake the following materials available for students to use. Items in bold are needed for the preliminary activity.data-collection interface non-germinating seeds of various types data-collection program warm waterVernier CO2 Gas Sensor ice cubesgerminating peas 1 L beaker250 mL respiration chamber thermometer or temperature probenon-germinating peas 100 mL beakersgerminating seeds of various types others as requested by studentsI Preliminary ActivityThis inquiry begins with an activity to reinforce prior knowledge of the use of Vernier data-collection technology and to introduce a method with which to collect respiration rate data. Students should use the auto-ID data-collection settings that are loaded when an auto-ID CO2 Gas Sensor is connected to a data-collection interface. If you are using non-auto-ID CO2 Gas Sensors, you will need to instruct the students to set up the data-collection program to collect data once every ten seconds for a ten minute period.Sample ResultsFigure 1CO2 respired by germinating peas at 22o CAnswers to the Questions1. Yes, the carbon dioxide concentration vs. time graph clearly indicates that carbon dioxide isbeing produced at a steady rate when germinating peas are in the respiration chamber.2. Germination greatly accelerates the rate of cell respiration. This reflects a higher rate ofmetabolic activity in germinating seeds. In most experiments, non-germinating seeds do not seem to be respiring. Occasionally, however, some respiration is detectable.3. It is necessary for germinating seeds to undergo cellular respiration in order to acquire theenergy they need for growth and development. Unlike their mature relatives, seeds do not yet have the necessary photosynthetic abilities needed to product their own energy sources.Cell Respiration4. Answers will vary.5. Answers will vary but may include temperature, type of seed, number of peas, and more.II Generating Researchable QuestionsSee page xiii in the Doing Inquiry Experiments section for a list of suggestions for generating researchable questions. Some possible researchable questions for this experiment are: •How does temperature affect pea respiration rate?•Is there a maximum permitted temperature for pea respiration?•What is the optimal temperature for pea respiration?•Do germinating peas respire at temperatures below 0ºC?•How would an O2-rich environment affect cellular respiration rate?•How would an O2-deprived environment affect cellular respiration rate?•How does the rate of O2 consumption compare with the rate of CO2 production duringcellular respiration?•How does salinity of the germination medium affect cellular respiration in peas?•How does the pH of the germination medium affect cellular respiration in peas?•How does the DO of the germination medium affect cellular respiration in peas?•How does the presence of gibberellin (kinetin, zeatin) in the germination medium affectcellular respiration in peas?•How does the presence of mannitol (sucrose) in the germination medium affect cellularrespiration in peas?•How does germination duration affect the rate of CO2 production?There are many more possible researchable questions. Students should choose a researchable question that addresses the learning outcomes of your specific standards.III PlanningDuring this phase students should formulate a hypothesis, determine the experimental design and setup, and write a method they will use to collect data. Circulate among the student groups asking questions and making helpful suggestions.IV Carrying out the PlanDuring this phase, students use their plan to carry out the experiment and collect data. Circulate among the student groups asking questions and making helpful suggestions.V Organizing the DataSee page xiv in the Doing Inquiry Experiments section for suggestions concerning how students can organize their data for their inquiry presentations.VI Communicating the ResultsSee page xv in the Doing Inquiry Experiments section for a list of inquiry-presentation strategies.Experiment 14VII ConclusionSee page xv in the Doing Inquiry Experiments section for a list of suggestions concerning assessment and ways to utilize the results in subsequent instruction.SAMPLE RESULTSHere are some sample results of germinating peas at different temperatures. Student results will vary depending on experimental design.Figure 2 CO2 respired by peas at different temperaturesTable 1: The Effect of Temperature on Respiration RatePeas Rate of Respiration (ppm/s) Germinating, cool temperature (10°C) 0.37Germinating, room temperature (20°C) 0.63Germinating, cool temperature (30°C) 1.19Germinating, warm temperature (40°C) 2.28This investigation addresses the question, “How does temperature affect pea respiration rate?” Over the range of 10ºC to 40ºC, pea respiration rates increased with increasing temperature.Cell RespirationTable 2: CO2 Production Rates (ppm/s)NaCl Percent Trial 0 0.1 1 31 2.573 2.075 1.867 1.8972 2.495 1.984 1.871 1.8833 2.589 2.349 2.072 1.479Average 2.221 2.349 1.937 1.753Pea (pisum sativum) samples were soaked in distilled water and NaCl solutions (0.1, 1, and 3%) overnight. The peas were then allowed to germinate at room temperature in a dark cabinet forthree days while rolled in paper towels moisten with the same solution and placed in paper bags.The moistened paper towels were replaced after the first and second days. After three days of germination, data were collected using a procedure similar to that shown in the PreliminaryActivity. Twenty peas were used in each trial.This investigation was prompted by the question, “How does the salinity of the germinationmedium affect cellular respiration in peas?” Cellular respiration generally decreased as thesalinity of the germination medium increased.Table 3: Three-Day Germination ResultsPercent NaCl0 0.1 1 3Percent92 94 76 0GerminatedAverage sprout22.8 22.9 7.5 0length (mm)Percent germination and average sprout length were also determined after three days of germination.Experiment 14TIPSExperiment Information1. Allow the seeds to germinate for three days prior to the experiment. Prior to the first day,soak them in water overnight. On subsequent days, roll them in a moist paper towel and place the towel in a paper bag. Place the bag in a warm dark place. Check each day to be sure the towels remain very moist. If time is short, the peas can be used after they have soakedovernight. For best results, allow them to germinate for the full three days.2. The recommended numbers of peas for use in this experiment are 10–15 peas for the originalCarbon Dioxide Gas Sensor (range 0–5,000 ppm) and 25 peas for the newer dual-rangeCarbon Dioxide Gas Sensor (low range 0–10,000 ppm). These numbers can vary depending on the type of peas and the length of time they have been germinating. For best results, do a trial run and adjust the number of peas accordingly.3. Heavy condensation buildup in the respiration chamber can interfere with readings from theCO2 Gas Sensor. This can be a source of error if the peas are very wet when placed in the respiration chamber. If necessary, blot the peas dry with a paper towel before placing them in the respiration chamber.4. A hair dryer and a funnel can be used to expel excess CO2 prior to data collection.5. Avoid using the respiration chamber at temperatures above 50ºC. The respiration chamberwill deform.6. Graphs can be copied and pasted into a Microsoft Word document. If your students collectdata on a TI graphing calculator, have them import the data to Logger Pro or do a screen capture using TI Connect. If you students collect data on a Palm® handheld, have themimport the data in to Logger Pro.Sensor Information1. The CO2 Gas Sensor requires a 90 second warm-up period before data collection can begin.2. The calibration loaded with an auto-ID CO2 Gas Senor is ideal for this experiment.3. The older style CO2 Gas Sensor without a switch has a range of 0 to 5000 ppm. The sensorcannot take readings at a CO2 concentration higher than 5000 ppm. Once the CO2concentration reaches this level, the computer, calculator, or handheld will continue todisplay a reading of ~5000 ppm until the actual level drops below 5000 ppm again.4. The newer style CO2 Gas Sensor with a switch measures gaseous carbon dioxide levels in therange of 0 to 10,000 ppm (low range setting) or 0 to 100,000 ppm (high range setting). Use the low range setting for this experiment.5. Human breath has a concentration of approximately 50,000 ppm. Do not blow in therespiration chamber. To remove CO2 from the chamber, fill the bottle with water and dry with a paper towel or use a blow dryer or fan to expel the CO2.6. The stopper included with the older style CO2 Gas Sensor is slit to allow easy application andremoval from the probe. When students are placing the probe in the respiration chamber, they should gently twist the stopper into the chamber opening. It does not need to be perfectly air tight. Warn the students not to twist the probe shaft or they may damage the sensing unit.Cell Respiration 7. The CO2 Gas Sensor relies on the diffusion of gases into the probe shaft. Students shouldallow a couple of minutes between trials so that gases can diffuse from the probe.Alternatively, the students can use a firm object such as a book or notepad to fan air through the probe shaft.Sensor CheckHere is an easy way to check that your CO2 Gas Sensor is working correctly:1. Connect the sensor to the interface.2. Start the data-collection program and allow the sensor to warm up for at least 90 seconds.3. Blow directly on the shaft of the sensor. You should see an increase in CO2 levels.4. Older style sensors will max out at 5000 ppm.5. An increase in CO2 levels indicates the sensor is functioning correctlyExperiment14PRELIMINARY ACTIVITY FORCell RespirationCell respiration refers to the process of converting the chemical energy of organic molecules intoa form immediately usable by organisms. Glucose may be oxidized completely if sufficientoxygen is available by the following equation:C6H12O6 + 6O2(g) → 6 H2O + 6 CO2(g) + energyAll organisms, including plants and animals, oxidize glucose for energy. Often, this energy isused to convert ADP and phosphate into ATP. It is known that peas undergo cell respirationduring germination.PROCEDURE1. If CO2 Gas Sensor your sensor has a switch, set it tothe low range setting. Connect the sensor to the data-collection interface.2. Place 10–15 germinating peas in the respirationchamber that ships with the CO2 Gas Sensor. Insertthe CO2 Gas Sensor in the neck of the respirationchamber. Start the data-collection program. Wait90 seconds for the sensor to warm up and begincollecting data.3. After data are collected, use the linear regression function to determine the rate of respiration.After completing the Preliminary Activity, you will first use reference sources to find out moreabout germination and cell respiration before you choose and investigate a researchable question. Some topics to consider in your reference search are:•germination •cell respiration •carbohydrates •glucoseQUESTIONS1. Do the results of this experiment verify that germinating peas respire? How do you know?2. What do you expect would happen to the rate of respiration if you repeated this experimentwith non-germinating peas?Experiment 143. Why do germinating peas undergo cell respiration?4. Use a concept map or other organizing tool to list your prior knowledge of cell respiration.5. List at least one researchable question for this experiment.。

相关文档
最新文档