Auto--Wound healing assay
Wound Healing Methods创伤动物模型总结
Wound Healing In Vitro MethodsIn vitro scratch assay.pdfECIS Handbook.pdfWound Healing In Vivo Methods (Experimental Animal Wound Models)A variety of normal and pathological injury paradigms can be applied in both small and large animal species.To mimic clinical problems, rates of repair can be compromised by surgical impairment of blood supply or metabolic manipulations such as the diabetic state. Although animal wound repair is an imperfect reflection of human wound healing and its clinical challenges, these models continue to be crucial tools for the development of new strategies and approaches to rational wound therapy.Methods (Protocols)1.Partial thickness thermal burns (11)Partial thickness thermal burns were created on the dorsal skin of pigs as described (12). Briefly, two domestic male pigs, each weighing 35 pounds, were anesthetized with ketamine and xylazine, and anesthesia was maintained with halothane inhalation. The dorsal skin was chemically depilitated, and six partial thickness burns measuring 3X3 cm were created by contact for 10 s with a solid brass block weighing 714 g heated previously in a water bath maintained at 70 °C. The six burns were arranged in two rows of three burns on each side of the spine. Blister roofs were removed and two burns were treated topically with 3 ml of Silvadene ® cream (Marion Laboratories, Kansas City, MO) containing 20 mg/mltyrphostin1478(Calbiochem) (13). Two burns were treated with Silvadene ® cream alone, and two burns were left untreated. Each burn was individually covered with a 6 3 6-cm 2 adhesive occlusive dressing(Steri-Drape™ 2, 3M, St. Paul, MN). Dressings were removed daily, the burns were retreated, and fresh dressings were applied. Five days after injury, the pigs were sacrificed and a full thickness biopsy was taken diagonally across each burn, fixed in 10% buffered formalin, and embedded in paraffin. Sections were stained with hematoxylin and eosin. The extent of epithelial healing for each burn was calculated by measuring the distance between intact epithelial edges divided by the total length of the wound and was expressed as percentage of re-epithelialization. Epithelial healing for each of the three treatment groups was averaged and compared for statistical significance by ana lysis of variance and Tukey’s HSD post-test.je16351319.pdf2.Sponge implantation on the site of skin incisions would model (14)AnimalsMale Sprague-Dawley rats (250–300 g; Harlan Sprague Dawley Inc., Indianapolis, IN) were housed in the VA Animal Care Facility maintained according to the American Association for Accreditation for Laboratory Animal Care standards. The animals were allowed food and water ad libitum. All surgical procedures were carried out under general anesthesia with ketamine and xylazine. All animal procedures were approved under the guidelines of the local Animal Care and Use Committees.Tensile StrengthTwo rows of three sites per row distributed transversely across the ventral surface of the rat. And then, full thickness, transverse incisions measuring 1 cm were made through the center of the target area, and the skin margins were approximated with three 11-mm Michel wound clips. Animals were killed at the specified time points by CO2 poisoning, after which the pelts were excised and the clips removed. Skin strips bisecting the long axis of the scar were prepared and tensile strength was determined using an Instron tensiometer. The results were normalized for the cross-sectional area of the wound and reported as a ratio. The significance of the difference between treatment groups was determined by the Student’s t test.Sponge implantationIn anesthetized rats, four polyvinyl alcohol (PVA) sponges fabricated from blocks of Ivalon made by Unipoint Industries, (High Point, NC) and measuring 10 mm in diameter and 2 mm in depth, were implanted subcutaneously beneath the panniculus carnosus adjacent to the abdominal wall fascia as previously described (15,16).Histology and immunocytochemistryTissue biopsies were fixed in 10% phosphate buffered formalin and embedded in paraffin for routine staining in hematoxylin and eosin. Tissue specimens for immunohistochemistry were performed.3.Full-thickness wound model (14)Mice of both genders were anesthetized with intraperitoneal pentobarbital (45 mg/kg), the dorsum was clipped free of hair, and the skin was prepped with providine-iodine and sterile H2O. A full-thickness wound (1.5X1.5 cm) was created, and the surface area was measured after the skin edges had retracted. Wounds were covered with a bioocclusive dressing to maintain sterility. Wound closure rates were measured every other day by tracing the wound area on acetate sheets. Dressings were changed at these times. Tracings were digitized, and areas were calculated in a blinded manner using a computerized algorithm (Sigma Scan™; Jandel Sc ientific, San Raphael, CA).4.Full-thickness wound model 2 (17,18)Mice were anesthetized with a single intraperitoneal injection of Ketamin / Xylazin. The hairs on the back of these mice were cut, and the back was subsequently wiped with 70% ethanol. Six full-thickness wounds (5mm diameter, 3-4 mm apart) were made on the backs of these mice by excising the skin and the underlying panniculus carnosus. The wounds were allowed to dry to form a scab. An area of 7-8 mm in diameter, which included the scab and the complete epithelial margins, was excised at each time point. As a control, a similar amount of skin was taken from the backs of four nonwounded mice. For every experimental time point, four wounds each from four animals (n=16 wounds) and the nonwounded back skin form four animals, respectiviely, were combined, frozen immediately in liquid nitrogen, and stored at -80C until used for RNA isolation. All animal experiments were carried out according to the guidelines and with the permission from the local government of Hessen.5.Blisters wound model (19)Experimental preparationTwelve male Hampshire/Yorkshire swine (SPF, 10-15 kg) obtained from Bio Medical Alternatives International, Inc., Raleigh, NC, U.S.A., were used in the study. Swine were sedated with an intramuscular injection of ketamine (10 mg/kg) and anaesthetized by intravenous administration of sodium pentobarbitone (30 mg/kg). Animals were placed in a respiratory ventilator with a positive pressure pump (Harvard Instrument). The common carotid artery and the left femoral vein were exposed and cannulated for monitoring heart rate/arterial blood pressure and blood samplings, respectively. Azapropazone was dministered intravenously via the marginal ear vein.Skin chamber deviceThe skin suction and chamber units were obtained from Neuro probe (Neuro probe, Cabin John, MD, U.S.A.). The suction unit has two rows of four wells in each row. Each well has a diameter of 7mm, allowing blisters to form with a maximal area of 0.38 cm2. Each row can be connected separately or together (with a Y-tube) to a vacuum pump. The second chamber unit which covers the blister lesions is very similar to the suction unit, but has bigger wells with a diameter of 12mm and a volume of 1 ml. A 2mm wide and 1 mm deep trough filled with a plastic ring surrounds the wells in order to prevent the leakage of the serum. A hole in the top of each well allows the introduction of a 20 gauge subcutaneous needle for the injection and the withdrawal of fluid.Blisters woundThe flat surface of the swine's thigh muscle was shaved and disinfected with ethanol. The suction unit was secured in place with a velcro bandage. A constant suction of 300 mmHg was applied using a vacuum pump. The suction field was warmed up in order to increase the rate of the blister formation. After 1.5 h, the blisters were complete. The blister fluid was removed and frozen for further analysis of myeloperoxidase (MPO) activity. The blister roofs were then gently removed with scissors. The eight lesions (0.38 cm2 area) were covered with the skin chamber unit and filled with 0.5 ml autologous serum.6.Split-thickness Epidermal Wound model (12)84 split-thickness wounds (0.005 inches thick, 1×1 cm) were made on the dorsal thorax of four adult miniature pigs (Vim-Vet Laboratories, Marion, ID). Four wounds from each group on each pig were randomly selected daily and entirely excised at a depth of 0.007 inches, including 5 mm of surrounding normal skin. Epidermis and dermis were readily separated from excised specimens after incubation in trypsin, and wounds were considered healed when no defect was present.References1. Todaro, G. J., Lazar, G. K., and Green, H. (1965) The initiation of cell division in a contact-inhibited mammalian cell line. J Cell Physiol66, 325-3332. Haudenschild, C. C., and Schwartz, S. M. (1979) Endothelial regeneration. II. Restitution of endothelial continuity. Lab Invest41, 407-4183. Etienne-Manneville, S., and Hall, A. (2001) Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta. Cell106, 489-4984. Fukata, Y., Oshiro, N., Kinoshita, N., Kawano, Y., Matsuoka, Y., Bennett, V., Matsuura, Y., and Kaibuchi, K. (1999) Phosphorylation of adducin byRho-kinase plays a crucial role in cell motility. J Cell Biol145, 347-3615. Abbi, S., Ueda, H., Zheng, C., Cooper, L. A., Zhao, J., Christopher, R., and Guan, J. L. (2002) Regulation of focal adhesion kinase by a novel proteininhibitor FIP200. Mol Biol Cell13, 3178-31916. Schiller, K. R., Maniak, P. J., and O'Grady, S. M. (2010) Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial woundrepair. Am J Physiol Cell Physiol299, C912-9217. Chan, C. M., Huang, J. H., Chiang, H. S., Wu, W. B., Lin, H. H., Hong, J. Y., and Hung, C. F. (2010) Effects of (-)-epigallocatechin gallate on RPE cellmigration and adhesion. Mol Vis16, 586-5958. Wu, N. L., Chiang, Y. C., Huang, C. C., Fang, J. Y., Chen, D. F., and Hung, C. F. (2010) Zeaxanthin inhibits PDGF-BB-induced migration in human dermalfibroblasts. Experimental dermatology19, e173-1819. Hsu, C. C., Tsai, W. C., Chen, C. P., Lu, Y. M., and Wang, J. S. (2010) Effects of negative pressures on epithelial tight junctions and migration in woundhealing. Am J Physiol Cell Physiol299, C528-53410. Jiang, W. G., Martin, T. A., Lewis-Russell, J. M., Douglas-Jones, A., Ye, L., and Mansel, R. E. (2008) Eplin-alpha expression in human breast cancer, theimpact on cellular migration and clinical outcome. Mol Cancer7, 7111. Pilcher, B. K., Dumin, J., Schwartz, M. J., Mast, B. A., Schultz, G. S., Parks, W. C., and Welgus, H. G. (1999) Keratinocyte collagenase-1 expressionrequires an epidermal growth factor receptor autocrine mechanism. J Biol Chem274, 10372-1038112. Brown, G. L., Curtsinger, L., 3rd, Brightwell, J. R., Ackerman, D. M., Tobin, G. R., Polk, H. C., Jr., George-Nascimento, C., Valenzuela, P., and Schultz, G.S. (1986) Enhancement of epidermal regeneration by biosynthetic epidermal growth factor. J Exp Med163, 1319-132413. Levitzki, A., and Gazit, A. (1995) Tyrosine kinase inhibition: an approach to drug development. Science267, 1782-178814. Benn, S. I., Whitsitt, J. S., Broadley, K. N., Nanney, L. B., Perkins, D., He, L., Patel, M., Morgan, J. R., Swain, W. F., and Davidson, J. M. (1996)Particle-mediated gene transfer with transforming growth factor-beta1 cDNAs enhances wound repair in rat skin. J Clin Invest98, 2894-2902 15. Buckley, A., Davidson, J. M., Kamerath, C. D., and Woodward, S. C. (1987) Epidermal growth factor increases granulation tissue formation dosedependently. J Surg Res43, 322-32816. Broadley, K. N., Aquino, A. M., Hicks, B., Ditesheim, J. A., McGee, G. S., Demetriou, A. A., Woodward, S. C., and Davidson, J. M. (1989) The diabeticrat as an impaired wound healing model: stimulatory effects of transforming growth factor-beta and basic fibroblast growth factor. Biotechnology therapeutics1, 55-6817. Stallmeyer, B., Kampfer, H., Kolb, N., Pfeilschifter, J., and Frank, S. (1999) The function of nitric oxide in wound repair: inhibition of inducible nitricoxide-synthase severely impairs wound reepithelialization. J Invest Dermatol113, 1090-109818. Frank, S., Stallmeyer, B., Kampfer, H., Kolb, N., and Pfeilschifter, J. (2000) Leptin enhances wound re-epithelialization and constitutes a directfunction of leptin in skin repair. J Clin Invest106, 501-50919. Mousa, S. A., Brown, R., Chan, Y., Hsieh, J., and Smith, R. D. (1990) Evaluation of the effect of azapropazone on neutrophil migration inanaesthetized swine using a multichamber blister suction technique. Br J Pharmacol99, 233-236。
压疮愈合监测工具
压疮愈合监测工具（一）为了精确了解压疮愈合过程并进行数据精细化管理，有必要对压疮愈合进行监测。
目前的临床实践中，压疮愈合监测是由医疗专业人员执行，辅以压疮评估工具和数字成像。
临床条件允许时，还可使用数据采集设备。
2014年，美国压疮咨询委员会、欧洲压疮咨询委员会、及泛太平洋压力损伤联盟联合制定的《压疮的预防与治疗快速指南》指出，应当使用有效而可靠的压疮评估量表来评估压疮的愈合过程。
目前已设计很多压疮评估量表/工具，来帮助评估压疮愈合过程，在上述指南中提到的量表/工具有：Bates-Jensen伤口评价工具（Bates-JensenWound Assessment Tool,BWAT），压疮状态评价工具（PressureSore Status Tool,PSST），压疮愈合量表（PressureUlcer Scale for Healing ,PUSH），和DESIGN/DESIGN-R（Depth，Exudate，Size，Inflammation/Infection，Granulation，Necrotictissue，DESIGN）等[1]。
现对以上四个量表进行阐述。
1.Bates-Jensen伤口评价工具（BWAT）BWAT最初叫压疮状态评价工具（PressureSore Status Tool,PSST），PSST是1990年Bates-Jensen与20名多学科的伤口专家采用德尔菲法开发出来[2]。
包括15个条目，其中13个条目描述伤口特点并采用Likert计分，另外有2个附加的不计分条目。
计分条目有：大小、深度、边缘、潜行、坏死组织类型、坏死组织量、渗液类型、渗液量、伤口周围皮肤颜色、周围组织水肿、周围组织硬化、肉芽组织、上皮化，每个条目赋值1~5分。
总分范围是13~65分，分值越高，压疮越严重。
不计分条目：伤口部位与形状。
源量表文献报道其内容效度为0.91；2位造口治疗师使用该工具分别对10例急诊医院的成人手术患者的20处压疮进行2次评价。
wound healing assay原理
wound healing assay原理"Wound healing assay原理"是指一种常用的实验方法，用于研究细胞和组织修复过程中的细胞迁移和扩增。
本文将逐步解释wound healing assay的原理，从实验设计到结果分析，以帮助读者深入理解该方法。
第一部分：实验前准备和实验设计在进行wound healing assay之前，首先需要做一些实验前准备工作。
这包括选取合适的细胞系、培养和处理细胞、准备实验所需的试剂和材料等。
1.1 细胞系的选择：细胞系的选择应根据研究目的而定。
常用的细胞系有人类肺癌细胞系A549、乳腺癌细胞系MCF-7等。
另外，有一些细胞系在演化过程中丧失了细胞迁移能力，因此需要选择具有细胞迁移能力的细胞系。
1.2 细胞的培养和处理：将选定的细胞系在无菌条件下培养至对数生长期。
然后，使用胰酶等消化酶将细胞从培养皿中释放，并进行必要的处理，如染色、转染等。
1.3 准备实验所需的试剂和材料：为了进行wound healing assay，我们需要准备一些必要的试剂和材料。
其中包括细胞培养基、细胞传代液、胰酶、无菌培养皿、培养皿标尺、吸管、显微镜镜片、组织病理刀片、无菌垫片等。
在实验设计方面，需要合理地确定参照组和实验组。
参照组通常是无任何处理的对照组，通过与实验组的对比，我们可以看到细胞迁移的差异。
实验组则是接受特定处理的组。
第二部分：实验操作步骤在进行wound healing assay之前，我们需要先在细胞培养皿中制作一个“伤口”（wound）。
这可以通过多种方法实现，最常用的是使用细胞刮痕方法。
2.1 细胞刮痕方法：在将细胞接种在培养皿中后，可以使用无菌的组织病理刀片或刮痕器在培养皿中划出一条直线，以制作一个均匀的伤口。
划痕的长度可以根据需要进行调整，但保持一致性是非常重要的。
2.2 吸走碎片细胞：制作好伤口后，将培养皿中的游离细胞碎片用吸管吸走。
β-榄香烯对人胃癌细胞SGC7901的抑制作用及部分机制
β-榄香烯对人胃癌细胞SGC7901的抑制作用及部分机制赵晓晓;张蕾;史天陆;邓明影;汪国玉;姜玲;魏伟【期刊名称】《安徽医科大学学报》【年(卷),期】2016(51)12【摘要】目的：研究β-榄香烯对胃癌细胞SGC7901的增殖、凋亡、迁移及赖氨酰氧化酶( LOX)的影响。
方法体外培养人胃癌SGC7901细胞, MTS法及流式细胞术检测不同浓度β-榄香烯对SGC7901细胞增殖与凋亡的影响,划痕实验、Tr-answell小室迁移实验检测β-榄香烯对细胞迁移的影响,酶标仪法检测β-榄香烯对LOX酶活性的影响,Western blot法检测 LOX 蛋白表达的变化。
结果β-榄香烯(50~800μmol/L)对SGC7901细胞的抑制作用呈时间依赖和浓度依赖性。
β-榄香烯(100、200、400、800μmol/L)处理 SGC7901细胞24 h后,凋亡率分别为8．1%、8．2%、18．7%、41．9%,与对照组(4．9%)比较,凋亡率明显升高( P<0．05)。
划痕实验、Transwell 实验结果显示β-榄香烯对 SGC7901细胞的迁移有明显抑制作用( P<0．05)。
β-榄香烯浓度依赖性地降低SGC7901细胞LOX酶活性(P<0．05)。
Western blot结果显示β-榄香烯可下调SGC7901细胞LOX的蛋白表达。
结论β-榄香烯对胃癌细胞 SGC7901有明显抑制增殖、迁移及促进凋亡作用,抑制LOX酶活性和蛋白表达可能是其发挥作用的机制之一。
%Objective To investigate the i nhibitory effect ofβ-elemene on the proliferation,apoptosis,migration and LOX activity of the gastric cancer SGC7901 cell line. Methods MTS was performed to reveal the inhibitory effect on cell proliferation with different concentrationsβ-elemene on SGC7901 cell,flow cytometry was used to determine the apoptosis of thegastric cancer SGC7901 cell line after the treatment ofβ-elemene. Effectsofβ-elemene on the migration of SGC7901 cells were detected by wound healing assay and transwell assay respectively. Changes of LOX activity were determined by colorimetric method,and changes of LOX expression were determined by Western blot. Results β-elemene(100,200,400,800 μmol/L) significantly inhibited the proliferationon SGC7901 cells in a time and dose-depen dent manner. After β-elemene treatment for 24 h in SGC7901 cells,the apoptotic ratios were 8. 1%,8. 2%,18. 7% and 41. 9% with concentrations ofβ-elemene at100,200,400,800μmol/L. Compared with the control(4. 9%) ,apoptosis rate increased obviously. Effects of migration on SGC7901 cells were significantly in-hibited by Wound healing assay and Transwell assay. LOX activity was decreased in a dose-dependent manner. Western blot showed thatβ-elemene SGC7901 cells could down-regulate expression of LOX. Conclusion The pro-liferation,apoptosis and migration of the gastric cancer SGC7901 cell line can be inhibited after the treatment ofβ-elemene andthe molecular mechanism for those effects may be related to its down-regulated LOX activity and the express of LOX.【总页数】6页(P1717-1721,1722)【作者】赵晓晓;张蕾;史天陆;邓明影;汪国玉;姜玲;魏伟【作者单位】安徽医科大学临床药理研究所抗炎免疫药物教育部重点实验室抗炎免疫药物安徽省协同创新中心，合肥230032;安徽医科大学附属省立医院药剂科，合肥 230001;安徽医科大学附属省立医院药剂科，合肥 230001;安徽医科大学附属省立医院药剂科，合肥 230001;安徽医科大学临床药理研究所抗炎免疫药物教育部重点实验室抗炎免疫药物安徽省协同创新中心，合肥230032;安徽医科大学临床药理研究所抗炎免疫药物教育部重点实验室抗炎免疫药物安徽省协同创新中心，合肥230032; 安徽医科大学附属省立医院药剂科，合肥 230001;安徽医科大学临床药理研究所抗炎免疫药物教育部重点实验室抗炎免疫药物安徽省协同创新中心，合肥230032【正文语种】中文【中图分类】R966;R735.2;R285.5【相关文献】1.细香葱提取物对人胃癌细胞SGC7901抑制作用 [J], 尤亚林;李慧;潘思轶;徐晓云2.miR-205对人胃癌细胞株SGC7901增殖的抑制作用 [J], 赵崇晖;王梅;沈俐;顾健美;史云燕;钱晖;许文荣3.β-榄香烯对人胃癌细胞 SGC7901作用的蛋白质组学研究 [J], 刘俊松;车向明;仇广林;樊林;赵伟;贺仕才;常帅;王曙逢4.桔梗皂甙D对人胃癌细胞SGC7901增殖的抑制作用和机制研究 [J], 代群;黄宣;陈喆5.柠檬酸盐对人胃癌细胞株SGC7901糖酵解的抑制作用及其机制研究 [J], 周正;许晓巍;孟祥军;宛新建因版权原因，仅展示原文概要，查看原文内容请购买。
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.。
细丝蛋白A抑制人结肠癌SW480细胞体外侵袭转移能力
2011年9月第31卷第9期基础医学与临床Basic ＆Clinical Medicine September 2011Vol．31No．9收稿日期：2010－－修回日期：2011－01－18基金项目：河北省卫生厅医学科学研究重大课题（20090010）；河北省普通高校强势特色学科建设基金（200552）*通信作者（corresponding author ）：tizq12@vip．163．com文章编号：1001-6325（2011）09-1000-06研究论文细丝蛋白A 抑制人结肠癌SW480细胞体外侵袭转移能力史建伟1，2，于跃明2，王士杰3，杨珊4，孔繁龙5，张娟2，王贵英2*（1.河北大学附属医院肿瘤外科，河北保定071000；河北医科大学第四医院2.外二科；3.肿瘤研究所；4.外一科；河北石家庄050011；5.山西医科大学临床二系07级，山西太原030001）摘要：目的研究细丝蛋白A （FLNa ）对人结肠癌SW480细胞侵袭转移行为的影响，并初步探讨其抑癌的机制。
方法将FLNa 基因质粒载体pcDNA3.1/V5-His-TOPO /FLNa ，以脂质体介导方式转染SW480细胞。
经G418筛选，获得FLNa 基因稳定表达的SW480/FLNa 细胞。
反转录聚合酶链反应（RT-PCR ）和Western blot 检测FLNa 基因导入；RT-PCR 和Western blot 检测细胞中基质金属蛋白酶-9（MMP-9）的表达；MTT 法检测细胞的增殖能力；划痕损伤实验、Transwell 小室和Matrigel 侵袭模型评价FLNa 对细胞侵袭和转移能力。
结果在SW480细胞中，FLNa 的表达载体pcDNA3.1/V5-His-TOPO /FLNa 获得稳定表达，SW480/FLNa 细胞FLNa 的mRNA 和蛋白的表达水平均较SW480细胞高，分别为［（1.27ʃ0.03）vs （0.14ʃ0.02），P ＜0.01］和［（1.18ʃ0.03）vs （0.25ʃ0.02），P ＜0.05］；SW480/FLNa 细胞MMP-9的mRNA 和蛋白表达水平均低于SW480细胞，分别为［（0.19ʃ0.02）vs （1.63ʃ0.04），P ＜0.05］和［（0.12ʃ0.02）vs （0.79ʃ0.04），P ＜0.05］。
【图片分析】如何应用imageJ进行细胞划痕面积分析？
细胞划痕（wound healing）法是简捷测定细胞迁移运动和修复能力的方法，常用于肿瘤细胞运动的研究。
当我们人为的划开一个“伤口”后，划痕边缘的细胞便努力迁移覆盖中间的空白区域，使伤口愈合。
然而迁移的形状并不是规律的，通过细胞迁移会形成各异的形状。
在细胞划痕实验结束后，我们需要对细胞划痕实验数据统计分析处理。
然而，面对细胞划痕形态多样的“愈合伤口”，统计二维的面积是目前最常用最适合的方法。
细胞划痕实验中常用的获取选区的方法有方差（variance）滤波法（插件法），查找边缘（find-edges）法。
imageJ软件，在面积统计方面非常适合，对于划痕实验数据的统计利用imageJ即可轻松实现。
今天小编就基于imageJ软件来详细介绍下如何进行划痕实验分析。
01插件法1、插件安装插件下载地址：rs.fr/attachments/download/1992/MRI_Wound_Healing_Tool.ijm将下载的插件“MRI_Wound_Healing_Tool.ijm”文件放到安装目录下的macros/toolsets文件夹。
2、重启ImageJ，然后点击ImageJ工具栏的>>按钮，可得插件位置；3、单击后在新工具栏中出现两个图标，第一个按钮是打开插件官网的帮助界面，“m”是用来测量的。
左键单击第二个按钮“m”开始测量，右键单击“m”设置参数。
4、插件内置有两种方法，默认是方差滤波法；（1）打开需要测量的图片，直接左键单击m图标，秒出结果，不过从本次结果来看，红色选区轮廓线离细胞有点距离，细胞划痕面积会偏小。
（2）以上为默认参数。
如果效果不好，可以根据下面的参数说明进行调参：右键点击“m”按钮设置参数。
比如将Variance filter radius值改小一点，将Threshold改大一点，点击OK；再次点击m 图标，这回划痕面积和选区轮廓线也更贴近细胞一点；●Variance filter radius（方差滤波器半径）：要根据图片分辨率来设置，默认是10px，主要是用来识别划痕边缘，越大计算越慢，而且会导致测量得到划痕面积偏小；●Threshold（阈值）：将由方差滤波器得到的图像转换为蒙版；如果调小一点选区会更精细，但会丢失掉一部分选区；调大一点，对于划痕区域识别会更准，但对边缘轮廓线会进行近似处理；具体可用查找边缘法中的threshold建立选区的操作体验一下。
百克瑞纱布创面敷料对伴甲床损伤手外伤患者的疗效观察
doi：12・3969/j・issn・1022-7396.2229.36229-论著-百克瑞纱布创面敷料对伴甲床损伤手外伤患者的疗效观察郑红志张桂荣张硕王芝云杨帆白雪景【摘要】目的探讨百克瑞纱布创面敷料对伴有甲床损伤的手外伤患者的临床疗效。
方法回顾性分析自204年2月至20-年2月门诊伴有甲床损伤的手外伤患者，并选取获得完全随访的患者40例，随访时间为2~4个月。
患者均给予正规清创缝合术，其中60例(对照组)患者为术后凡士林无菌纱布2层覆盖伤口，外覆无菌纱布包扎，换药至伤口干燥无渗出，再单独以碘伏消毒，无菌纱布包扎，直至伤口愈合拆线。
64例(研究组)患者为术中给予应用百克瑞纱布创面敷料擦拭伤口，术后换药过程中给予盐水冲洗伤口，覆百克瑞纱布创面敷料于伤口处，无菌纱布包扎，直至伤口干燥无渗出，再单独以碘伏消毒，无菌敷料包扎，直至伤口愈合拆线。
结果研究组患者的伤口愈合时间及换药次数明显短于对照组，差异有统计学意义(P<0.07);研究组患者的术后感染率明显少于对照组，差异有统计学意义(P<0.07);研究组患者术后渗出及疼痛指数明显少于对照组，差异有统计学意义(P<0.07)。
结论百克瑞纱布创面敷料应用于对伴有甲床损伤的手外伤患者的治疗中预防感染疗效确切，伤口渗出少，减少换药次数，换药过程中疼痛痛苦小，愈合时间短，治疗无不良反应，值得临床推广。
【关键词】百克瑞纱布创面敷料;凡士林无菌纱布；甲床损伤；伤口感染；伤口愈合【中图分类号】R64【文献标识码】A【文章编号】-02-7336(2021)03-412-04The crrutive effectn of Bacre garze wornd dressing in UeatmenU of hand trurma camplicateC by naii beC injrruZHENG Hongzhi*,ZHANG Guhong,ZHANG Shan,et aO*Depavtm,en1tf Emergency MePicine,flip Foutt Hospital of HengsPaiC o/,HePei,HengsPai073400,COina[Abstruct i Objective To isves/gate the csra/ve eTects of Bacre gauze wousd dressing is Weatmeot of haod trauma complicated by sail bed Ojury.Methods A total of40pa/eots with hand trauma complicated by sail bed injum who weretreated is our hospiml from Fedsary204to Fedruarp2019were eorolleP is the study,who were raodomly divided into contslgsup and research gsup,with64cases is each gmup and followed up for6~4months.All the pa/eots were treated byregular dePdPemeot and suture,aod the patieots is conWol gsup were treated by two layers of vasePse sterile gauze coveringthe wound Oter the operaPon,theo covered with sterile gauze,the dressing was changed us/i the wound was dry withoutexyda/on.And the patieots is research gmup were treated by Bacre gauze wousd dressing du/ng opera/on to wipe the wound,aod washing the wousd with sa/se du/ng the dressing change Opr operaPon.The wousd was covered with Bacre gauze wounddressing and Uandaged with sterile gauze until wound healing aod disconsectOg.Resnltt The wousd healing Kmc and thefsqneoch of dressing changes is research gsup were significanKy skoder thas those is conWol gsup(P<0.07).And the posmpemOve infection rate is research gsup was significanKy lower thas that is control gsup(P<4.47).Moreover the posmpemOve exxda/on aod pais index is research gsup were significanKy lower thas those is conWol gsup(P<4.47).Conclrsion The app/ca/on of Bacre gauze wousd dressing is Weatmeot of hasp trauma comp/cated Uy sail bed injum ReTective is preveoKng infection,reXucing wousd exxdaKon,aod fsqneoch of dressing chasges aod the pais durOg dressingchange,which cas skodeo the hea/ng Kmc,without adverse reactions,thus,which is wodhy of clinical promo/on.【Key words】Bacre gauze wousd dressing；vasePse sterile gauze；sail bed injury；wound infection;wousd hea/ng由于手部在多数情况下没有太多的保护,而又需要不断地接触各种工具和物件，使得它成为全身最易受伤的一个部分「4/。
IL-6在角膜损伤修复中作用的研究进展
IL-6在角膜损伤修复中作用的研究进展曾卫娟;罗红【摘要】角膜损伤是眼科临床常见疾病,角膜愈合过程对视力恢复至关重要.角膜损伤后的愈合过程是多种细胞及细胞因子的共同作用,IL-6在介导炎症反应及组织修复及角膜损伤修复过程中均发挥重要作用.【期刊名称】《眼科学报（英文版）》【年(卷),期】2018(033)001【总页数】4页(P50-53)【关键词】角膜损伤;白介素-6;创面愈合【作者】曾卫娟;罗红【作者单位】武汉大学中南医院眼科,武汉430071;武汉大学中南医院眼科,武汉430071【正文语种】中文角膜损伤在临床较为常见，角膜损伤修复过程受阻可导致视力受损甚至失明。
多种细胞及细胞因子参与角膜损伤修复过程。
IL-6在免疫调节、促进血管新生及促进损伤修复过程中发挥重要作用。
近年来，IL-6参与角膜损伤修复的相关研究已取得进展，本文就IL-6在角膜损伤修复中的作用及其机制作一综述。
1 角膜的结构角膜是一层透明且有弹性的无血管组织，其中心部位厚约0.5 mm，由前向后分为5层，依次为上皮细胞层、前弹力层、基质层、后弹力层和内皮细胞层。
角膜上皮细胞层由5～6层细胞及基底膜组成。
基质层由角膜基质细胞、I，IV，VI型胶原纤维、黏蛋白和糖蛋白等组成。
内皮细胞由单层内皮细胞组成。
正常角膜组织封闭且完整，被泪膜及房水包裹，其营养主要由房水与角巩膜缘毛细血管渗透供给。
因此，角膜损伤后，角膜上皮细胞、间质细胞、内皮细胞及角膜基质在其损伤修复过程中至关重要；同时，泪膜、房水及角巩膜缘内营养物质、生长因子及细胞因子在角膜损伤修复过程中也发挥重要作用。
2 角膜损伤修复过程角膜组织损伤可由外伤、肿瘤、炎症、感染等多种原因导致。
组织损伤后机体启动修复过程。
组织修复与伤口愈合大致经历3个基本阶段：炎症反应、组织增生与肉芽形成、伤口收缩与瘢痕形成。
也有文献[1]将其分为凝血期(有血管组织)、炎症期、上皮化、血管新生、纤维增生、细胞外基质沉积及重塑期。
(完整word版)病理题库(word文档良心出品)
选择题1. 全身营养不良时，首先发生萎缩的组织或器官是：A. 骨骼肌B. 脂肪组织C. 肝D. 脑E. 心肌【答案】B2. 一种成熟组织由另一种成熟组织取代的现象称：A．间变B．机化C．化生D．再生【答案】C3. 细胞水肿和脂肪变性常发生在：A. 肺、脾、肾B. 心、脾、肺C. 心、肝、肠D. 肝、肾、脾E. 心、肝、肾【答案】E4. 下列哪个器官的梗死灶为地图形A. 小肠B. 脾C. 肾D. 肺E.心【答案】E5. 易发生干性坏疽的器官是：A．肺B．阑尾C．膀胱D．四肢E．子宫【答案】D6. 一种成熟组织由另一种成熟组织取代的现象称：A．间变B．机化C．化生D．再生E．分化【答案】C7. 缺碘所致的甲状腺肿大属于A．过再生性增生B．再生性增生C．甲状腺肥大D．内分泌障碍性增生E．甲状腺增生合并肥大【答案】D8. 肠上皮化生的概念是：（）A．原来无上皮部位出现肠上皮B．肠上皮转变为其他上皮C．肠上皮转变为胃黏膜上皮D．胃粘膜上皮转变为肠上皮E．呼吸道上皮转变为肠上皮【答案】D9. 虎斑心见于：A．白喉B．中毒C．严重贫血D．肥胖E．高脂血症【答案】B10. 浊肿和脂肪变性主要发生于A、肺、肾、脾B、心、肝、肾C、心、肺、脾D、心、肝、脑【答案】B11. 属不可逆性的病变是：（）A．水样变性B．脂肪变性C．线粒体肿胀D．内质网扩张E．核碎裂【答案】E12. 四肢骨折石膏固定后引起的骨骼肌萎缩．主要属于：A．神经性萎缩B．营养不良性萎缩C．压迫性萎缩D．废用性萎缩E．生理性萎缩【答案】D13. 按组织再生能力的强弱来比较．下列各组的排列哪个是正确的?A．结缔组织＞神经细胞＞肝细胞B．软骨＞腱＞肾小球C．骨＞平滑肌＞神经细胞D．鳞状上皮细胞＞横纹肌＞周围神经纤维E．肾小管上皮细胞＞骨髓细胞＞脂肪细胞【答案】D14. 坏死细胞的微细结构消失，但细胞和组织结构轮廓仍存在，这种坏死称A、液化性坏死B、干酪样坏死C、坏疽D、凝固性坏死【答案】D15. 纤维素样变性最常见于哪种疾病中A、急性细菌性炎症B、病毒性炎症C、变态反应性炎症D、寄生虫性炎症【答案】A16. “肥大”是指：A．实质细胞数目增多B．实质细胞体积增大C．组织、器官体积的增大D．是细胞、组织、器官体积的增大E．间质增生【答案】D17. 下列变性病变应除外：A．气球样变B．Russell小体C．虎斑心D．蜡样变性E．透明变性【答案】D18. 1. 急性肠扭转最可能引起：（）A．凝固性坏死B．湿性坏疽C．气性坏疽D．液化性坏死E．以上都不是【答案】D19. 血管壁的玻璃样变性主要发生于A、小动脉B、中等动脉C、大动脉D、细动脉【答案】D20. 细胞萎缩在电镜下最显著的特点是：A．肌丝增多B．线粒体增多C．滑面内质网增多D．粗面内质网增多E．自噬泡增多【答案】E21. 下述哪项与液化性坏死无关？（）A．脑组织坏死B．化脓菌感染C．乳房的外伤性脂肪坏死D．急性胰腺炎引起的脂肪坏死E．肾梗死【答案】E22. 浊肿是指A、器官体积增大B、细胞体积增大C、组织水种D、细胞颗粒变性【答案】A23. 细胞水肿和脂变常发生在：A．肺、脾、肾B．心、脾、肺C．心、肝、肠D．肝、肾、脾E．心、肝、肾【答案】E24. 慢性萎缩性胃炎时，胃粘膜上皮可化生为（）A、鳞状上皮B、移行上皮C、软骨D、骨E、肠上皮【答案】E25. 细胞坏死（necrosis）的主要形态标志是（）A．线粒体肿胀B．核碎裂C．胞质嗜酸性增强D．胞质脂滴增加E．间质粘多糖积聚【答案】B26. 在组织学上看到有细胞核浓缩、碎裂、溶解时．说明：A．细胞正开始死亡B．细胞的功能还有可能恢复C．细胞的功能虽然可能恢复．但已极为困难D．细胞已经死亡了一段时间E．细胞浆可能还没有发生改变【答案】D27. 易发生干性坏疽的器官是A、肺B、四肢C、心肌D、肠道【答案】B28. 全身营养不良时．首先发生萎缩的组织或器官是：A．骨骼肌B．脑C．肝D．脂肪组织E．心肌【答案】D29. 一种成熟的组织变成另一种成熟组织的过程称：A．机化B．钙化C．分化D．化生E．适应【答案】D30. 四肢骨折石膏固定后引起的骨骼肌萎缩．主要属于：A．神经性萎缩B．废用性萎缩C．压迫性萎缩D．营养不良性萎缩E．生理性萎缩【答案】B31. “肥大”是指：A．实质细胞数目增多B．实质细胞体积增大C．组织、器官体积的增大D．是细胞、组织、器官体积的增大E．间质增生【答案】D32. 一种成熟组织由另一种成熟组织取代的现象称：A．间变B．机化C．分化D．再生E．化生【答案】E33. 下列哪种组织再生能力最强A．腺体B．骨骼肌C．神经细胞D．软骨E．平滑肌【答案】A34. 支气管粘膜上皮出现鳞状上皮化生应属于A、分化不良B、不完全再生C、癌前期病变D、适应性改变【答案】D35. 下列各项中哪个是错误的?A．机化乃是用肉芽组织来取代坏死组织或异物的过程B．单核细胞的溶酶体如果功能发生异常则不能很好的将异物排除或机化C．包裹从本质上说也是一种机化过程D．机体对外界侵入体内的异物．总是以机化的方式加以处理E．机化对机体是有利的．但有时也可带来不良后果【答案】D36. 坏死与坏疽的主要区别在于：（）A．动脉阻塞的程度B．静脉回流的好坏C．有无腐败菌感染D．发生部位E．病变深度【答案】C37. 支气管粘膜上皮出现鳞状上皮化生．常属于下列哪一种改变A．分化不良B．不典型增生C．不完全再生D．癌前期改变E．适应性改变【答案】E38. 虎斑心的原因是A、长期严重贫血B、营养不良C、中毒D、严重感染【答案】A39. 梗死的形状取决于：（）A．脏器的外形B．动脉阻塞的部位C．动脉阻塞的程度D．血管的分布E．有无淤血的基础【答案】D40. 组织和细胞的变性是指细胞内或细胞间质中A、变质B、出现异常物质C、正常物质积聚过多D、B＋C【答案】D41. 本质是坏死的“变性”是下列哪一种病变A、水样变性B、颗粒变性C、粘液样变性D、纤维素样变性【答案】D42. 肠上皮化生的概念是：（）A．原来无上皮的部位出现肠上皮B．肠上皮转变为其他上皮C．肠上皮转变为胃粘膜上皮D．胃粘膜上皮转变为肠上皮E．呼吸道上皮转变为肠上皮【答案】D43. 下列哪一种病变不见于浊肿A、细胞内水分增多B、胞浆内出现红染的颗粒C、内浆网扩张脱颗粒D、糖原及自噬泡增多【答案】D44. 下列哪一种最适合肉芽组织?A．巨细胞和胶原纤维B．纤维母细胞和巨细胞C．毛细血管和淋巴细胞D．纤维母细胞和巨噬细胞E．纤维母细胞和新生毛细血管【答案】E45. 慢性消耗性疾病首先发生萎缩的组织是A、心肌B、骨骼C、脂肪组织D、肌肉组织【答案】C46. 细胞水肿发生的机理是：A．内质网受损B．线粒体受损C．高尔基氏器受损D．核糖体受损E．中心体受损【答案】B47. 肉芽组织是由……组成：A．吞噬细胞和成纤维细胞B．新生毛细血管和成纤维细胞C．新生毛细血管和单核细胞D．单核细胞、巨细胞和淋巴细胞形成的结节E．以上都不是【答案】B48.下列肾小管变性中．哪种损害最轻?A．气球样变B．水变性C．脂变D．细胞内钙盐沉着E．细胞内玻变【答案】A49. 支气管鳞状上皮化生是A、由纤毛柱状上皮直接转变为鳞状上皮B、从咽部鳞状上皮增生延伸覆盖到支气管纤毛柱状上皮C、支气管腺体细胞转化鳞状上皮D、由基底储备细胞增生向鳞状上皮分化【答案】D50. 液化性坏死主要发生于：A．肺B．肾C．脑D．心E．肝【答案】C51. 以下哪一项原因不引起萎缩?A．肾盂积水B．慢性肝淤血C．垂体功能低下D．四氯化碳中毒E．脊髓前角神经细胞坏死【答案】D52. 脑动脉粥样硬化引起脑萎缩属于（）A、营养不良性萎缩B、废用性萎缩C、神经性萎缩D、压迫性萎缩【答案】A53. 肉芽组织是由……组成：A. 吞噬细胞和成纤维细胞B. 新生毛细血管和成纤维细胞C. 新生毛细血管和单核细胞D. 单核细胞、巨细胞和淋巴细胞形成的结节 E. 以上都不是【答案】B54. 有利于创伤愈合的因素是（）A、局部血供不足B、局部感染C、清除异物D、神经损伤【答案】C55. 下列各种组织哪一种再生力最强?A．骨骼肌B．神经节细胞C．心肌D．神经胶质细胞E．软骨【答案】D56. 下列哪种细胞再生能力最弱（）A、汗腺上皮细胞B、横纹肌细胞C、神经节细胞D、表皮细胞【答案】C57. ．大块疤痕可引起：A．关节运动障碍B．器官表面凹陷C．器官变形D．腔室狭窄E．以上均可【答案】E58. 再生能力最强的细胞是A、结缔组织细胞B、肝细胞C、神经细胞D、心肌细胞【答案】B59. 全身营养不良时．首先发生萎缩的组织或器官是：A．骨骼肌B．脂肪组织C．肝D．脑E．心肌【答案】D60. 患者脚趾呈黑褐色、干缩与正常组织界限清楚的病变，最可能是（）A、严重淤血B、恶性黑色素瘤C、干性坏疽D、出血性坏死【答案】C61. 机化是指A、组织完全再生B、组织化生C、结缔组织及血管增生D、肉芽组织替代坏死组织或异物的过程【答案】D62. 只有一个开口的病理性盲管是（）A、糜烂B、溃疡C、窦道D、瘘管E、空洞【答案】C63. 下列哪种是肉芽组织的特征?A．新生毛细血管及纤维母细胞B．巨细胞和纤维母细胞C．巨细胞增生和淋巴细胞浸润D．巨细胞增生．淋巴细胞和浆细胞浸润E．新生的毛细血管及组织细胞增生【答案】A64. 除下列哪一项外，均为一期愈合的特点：（）A．组织破坏极少B．无或极少细菌感染C．过多的疤痕组织D．创缘整齐，对合紧密E．适量的肉芽组织【答案】C65. 组织损伤后由结缔组织增生来修补的过程称：A．再生B．增生C．化生D．机化E．不完全再生【答案】E66. 下列各种细胞再生能力最强的是：（）A．表皮细胞B．平滑肌细胞C．肾小管上皮细胞D．血管内皮细胞E．软骨母细胞【答案】A67. 下列各种细胞中，哪种再生能力最强？A．神经节细胞B．平滑肌细胞C．软骨细胞D．心肌细胞E．间皮细胞【答案】E68. 细胞缺氧．细胞膜受损．细胞内出现：A．钠多，钾多，水多B．钠少，钾多，水少C．钠多，钾少，水多D．钠多，钾少，水少E．钠少，钾多，水多【答案】C69. 肉芽组织是由……组成：A．吞噬细胞和成纤维细胞B．新生毛细血管和成纤维细胞C．新生毛细血管和单核细胞D．单核细胞、巨细胞和淋巴细胞形成的结节E．以上都不是【答案】B70. 1. 下列各种细胞再生能力最强的是：（）A．表皮细胞B．平滑肌细胞C．肾小管上皮细胞D．血管内皮细胞E．软骨母细胞【答案】A71. 1. 下述血栓中，哪种是白色血栓：（）A．风湿性心内膜炎的瓣膜赘生物B．心房纤颤时心耳内球状血栓C．心肌梗死时的附壁血栓D．微循环内微血栓E．下肢深静脉内的延续性血栓【答案】A72. 右上肢静脉血栓脱落主要栓塞于A. 肠系膜动脉B. 脑动脉C. 肺动脉D. 肾动脉E. 心冠状动脉【答案】C73. 下列哪种血栓的名称不反映其组成成分A、白色血栓B、混合血栓C、附壁血栓D、红色血栓【答案】C74. “槟榔肝”的形成是由于：（）A、肝淤血和肝细胞脂肪变性B、肝淤血和肝细胞萎缩C、肝淤血和肝细胞坏死D、肝脂肪变和结缔组织增生E、肝出血和结缔组织增生【答案】A75. 下列哪项不是慢性淤血的后果A．实质细胞的增生B．出血C．含铁血黄素沉积D．组织间质增生E．可并发血栓形成【答案】A76. 下列对肺出血性梗死的叙述，哪一项是错误的？（）A、多发生在严重肺淤血的基础上B、由肺动脉分支阻塞引起C、梗死区与周围非梗死区界限不清D、梗死灶呈锥体形E、多由支气管动脉阻塞引起【答案】E77. 下述因素哪种与血栓形成无关? A．血管内膜损伤B．血流缓慢C．血小板数量增多D．癌细胞崩解产物E．纤维蛋白溶酶增加【答案】D78. 下列哪种镜下改变不属于流行性乙型脑炎的病变：（）A．血管套B．筛状软化灶C．噬神经现象D．蛛网膜下腔脓液聚积E．胶质小结【答案】D79. 肺栓塞时90%的血栓栓子来源于（）A、肝静脉B、脾静脉C、下肢深静脉D、上肢静脉【答案】C80. 循环血液中的血凝块，随血流运行，阻塞相应大小的血管叫（）A、血栓B、血栓形成C、血栓性栓塞D、血栓栓子【答案】C81. 原位癌是指：（）A．早期癌B．未突破基膜的癌C．癌前病变D．原发癌E．未发生转移的癌【答案】B82. 右上肢静脉血栓脱落主要栓塞于A．肺动脉B．脑动脉C．肝动脉D．心冠状动脉E．以上均不是【答案】A83. 栓子是A．循环血液内脱落的血栓B．循环血液内脱落的菌落C．循环血液内不溶于血液的异物D．循环血液内脂肪和空气E．以上都不是【答案】C84. 股静脉血栓形成时下述哪种结局不易发生?A．阻塞血流B．机化C．脱落D．钙化E．血流完全恢复正常【答案】E85. 下述关于肺淤血的描述中哪一项是错误的?A．肺泡壁毛细血管扩张B．肺泡内中性白细胞和纤维素渗出C．肺泡腔内有水肿液D．可发生漏出性出血E．常可见心衰细胞【答案】D86. 循环血液中的凝血块，随血流运行至相应大小的血管，引起管腔阻塞的过程叫做A．血栓B．血栓形成C．血栓栓塞D．梗塞E．血栓栓子【答案】C87. 脑动脉发生血栓性栓塞，其栓子最可能来自（）A. 下肢深静脉血栓B. 下肢浅静脉血栓 C. 盆腔静脉血栓D. 左心室附壁血栓E. 门静脉血栓【答案】D88. 有关慢性肝淤血的叙述中，下列哪一项不妥?A．中央静脉扩张B．肝窦扩张C．肝细胞有萎缩D．门静脉扩张E．部分肝细胞脂变【答案】D89. 混合性血栓对机体造成最严重的危害是A．血栓脱落引起的栓塞；B．阻塞血管引起局部淤血C．阻塞血管引起血管局部坏死D．血栓形成时消耗大量凝血因子造成机体出血E．以上均不是【答案】C90. 梗死灶呈不规则形的是下列哪一种梗死A、肾梗死B、肠梗死C、心肌梗死D、脾梗死【答案】C91. 右心衰竭时引起淤血的器官主要是A．肺、肝及胃肠道B．肝、脾及胃肠道C．脑、肺及胃肠道D．肾、肺及胃肠道E．脾、肺及胃肠道【答案】B92. 瘀血时血液主要瘀积于（）中A、小静脉丛B、大、小中等静脉C、小动脉及毛细血管D、小静脉及毛细血管【答案】D93. 混合性血栓可见于：A．静脉血栓的尾部B．毛细血管内血栓C．急性风湿性心内膜炎的疣状血栓D．动脉血栓的头部E．左心房内的附壁血栓【答案】E94. 下列哪个器官易发生出血性梗死?A．心B．肾C．肺D．脑E．脾【答案】C95. 下列血液性质的改变哪种不利于血栓形成A、新生血小板大量入血B、血液浓缩C、组织因子大量释放D、纤溶系统激活【答案】D96. 3．静脉内血栓脱落可引起：（）A．下肢坏疽B．肺静脉栓塞C．肺动脉栓塞D．脑动脉栓塞E．门静脉栓塞【答案】C97. 肺尖的一球形病灶，境界清楚，直径为3.5cm，应首先考虑：（）A．原发性肺癌B．转移性肺癌C．结核球D．肺脓肿E．矽肺【答案】C98. 风湿性心内膜炎在二尖瓣形成赘生物的条件是：A、内皮细胞受损脱落B、涡流形成C、炎症使血液凝固性增高D、心肌收缩力减弱致血流减慢【答案】A99. 慢性肺淤血的镜下改变，下列哪一项应该除外?A．肺泡腔内有心衰细胞B．肺泡壁增宽C．肺泡壁毛细血管扩张充血D．切面为棕红色E．肺内支气管扩张【答案】D100. 梗死形成的基本条件是：A、血管吻合支不丰富B、高度淤血C、动脉血供阻断D、组织致密【答案】C101. 股静脉内血栓脱落易引起：（）A．下肢坏疽B．肺动脉栓塞C．脑动脉栓塞D．门静脉栓塞E．肾动脉栓塞【答案】B102. 肺栓塞的后果包括以下几点，但________除外A．急死B．肺梗死C．间质性肺炎D．肺动脉高压E．右心房扩大【答案】E103.下列梗死中哪项属于液化性坏死?A．肺梗死B．脑梗死C．肠梗死D．肾梗死E．脾梗死【答案】B104. 右下肢静脉血栓脱落主要栓塞于A．肺动脉B．下腔静脉C．右心房D．右心室E．右下肢大静脉【答案】A105. 下述有关血栓形成的记述中，哪一项是错误的？（）A. 静脉血栓多于动脉血栓B. 下肢血栓多于上肢C. 动脉内血栓多为混合血栓D. 静脉内血栓多为红色血栓E. 毛细血管内血栓多为透明血栓【答案】C106. 1．槟榔肝是指A．肝脂变B．肝水变性C．门脉性肝硬化D．慢性肝淤血E．坏死后性肝硬化【答案】D107. 心衰细胞是由于A．心衰时肺泡内巨噬细胞吞噬了红细胞B．心衰时肺泡内巨噬细胞吞噬了尘埃颗粒C．心衰时肺泡内巨噬细胞吞噬了纤维素样坏死物D．心衰时巨噬细胞的集聚E．以上都不是【答案】A108. 下述哪项是错误的？A．双重血液循环的器官不易发生梗死B．全身血液循环状态对梗死的形成无影响C．动脉痉挛促进梗死的形成D．有效的侧枝循环的建立可防止梗死的发生E．梗死多由动脉阻塞引起【答案】B109. 含细菌的血栓脱落可引起：A．败血性梗死B．贫血性梗死C．出血性梗死D．单纯性梗死E．广泛性梗死【答案】A110. 22、哪种血栓形成涉及的面积最广A、心瓣膜白色血栓形成B、下腔静脉血栓形成C、心肌梗死附壁血栓形成D、DIC 形成的透明血栓【答案】D111. 肝小叶中央脂肪变性主要发生于A、慢性酒精中毒B、慢性磷中毒C、严重感染D、肝淤血【答案】D112. 混合性血栓可见于：（）A．静脉血栓的尾部B．毛细血管内血栓C．急性风湿性心内膜炎的疣状血栓D．动脉血栓的头部E．左心房内的附壁血栓【答案】E 113. 槟榔肝大体形态的组织学基础是（）A．肝小叶周边部肝细胞萎缩B．肝小叶结构破坏C．肝血窦扩张淤血，肝细胞脂肪变性D．出血性梗死E．门静脉分支扩张【答案】C114. 下列哪项不是慢性淤血的后果：A．漏出性出血B．实质细胞增生C．间质细胞增生D．间质细胞增生E．并发血栓形成【答案】B115. 新生毛细血管、成纤维细胞长入血肿的过程称为A、血凝块包裹B、血肿机化C、栓塞D、化生【答案】B116. 关于化脓性炎症的结局哪一项是错误的？A. 吸收消散B. 机化包裹C.脓汁引流排出，组织再生而痊愈D. 转为慢性E. 化脓菌入血引起菌血症【答案】B117. 急性炎症时组织肿胀的主要原因是：A. 组织增生B. 组织变质C.肉芽组织形成D. 静脉血栓阻塞E.充血及血液成分渗出【答案】E118. 下列哪一项不是浆液性炎A. 胸膜炎积液B. 早期感冒的鼻粘膜炎 C. 肾盂积水D. 昆虫毒素引起的皮下水肿E.皮肤Ⅱ度烧伤水疱【答案】C119. 下列哪一种疾病是以变质为主的炎症A、大叶性肺炎B、流行性脑脊髓膜炎C、肾小球肾炎D、病毒性肝炎【答案】D120. 下列哪项不是渗出性炎?A．大叶性肺炎B．钩体病C．阑尾炎D．绒毛心E．Pautrier微脓肿【答案】B121. 急性炎症时局部组织肿胀，其主要原因是A、动脉充血B、静脉阻塞C、富于蛋白的液体渗出D、组织增出【答案】C122. 1．在慢性炎症中下列哪种细胞常见?A．嗜酸性粒细胞B．淋巴细胞C．嗜中性粒细胞D．肥大细胞E．巨噬细胞【答案】B123. 增生性炎（）A、都是急性炎B、都是慢性炎C、都是亚急性炎D、有急性炎也有慢性炎【答案】D124. 白喉、细菌性痢疾一般属于哪一类炎症？（）A．纤维素性炎B．化脓性炎C．卡他性炎D．浆液性炎E．出血性炎【答案】A125. 哪一项主要是由血管通透性增加引起的A、红B、肿C、痛D、热【答案】B126. 多核细胞不见于下列哪种疾病?A．伤寒B．结核C．霍奇金淋巴瘤D．血吸虫虫卵结节E．异物肉芽肿【答案】C127. 巨噬细胞、纤维母细胞和淋巴细胞最常见于A．急性炎症B．肉芽组织C．伤口愈合处D．慢性炎症E．化脓性炎症【答案】D128. 下列哪项不属于渗出性炎症?A．假膜性炎症B．大叶性肺炎C．卡他性炎症D．阿米巴肝脓肿E．流脑【答案】D129. 流行性脑脊髓膜炎属于（）A、浆液性炎B、纤维素性炎C、化脓性炎D、出血性炎【答案】C130. 病毒感染的病灶中，最常见的细胞是（）A、中性粒细胞B、嗜酸性粒细胞C、淋巴细胞、单核细胞D、浆细胞E、肥大细胞【答案】B131. 下列哪一种不是渗出性炎症?A．卡他性炎症B．乙型脑炎C．流行性脑膜炎D．肾盂肾炎E．脓肿【答案】D132. 溶血性链球菌最常引起A．蜂窝织炎B．假膜性炎C．坏死性炎D．脓肿E．出血性炎【答案】D133. 细菌性痢疾属于下列哪一种炎症A、纤维素性炎B、浆液性炎C、化脓性炎D、出血性炎【答案】A134. 败血症是指：A．细菌入血B．毒素入血C．细菌入血大量繁殖，产生毒素D．细菌和毒素入血E．化脓菌引起多发性脓肿【答案】C135. 假膜性炎症是指A、浆膜发生的纤维素性炎B、浆膜发生的化脓性炎症C、粘膜发生的纤维素性炎D、粘膜发生的化脓性炎【答案】C136. 炎症的渗出主要由于A．血液动力学改变B．血管壁通透性改变C．小静脉血栓形成D．循环血量增加E．组织间液比重降低【答案】B137. 炎症的防御反应在炎症的局部病灶为：（）A增生性变化 B 血管扩张充血 C炎细胞浸润D 肉芽组织形成E纤维化【答案】C138. 炎症的基本病变是A、组织细胞的变性坏死B、组织的炎性充血和水肿C、红、肿、痛、热、机能障碍D、变质、渗出、增生【答案】D139. 下列有关炎症的理解哪项不正确? A．血管反应是炎症的中心环节B．对机体损害的任何因素均可为致炎因子C．炎症对机体有利．又有潜在危害性D．凡是炎症都运用抗菌素抗炎E．炎症既有局部反应．又可有全身反应【答案】D140. 下列哪一项不属于渗出性炎症? A．卡他性炎症B．阿米巴肝脓肿C．假膜性炎D．绒毛心E．大叶性肺炎【答案】B141. 下列哪项不属于渗出性炎症?A．假膜性炎症B．卡他性炎症C．肾盂肾炎D．乙型脑炎E．流行性脑炎E．流行性脑炎【答案】D142. 下列哪项是变质性炎症A．肾盂肾炎B．菌痢C．大叶性肺炎D．阿米巴肝脓肿E．阑尾炎【答案】A143. 下列哪项不属于渗出性炎症?A．淋病B．绒毛心C．肾盂肾炎D．菌痢E．肠伤寒【答案】E144. 1. 在急性炎症早期哪种细胞多见：（）A．浆细胞B．嗜酸性粒细胞C．单核巨噬细胞D．淋巴细胞E．中性粒细胞【答案】E145. 渗出液的描述中，哪项是错误的？（）A、血管通透性增高B、液体比重高C、液体静置后凝固D、液体内含纤维蛋白原E、液体内含细胞极少【答案】E146. 哪一项不属于渗出性炎症?A．浆液性炎B．假膜性炎C．化脓性炎D．感染性肉芽肿性炎E．出血性炎【答案】D147. 在急性炎症的早期，下列哪种细胞多见A、中性白细胞B、嗜酸性白细胞C、单核巨噬细胞D、淋巴细胞【答案】A148. 急性炎症时血液动力学的变化一般按下列顺序发生A．血流速度减慢→血管扩张，血流加速→细动脉短暂收缩→白细胞附壁B．血管扩张，血流加速→细动脉短暂收缩→白细胞附壁→血流速度减慢C．细动脉短暂收缩→血流速度减慢→血管扩张，血流加速→白细胞附壁D．细动脉短暂收缩→血管扩张，血流加速→白细胞附壁→血流速度减慢E．细动脉短暂收缩→血流速度减慢→血管扩张，血流加速→白细胞附壁【答案】E149. 下列哪一种细胞是化脓性炎症时的特征性细胞A、浆细胞B、郎罕氏巨细胞C、嗜酸性白细胞D、变性坏死的中性白细胞【答案】D150. 寄生虫感染引起的炎症，下列哪种细胞多见A、中性白细胞B、嗜酸性细胞C、单核巨噬细胞D、淋巴细胞【答案】B151. 渗出液中应表现为：（）A．蛋白含量多，不易凝固，细胞数多。
mettl3介导的n6-甲基腺苷修饰对胃癌上皮-间充质转化和转移至关重要
• However, dysregulation of m6A and its functions in tumor epithelial-mesenchymal transition (EMT) and metastasis remain obscure.
• Control the lifetime and degradation of mRNA • Mediates the translation of cyclic RNAs
Background: m6A modification: dynamic reversible modification
m6A: The sixth N of adenosine is methylated. • plays a role in post-transcriptional regulation：
• Regulates gene expression, splicing, RNA editing, and RNA stability
METTL3 knockdown
Results: METTL3 maintained ZMYM11 expression in GC
• Relationship between ZMYM1 and METTL3 in mRNA and protein level
• Immunofluorescence staining • depletion of METTL3 induced loss of ZMYM1 expression • overexpression of METTL3 increased ZMYM1 levels
wound healing assay原理
wound healing assay原理伤口愈合分析（Wound Healing Assay）是一种常用的实验技术，用于研究细胞迁移和伤口愈合过程。
它可提供关于细胞迁移速率、迁移方向、细胞相互作用以及细胞信号传导等方面的信息。
伤口愈合分析的原理主要是利用细胞在宿主细胞层上的迁移能力来评估伤口愈合过程。
该实验技术可以通过创造一个“伤口”区域，使细胞迁移进入该区域，并通过观察和计算移动速率等参数来评估细胞迁移的能力。
具体步骤如下：1. 准备实验器材和试剂：实验器材包括离心管、细胞培养器皿、显微镜和细胞培养设备等。
试剂包括细胞培养液、缓冲液和荧光染料等。
2. 培养细胞：将感兴趣的细胞株培养在细胞培养器皿中，使其达到70-80%的密度。
3. 制作伤口区域：在培养皿中使用P200移液枪等工具，在细胞层上划定一个直径约为1 mm的伤口区域。
也可以使用自动化仪器，如细胞迁移孔（Cell Migration Plate）来方便制作伤口。
4. 拍摄图像：在制作伤口之后，将培养皿放入组织培养箱中，在预定时间点上使用显微镜拍摄伤口附近的图像。
最好能够每隔一段时间拍摄一次，以便观察细胞的迁移过程。
5. 分析图像：使用图像分析软件，定量地分析细胞迁移的过程。
可以通过测量细胞边缘的距离来计算移动速率，并绘制细胞迁移距离的时间曲线。
6. 统计分析：将数据导入统计软件，进行统计学分析，如t检验或方差分析，以确定不同组之间的显著差异。
伤口愈合分析的关键是制作一个均匀的伤口，并且要避免干扰因素。
细胞要在伤口附近迁移，需要一定的细胞自由空间和足够的时间。
此外，尽量避免细胞产生严重的应力，例如由于培养皿的振荡、温度变化或机械刺激等。
伤口愈合分析可以用于研究多种细胞类型，包括上皮细胞、成纤维细胞和免疫细胞等。
它在组织工程、药物筛选和疾病研究等领域中广泛应用。
总结起来，伤口愈合分析是利用细胞迁移的能力来评估细胞在创伤愈合过程中的动态行为。
负压封闭引流技术在胆管癌术后患者切口感染中的应用体会及文献分析
•病例报告窑---------中国医药导报2020年10月第17卷第30期负压封闭引流技术在胆管癌术后患者切口感染屮的应用体会及文献分析陈金梅1黄仲海2俞渊2吴华帅1甘苡榕1李承积11.广西中医药大学研究生院，广西南宁530022；2.广西中医药大学第一附属医院肝胆外科，广西南宁530023［摘要］近年来,随着医疗卫生事业的发展,负压封闭引流技术作为促进创面修复的新型方法在外科创面愈合中应用广泛,发挥着独特的优势。
2019年3月广西中医药大学第一附属医院收治了1例因“重度黄疸”行经皮肝穿胆道造影+胆道内外引流术，后又行胆管癌根治术,术后发生吻合口漏导致切口感染的老年男性患者，在应用负压封闭引流技术持续引流切口渗液后,切口逐渐改善，愈合，效果良好。
本文报道了该例患者的临床资料,就使用负压封闭引流技术在腹部术后切口感染中的应用体会及文献分析进行阐述，以期为临床提供指导。
［关键词］负压封闭引流技术;胆管癌术后；切口感染；吻合口漏；应用［中图分类号］R635［文献标识码］A［文章编号］1673-7210(2020)10(C)-0178-04Application experience and literature analysis of vacuum sealing drainage in incision infection in patients with cholangiocarcinomaCHEN Jinmei1HUANG Zhonghoi2YU Yuan2WU HucishuoL1GAN Yirong1LI Chengji1[.Research SLudenL Academy of Guangxi University of Chinese Medicine,Guangxi Zhuang Autonomous Region,Nanning530022,China; 2.DeparLmenL of HepaLobiliary Surgery,Lhe FirsL AffiliaLed HospiLal of Guangxi UniversiLy of Chinese Medicine,Guangxi Zhuang AuLonomous Region,Nanning530023,China[Abstract]In recenL years,wiLh Lhe developmenL of medical and healLh services,vacuum sealing drainage has been widely used in surgical wound healing as a new meLhod Lo promoLe wound repair,and has played a unique advanLage.In March2019,Lhe FirsL AffiliaLed HospiLal of Guangxi UniversiLy of Chinese Medicine underwenL a percuLaneous Lran-shepaLic biliary angiography+inLernal and exLernal drainage of Lhe biliary LracL for野severe jaundice冶,followed by radical cholangiocarcinoma.PosLoperaLive anasLomosis caused an incision infecLion of elderly male paLienLs,afLer applying Lhe vacuum sealing drainage Lo conLinuously drain Lhe incision,Lhe incision gradually improved,healed,and Lhe effecL was good.This arLicle reporLs Lhe clinical daLa of Lhis paLienL,and expounds Lhe applicaLion experience and liLeraLure analysis of using vacuum sealing drainage in abdominal posLoperaLive incision infecLion in order Lo provide clinical guidance.[Key words]Vacuum sealing drainage;PosLoperaLive cholangiocarcinoma;Incision infecLion;AnasLomoLic leakage;Ap-plicaLion近年来,社会经济飞速发展，人民生活水平提高,我国的基本医疗卫生制度不断完善，医疗卫生事业已进入快速发展阶段,医疗技术、科研水平、临床能力在不断提高叫以往临床对于术后发生切口感染的患者,选用的是常规抗感染、换药、清洗创面坏死组织、引流［基金项目］国家自然科学基金资助项目(81760870)；广西自然科学基金资助项目(2017GXNSFAA198120)；广西科学研究与技术开发计划项目(重点研发计划)(桂科AB17195071)遥［作者简介］陈金梅(1992-),女，广西中医药大学研究生院2018级中医外科学在读硕士研究生;研究方向：肝胆疾病中西临床与研究。
PD-L1下调对口腔鳞癌细胞生物学行为的影响
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２Ｄ０ＤＪ ．Ｃｈｉｎａ
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， ＷＡＮＧ Ｘｕ，ＣＨＥＮ ｎ—ｔａｏ．Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｏｒａｌ ａｎｄ Ｍａｘｉｌｌｏｆａｃｉａｌ—Ｈｅａｄ ａｎｄ Ｎｅｃｋ Ｏｎｃｏｌｏｇｙ，Ｃｏｌｌｅｇｅ ｏ ｆ Ｓｔｏｍａｔｏｌｏｇｙ，
肌肽对人乳腺癌MCF-7细胞增殖、迁移侵袭抑制作用及其机制的研究
Dissertation Submitted to North China University of Science and Technology
in partial fulfillment of the requirement for the degree of
自授予学位之日起；
□自
年 月 日起。
作者签名： 签字日期：
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年月 日
摘要
目的 通过研究肌肽对人乳腺癌细胞 MCF-7 的增殖抑制以及迁移和侵袭抑制现 象，探讨肌肽的可能作用机制。 方法 通过 MTT 实验分别检测实验组及对照组细胞的存活率；细胞平板克隆形成 实验分别检测各分组细胞的克隆形成数；细胞划痕愈合实验、Transwell 迁移和侵 袭实验分别检测各组细胞的迁移和侵袭能力；实时荧光定量 PCR 法检测目的基因 （PTBP1、PKM1、PKM2 和 MTA1） mRNA 的转录水平；Western blot 检测丙酮 酸激酶 PKM1 和 PKM2、肿瘤转移相关蛋白 MTA1 的表达水平。应用 GraphPad PRISM 6.0 软件及 SPSS 23.0 软件进行统计学分析。 结 果 1 MTT 实 验 结 果 显 示 ， 不 同 肌 肽 浓 度 （ 12.5mM 、 25mM 、 50mM 和 100mM）处理 24、48 和 72h 后，乳腺癌细胞的存活率随浓度的增大而降低 （ F=4.852 ， P<0.05 ） ， 具 有 一 定 的 浓 度 依 赖 ， 但 是 与 时 间 的 关 系 并 不 显 著 （F=1.825，P>0.05），并且经统计学分析发现时间与肌肽浓度之间并不存在交互 效应（F=1.829，P>0.05）。2 细胞平板克隆形成实验结果显示，人乳腺癌 MCF-7 细胞经肌肽处理后，与对照组相比可以发现细胞克隆集落数明显减少（F=746.5， P<0.05），各组平板克隆集落数分别为：25mM 组克隆集落数为 135.00±6.40， 50mM 组 为 62.00±7.97 ， 100mM 组 为 7.4±1.34 ， 对 照 组 为 223.60±11.35 。 3 Transwell 迁移实验结果数据显示，对照组和实验组（25mM、50mM 和 100mM） 平均穿过小室膜的细胞数分别为 313.00±23.35、220.60±21.54、132.40±7.77 和 90.80±5.50 个，实验组平均穿膜细胞数量较对照组平均穿膜细胞数显著减少，差 异具有统计学意义（F=177.20，P<0.05）。4 Transwell 侵袭实验结果显示，空白 对照组和实验组（25mM、50mM 和 100mM）平均穿膜细胞数分别为 258.60± 21.93、178.40±11.26、123.40±13.72 和 63.60±13.50 个，经统计分析后发现，各 组间的穿膜细胞数差异明显（F=140.6，P<0.05）；与对照组相比较，实验组的穿 膜细胞数均明显减少，且随着肌肽浓度增高，穿膜细胞数随之下降（P<0.05）。5 细胞划痕愈合实验结果显示，空白对照组、25mM 组、50mM 组和 100mM 组细胞 愈合率分别为 0.46±0.02、0.36±0.02、0.27±0.05 和 0.03±0.02，显著抑制乳腺癌 细胞的划痕愈合率（F=99.20，P<0.05）。6 实时荧光定量 PCR 结果显示，乳腺癌 细胞经肽处理 24h 后，PTBP1，PKM2 和 MTA1 mRNA 转录水平明显降低（P 均
miR-200a_对宫颈癌细胞系HeLa_增殖、迁移、侵袭调控作用观察及其靶向mRNA_和lncRN
miR -200a 对宫颈癌细胞系HeLa 增殖、迁移、侵袭调控作用观察及其靶向mRNA 和lncRNA 预测分析蔡添娥，陈丽婷，于英上海交通大学医学院附属上海儿童医学中心海南医院产前诊断中心，海南三亚572000摘要：目的 观察微小RNA -200a （miR -200a ）对宫颈癌细胞系HeLa 增殖、迁移和侵袭的调控作用，对miR -200a 的靶向mRNA 及靶向长链非编码RNA （lncRNA ）进行预测分析。
方法 取人宫颈癌细胞系HeLa 分为1、2、3及4组，分别转染miRNA 模拟物miR -200a mimics 、miRNA 抑制物miR -200a inhibitor 、对照物mimics NC 及inhibitor NC 。
培养48 h 时采用qRT -PCR 法检测各组细胞miR -200a 表达；采用CCK8法检测各组细胞增殖能力，采用Tran⁃swell 侵袭实验检测细胞侵袭能力，采用Transwell 迁移实验和细胞划痕实验检测各组细胞迁移能力。
使用miRNA 数据库在线预测miR -200a 靶向mRNA 和靶向lncRNA ，采用基因本体论功能注释和京都基因和基因组百科全书富集分析法分析宫颈组织中miR -200a 的靶向mRNA 和靶向lncRNA 的生物学功能。
结果 与3组比较，1组细胞miR -200a 相对表达量高；与4组比较，2组细胞miR -200a 相对表达量低（P 均<0.05）。
与3组比较，培养72、96 h 时1组细胞增殖能力低，培养48 h 时侵袭细胞数及细胞穿膜数少，细胞迁移率低（P 均<0.05）；与4组比较，培养72、96 h 时2组细胞增殖能力高，培养48 h 时侵袭细胞数及细胞穿膜数多，细胞迁移率高（P 均<0.05）。
miR -200a 靶向mRNA 及lncRNA 分别有83、6个， miR -200a 靶向mRNA 功能主要有与转录调控、自噬体组装、神经元迁移和轴突导向等生物学过程，信号通路主要有Wnt 信号通路、Hippo 信号通路及内质网蛋白加工信号通路等。
沉默FOXK1_基因对胃癌HGC-27_细胞增殖、迁移和侵袭的影响
第 50 卷第 2 期2024年 3 月吉林大学学报（医学版）Journal of Jilin University（Medicine Edition）Vol.50 No.2Mar.2024DOI：10.13481/j.1671‐587X.20240210沉默FOXK1基因对胃癌HGC-27细胞增殖、迁移和侵袭的影响陈爽, 李红（锦州医科大学基础医学院生物化学与分子生物学教研室，辽宁锦州121000）［摘要］目的目的：探讨沉默叉头框K1（FOXK1）对胃癌HGC-27细胞增殖、迁移和侵袭的影响，并阐明其可能的机制。
方法方法：基于基因表达水平值的交互式分析平台（GEPIA）数据库查询在胃癌组织和正常胃组织中FOXK1 mRNA表达水平；利用化学合成的si-FOXK1体外转染胃癌HGC-27细胞，实验分为空白对照组、nc-FOXK1组和si-FOXK1组，采用Western blotting法评估各组的转染效率，MTT法检测si-FOXK1转染后各组胃癌HGC-27细胞的增殖能力，克隆形成实验检测si-FOXK1转染后各组胃癌HGC-27细胞的克隆形成数，细胞划痕实验检测si-FOXK1转染后各组胃癌HGC-27细胞的迁移率，Transwell小室实验检测si-FOXK1转染后各组胃癌HGC-27细胞的细胞迁移数和细胞侵袭数，Western blotting法检测si-FOXK1转染后各组胃癌HGC-27细胞中核因子κB（NF-κB）通路相关蛋白［NF-κB p65和磷酸化核因子κB p65 （p-NF-κB p65）］的表达水平。
结果结果：GEPIA数据库查询，在胃癌组织中FOXK1 mRNA表达水平高于正常胃组织（P<0.05）；Western blotting法检测，在胃癌HGC-27细胞中FOXK1蛋白表达水平高于人正常胃黏膜GES-1细胞（P<0.01）；与空白对照组和nc-FOXK1组比较，si-FOXK1组FOXK1蛋白表达水平明显降低（P<0.01）；MTT法检测，与空白对照组和nc-FOXK1组比较，si-FOXK1组胃癌HGC-27细胞的增殖能力降低（P<0.05）；克隆形成实验检测，与空白对照组和nc-FOXK1组比较，si-FOXK1组胃癌HGC-27细胞克隆形成数减少（P<0.05）；细胞划痕实验检测，与空白对照组和nc-FOXK1组比较，si-FOXK1组胃癌细胞的迁移率降低（P<0.05）；Transwell小室实验检测，与空白对照组和nc-FOXK1组比较，si-FOXK1组胃癌HGC-27细胞的迁移细胞数和侵袭细胞数明显减少（P<0.05）；Western blotting法检测，与空白对照组和nc-FOXK1组比较， si-FOXK1组胃癌HGC-27细胞中p-NF-κB p65蛋白表达水平降低（P<0.05）。
灵芝酸A_对人非小细胞肺癌PC-9_细胞增殖和凋亡的影响及其机制
第 49 卷第 6 期2023年 11 月吉林大学学报（医学版）Journal of Jilin University（Medicine Edition）Vol.49 No.6Nov.2023DOI：10.13481/j.1671‑587X.20230609灵芝酸A对人非小细胞肺癌PC-9细胞增殖和凋亡的影响及其机制任爱华1, 鞠欣达1, 柳骜飞1, 刘永超2, 刘岩峰1（1. 北华大学基础医学院解剖教研室，吉林吉林132013；2. 北华大学基础医学院免疫教研室，吉林吉林132013）［摘要］目的目的：探讨灵芝酸A（GAA）对PC-9细胞增殖、凋亡和迁移能力的影响，并阐明其作用机制。
方法方法：体外培养非小细胞肺癌（NSCLC）PC-9细胞，分为对照组（未加GAA）、低剂量GAA组（0.1 mmol·L－1 GAA）和高剂量GAA组（0.5 mmol·L－1 GAA）。
采用噻唑蓝（MTT）法检测各组PC-9细胞增殖率，流式细胞术检测各组PC-9细胞凋亡率，细胞划痕实验检测各组PC-9细胞划痕愈合率，Transwell小室实验检测各组PC-9细胞迁移能力，实时荧光定量PCR（RT-qPCR）法和Western blotting法检测各组PC-9细胞中血管内皮生长因子（VEGF）和缺氧诱导因子1α（HIF-1α）mRNA及蛋白表达水平。
结果结果：MTT法检测，与对照组比较，处理48和72 h时低剂量GAA组细胞增殖率均降低（P<0.05），处理24、48和72 h时高剂量GAA组细胞增殖率均降低（P<0.05）；与低剂量GAA组比较，处理24、48和72 h时高剂量GAA组细胞增殖率均降低（P<0.05）。
流式细胞术检测，与对照组和低剂量GAA组比较，高剂量GAA组细胞凋亡率升高（P<0.05）。
细胞划痕实验，与对照组和低剂量GAA组比较，高剂量GAA组细胞划痕愈合率降低（P<0.05）。
尿路上皮癌抗原1促进滋养细胞功能及对上皮-间质转化转化的调控作用
异位妊娠是妇产科常见的急腹症，也是导致妊娠早期孕妇出血性死亡的主要原因，发病率约2%，其中输卵管妊娠（TP ）最为常见［1,3］。
做为二氢叶酸还原酶抑制剂，甲氨蝶呤可以抑制滋养细胞增殖，破坏绒毛活性，是目前治疗TP 的一线用药，但存在肝、肾功能损害、脱发、骨髓抑制等不良反应［1］。
因此，深入了解TP 发生、发展的分子机制，探索新的治疗靶点仍至关重要。
LncRNA 是一种长度大于200nt 的RNA 分子，不参与蛋白质编码，但可以调控细胞增殖、分化、侵袭、迁移、凋亡等多种生物学功能的发挥［4,5］，LncRNA 表达异常和胃癌、食管鳞状细胞癌、膀胱癌等多种肿瘤疾病的发生有关［6-8］，还可引起复发性流产、先兆子痫等妇产科疾病［9-13］。
尿路上皮癌抗原1（UCA1）是一种首次在膀胱癌中发现并得名的lncRNA ，其表达失调不仅可以促进肿瘤的发生、发展［8,14］，还和子痫前期、妊娠期高血压、妊娠丢失等多种妇产科疾病的发生有关［15-18］。
研究表明，UCA1低表达可使滋养细胞侵袭、迁移能力减弱，造成滋养细胞侵袭不足，从而导致流产等疾病发生［17,18］。
而High expression of long noncoding RNA UCA1promotes invasion,migration and epithelial-mesenchymal transition of trophoblasts in vitroSUN Jianhua 1,2,ZHANG Yi 1,YANG Liping 1,ZHOU Li 1,2,LU Xiyang 2,LI Jiuxian 2,CHEN Ping 11First Clinical College of Medicine,Henan University of Traditional Chinese Medicine,Zhengzhou 450046,China;2Department of Obstetrics and Gynecology,First Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450099,China摘要：目的研究尿路上皮癌抗原1（UCA1）对滋养细胞（HTR-8/SVneo ）侵袭和迁移能力及上皮-间质转化（EMT ）特性的影响，探索UCA1在输卵管妊娠（TP ）发生发展过程中的作用及机制。
Wound-Healing Assay
3Wound-Healing AssayLuis G.Rodriguez,Xiaoyang Wu,and Jun-Lin GuanSummaryThe wound-healing assay is simple, inexpensive, and one of the earliest developed methods to study directional cell migration in vitro. This method mimics cell migration during wound healing in vivo. The basic steps involve creating a “wound” in a cell mono-layer, capturing the images at the beginning and at regular intervals during cell migra-tion to close the wound, and comparing the images to quantify the migration rate of the cells. It is particularly suitable for studies on the effects of cell–matrix and cell–cell interactions on cell migration. A variation of this method that tracks the migration of individual cells in the leading edge of the wound is also described in this chapter.Key Words: Cell migration; wound healing; extracellular matrix; transfection; image capture and analysis; time-lapse microscopy.1.IntroductionThe wound-healing assay is one of the earliest developed methods to study cell migration in vitro (1). This method is based on observation of cell migra-tion into a “wound” that is created on a cell monolayer. Although not an exact duplication of cell migration in vivo, this method mimics to some extent migra-tion of cells in wound healing. For example, denuding part of the endothelium in the blood vessels will induce endothelial cell migration into the denuded area to close the wound (2). Depending on the cell type, cells migrate into the wound as loosely connected populations (e.g., fibroblasts) or as sheets of cells (e.g., epithelial and endothelial cells), which also mimic the behavior of these cells during migration in vivo.In comparison with other popular in vitro methods, such as time-lapse micro-scopy and Boyden chamber assays, the wound-healing assay is particularly suit-able for studies of directional cell migration and its regulation by cell interaction From:Methods in Molecular Biology, vol. 294: Cell Migration: Developmental Methods and ProtocolsEdited by: J-L. Guan © Humana Press Inc., Totowa, NJ23with extracellular matrix (ECM) and cell–cell interactions. Migration of the cells is regulated by both the ECM under the cells and soluble factors (3,4) as well as intercellular interactions in the case of epithelial and endothelial cells (5). The effects of each of these factors on directional cell migration can be studied using the wound healing method. More recently, this assay also has been combined with microinjection or transfection to assess the effects of expression of exog-enous genes on migration of individual cells (6–8). This is probably the sim-plest method to study cell migration in vitro and the only that requires common and inexpensive supplies found in most labs that are capable of cell culturing. The basic steps involve creation of a “wound” on monolayer cells, capture of images at the beginning and regular intervals during cell migration to close the wound, and comparison of the images to determine the rate of cell migration.More recently, a wound-healing assay has been used to track migration of individual cells at the leading edge of the wound to determine the role of par-ticular genes in the regulation of directional migration (8). This requires the aid of time-lapse microscopy and image analysis software and differs from the con-ventional use of time-lapse microscopy in that it measures directional migration of the cells in the population of migrating cells into the wound instead of ran-dom migration of sparsely plated cells. This variation will be described subse-quently (see Subheading 3.).2.Materials1.60-mm or other size tissue culture dishes.2.Razor or extra fine Sharpie® marker.3. 1 mg/mL fibronectin stock.4. 1 mg/mL poly-L-lysine (PLL) stock.5.Phosphate-buffered saline (PBS).6. 2 mg/mL Bovine serum albumin (BSA).7.Versene with trypsin.8.Dulbecco’s modified Eagle’s medium cell culture media with supplements (serum,antibiotics).9.p200 Pipet tips.10.Phase contrast microscope.11.Camera.Optional and for tracking individual cells:12.Video camera.13.Image analysis software.14.Stage incubator.15.Fluorescence microscope.16.LipofectAmine and PLUS transfection reagents (Life Technologies).17.Plasmid-encoding green fluorescent protein (GFP) or other markers.18.CO2 independent media.3.Methods3.1.Measurement of Migration as Population of Cells1.Prepare 60-mm dishes with markings on the outer bottom of the dish to be usedas reference points during image acquisition. Acquisition of images requires the matching of the first image with the second image acquired in steps 7 and 9.Marking the tissue culture dish can be achieved by numerous methods to obtain the same field during the image acquisition. Etching lines lightly with a razor blade on the bottom of the dish provides a good reference under the microscope and can be visualized with the naked eye for creating the wound itself. Keep in mind when using a razor not to separate the lines too much because the goal is to observe two lines in the same field under magnification. Matching the images is essential for quantitative analysis (see Note 1).2.Coat the dishes with 3 mL of either 10 μg/mL fibronectin or 50 μg/mL poly-L-lysine as a control by incubating the dishes overnight at 4°C or several hours at 37°C without rotation or shaking. These are for studies of fibroblast migration.For other cell types, other appropriate ECM substrates can be used.3.When ready to proceed, aspirate off unbound ECM substrate and block the coateddishes with 3 mL of 2 mg/mL bovine serum albumin for 1 h at 37°C.4.Resuspend subconfluent cells growing in a tissue culture dish by washing cellsonce with PBS, add trypsin containing versene, resuspend cells with complete media, and obtain cell counts for all cells to be plated. Cells should be dispersed gently by pipetting and/or rocking the dish before incubation so that cells are distributed equally among the plate.5.Wash the prepared dishes once with PBS and fill the dish with 3–5 mL of media(see Note 2). Plate 1 × 106 NIH 3T3 cells to create a confluent monolayer onto the prepared 60 mm dish. Allow cells to adhere and spread completely for approx4 h under proper incubation. If different cell types or different size dishes areused, adjust the number of cells used to create a confluent monolayer.6.Create a wound by manually scraping the cell monolayer with a p200 pipet tip.Wash the cells once with 1 mL of desired media and replace with 5 mL of the same media. The wound should be created relative to the marking/reference point on the dish. For example, the razor blade method, being the least expensive and most popular, creates a point of reference. The wound is created perpendicular to the marking on the dish.7.Acquire the first image by using the markings on the culture dish as a referencepoint. Place the reference mark outside the capture image field but within the eyepiece field of view such that the reference mark can be aligned later for the subsequent image acquisition.8.Incubate dishes in a tissue culture incubator for 8–18 h (see Note 3).9.Match the photographed region acquired in step 7 and acquire a second image(Fig. 1).10.The images acquired above can be analyzed quantitatively by several methods.The migration of cells can be determined by the number of cells that cross into the wound area from their reference point at time zero. This method provideslarge sample sizes that are easily quantified statistically. Alternatively, various methods can be used to measure the distance or area of the wounded region lack-ing cells. Free software is available that simplifies this process with convenient tools (Website: /ij/). Additionally, more elaborate commer-cial software is available that can automate the measurement process (Media Cybernetics; Carlsbad, CA).3.2. Tracking Migration of Individual Cells in the Wound-Healing Assay1.Prepare 60-mm culture dishes as described in step 1 (Subheading 3.1.) to be used to match wounds during image acquisitions. If time-lapse microscopy will be used, this step is not necessary (see Note 4).2.Plate growing NIH 3T3 cells at 50–60% confluency 12 to 18 h prior to transfec-tion. Transfect NIH 3T3 cells with plasmid encoding the gene of your interest along with a marker plasmid (i.e., GFP) in a 7:1 ratio. Alternatively, a vector-Fig. 1. Example of images acquired at 0 and 18 h in wound-healing assay. Cells shown are uninduced (U) and induced (I), NIH 3T3 cells expressing wild-type 14-3-3β. The dotted lines define the areas lacking cells.encoding GFP fusion protein containing the gene of your interest can be used.Cells are transfected with LipofectAmine and PLUS transfection reagents (Life Technologies) per the manufacturers instructions.3.When cells reach 100% confluency (usually 24 to 48 h after transfection), createa wound with a p200 pipet tip, as described in step 6 (Subheading 3.1.). Washthe plates once and replace with the desired medium. If time-lapse microscopy will be used, CO2 independent media will be required.4.Observe the cells under a fluorescence microscope to ensure that enough cells inthe leading edge of the wound are positively transfected (i.e., as marked by GFP;see Note 5). Acquire both phase contrast and fluorescence images every 2 h by matching the wounded region until the wound has completely closed (usually about 10 h). Return cells to a 37°C cell culture incubator between images unless CO2-independent media was used.5.Calculate the rate of migration of the transfected cells by measuring the distancetraveled toward the center of the wound after 8 h using a motility program, like OMAware or similar, described previously (8). It is useful to draw an imaginary line in the middle of the wound in the images captured (Fig. 2).4.Notes1.Alternative to using a razor, marked culture dishes and stickers that attach to thebottom of the dish are available commercially for use as reference markers. 2.The amount of serum in the media needs to be determined for the particular celltypes used. It is recommended to use less than the amount in the regular growth media to minimize cell proliferation during the period of assay. However, using too little may lead to apoptosis and/or cell detachment for some cell types. For NIH 3T3 cells, which are normally cultured in 10% calf serum, we find that 1–2% calf serum is optimal for the assay.3.The amount of incubation time should be determined empirically for the particu-lar cell types under study. However, incubations longer than overnight are not recommended as differences in cell proliferation may start to contribute to the differences in the migration assay. The most desirable length of incubation time is one where the cells in the fastest migrating condition just reach complete clo-sure of the wound. The dishes can be taken out of the incubator and examined periodically and then put back to resume incubation.4.An automated time-lapse microscope equipped with a temperature control cham-ber can be used under CO2-independent media, as the same region would be acquired automatically. However, this usually is not very practical for an 18-h incubation as time constraints on a microscope usually interfere.5.The cells at the leading edge of the wounded monolayer are most migratory andusually are chosen as the ones that are examined for migration. The rate of their migration reflects the overall rate of wound closure of the migrating monolayer of cells. In addition, these cells are usually not dividing (whereas cells in the back could), thus not presenting the complication of single cells under tracking becom-ing double. Therefore, the effects of genes on migration are usually assessed usingFig. 2. Measurement of individual cell migration in wound-healing assay. NIH 3T3 cells grown on fibronectin (10 μg/mL) were with a plasmid-encoding GFP. One day after transfection, the cell monolayer was wounded with a pipet tip, incubated at 37°C, and fluorescent and phase contrast images were captured at regular intervals as indi-cated. The rate of migration was measured by quantifying the total distance that the GFP+ cells (as indicated by arrows) moved from the edge of the wound toward the center of the wound (marked by imaginary dotted lines). This figure is reprinted from ref.8 with permission of the American Society for Cell Biology.the cells at the leading edge, which would be the greatest. In the past, more com-plex microinjection methods have been used to introduce the genes (and proteins) along with a marker into the cells at the leading edge for this purpose. However, the high-transfection efficiency of the lipofectamine method coupled with a time-lapse microscope now allows such studies in many cell types, such as the NIH 3T3 cells, as outlined in this chapter. This simpler approach, which also avoids the use of sophisticated and expensive equipment, should permit more laboratories touse the method.AcknowledgmentsThis work was supported by the NIH grant GM48050 (J.G.) and a predoc-toral fellowship GM64086 (L. G. R.).References 1.Todaro, G. J., Lazar, G. K., and Green, H. (1965) The initiation of cell division in a contact-inhibited mammalian cell line. J. Cell Physiol.66, 325–333.2.Haudenschild, C. C. and Schwartz, S. M. (1979) Endothelial regeneration. II.Restitution of endothelial continuity. Lab Invest.41, 407–418.3.Han, D. C., Rodriguez, L. G., and Guan, J-L. (2001) Identification of a novel interaction between integrin beta1 and 14,-3-3beta. Oncogene 20, 346–357.4.Lipton, A., Klinger, I., Paul, D., and Holley, R. W. (1971) Migration of mouse 3T3 fibroblasts in response to a serum factor. Proc. Natl. Acad. Sci. USA 68,2799–2801.5.Underwood, P. A., Bean, P. A., and Gamble, J. R. (2002) Rate of endothelial expan-sion is controlled by cell:cell adhesion. Int. J. Biochem. Cell Biol.34, 55–69.6.Etienne-Manneville, S. and Hall, A. (2001) Integrin-mediated activation of Cdc42controls cell polarity in migrating astrocytes through PKCzeta. Cell 106, 489–498.7.Fukata, Y., Oshiro, N., Kinoshita, N., Kawano, Y., Matsuoka, Y., Bennett, V., et al.(1999) Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motil-ity.J. Cell Biol.145, 347–361.8.Abbi, S., Ueda, H., Zheng, C., Cooper, L. A., Zhao, J., Christopher, R., et al.(2002) Regulation of focal adhesion kinase by a novel protein inhibitor FIP200.Mol. Biol. Cell 13, 3178–3191.1234567。
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Wound healing assay
Kees Straatman Feb 2008
The wound healing assay allows the researcher to study cell migration and cell interactions. In some cases also single cell migration can be analyzed. This assay can be imaged using Nikon microscope 3 or the Olympus Cell^R/Scan^R system. Normally Nikon microscope 3 is used but if there is demand we can also write a protocol for the Olympus system. An automation is available for Nikon microscope 3 to image multiple positions in a multi well plate. At the moment we can image 60 positions. Normally we use a 10x Phase contrast objective, but recently we purchased a 4x objective that also can be used. CO2 is available or you can add some HEPES to your medium.
Don't forget to pre-heat the microscope system for several hours or over night before you start the experiment.
Wound images after T=0h, T=12h and T=23h
Protocol
1.Seed your cells in multi-well plates and culture until confluent. We normally
use 6 well plates, but a plate with a larger number of wells is possible. If you use different treatments or different mutants you might have to adapt the
seeding density. It is important that all the cultures are confluent at the start of the experiment.
ing a (yellow) pipette tip make a straight scratch, simulating a wound. Often
we make a scratch keeping the pipette tip under an angle of around 30 degrees to keep the scratch width limited. This allows imaging of both wound edges
using the 10x objective.
3.Check there is enough memory available on the HD; select MaCintosh HD, go
to 'File' ---> 'Get info'. The amount you need depends on the number of
positions you image and the number of time points. For 18 positions and 144 time points 7.2 GB is enough.
4.Start the automation 'BF-timeseries-focusdrive'.
5.Double click 'Save image document' (next to yellow box) and select the
subdirectory you want to save your files in.
6.Click 'run' to start running the automation. You will be asked a few questions
about the number of files etc. The number of time points you can save in one go depends on the time intervals between time points and the total number of time points! Maximal number of positions on Nikon 3 is 60.
7.Select the positions you want to image. We often take 3 images per well. The
first image you can focus using the focus knob on the microscope. After that USE ONLY THE FOCUS KNOB ON THE JOYSTICK UNIT to focus your image!!
8.When all positions are set the system will start imaging.
Analyze your data
Separate the different positions stored in single files in one file per position using the automation 'separate-timeseries' and save as *.tif files. Don't forget to select the directory your files are stored and to select a directory to save the new files as well as to change the file extension of the new files from *.lif to *.tif.
Open a *.tif file in ImageJ
Follow the next steps:
- Process ----> Find Edges
- Process ----> Sharpen
- Image ----> Adjust ----> Threshold; Select Black/White. Upper slider to 0 (left). Set lower slider in such a way that it is clear where the cells are. You might want to check a time point where the wound is partially closed to see if the difference between cells and wound are still visible.
- Process ----> Find Edges
- Image ----> Lookup Tables ----> Invert LUT
- Analyze Particles. Size: select a number that make sense if you look at a time point where the wound is partially closed (often a value between 100000 and 50000000; dependent on image size). Circularity: 0.00-1.00. Show: Outlines. Flag: Summarize. - Save both the Summarized data file and the file with outlines. The data file will show the percentage of wound area in your image on every time point.
Outline of the wound
- The data file can be further analyzed using Excel.
- The outline file can be used to analyze if the wound area found corresponds with the real wound by merging this file with the original *.tif file using Image ----> Color ----> RGB Merge (or using the Plugin 'RGB Gray Merge'). If necessary repeat
the analysis with a different setting at the
'Threshold' or 'Analyze Particles'.
Merged image to check accuracy of the
analysis.。