9第9章淋巴细胞

合集下载

组织学与胚胎学-第9章免疫系统

3．脾的功能
（1）滤血：主要部位是脾索和边缘区.
（2）进行免疫应答的场所
（3）造血：
（4）储血：约可储血40ml
思考题
• 1、何谓血－胸腺屏障？ • 2 、淋巴组织的分类。 • 3、简述淋巴结和脾脏的一般结构。 • 4、髓索和脾索的异同。
豆形，位于淋巴回流的通路上，成群分布于肺门、腹股沟及腋下等处，是滤过淋巴和产生免疫应答的重要器官。
1.淋巴结的组织结构
被膜：薄层致密结缔组织，数条输入淋巴管伸入实质形成小梁，小梁相互连接，形成淋巴结的粗支架。淋巴结门：CT+血管＋神经＋2-3输出淋巴管。
实质：分为皮质和髓质两部分。
(1)皮质位于被膜下方，由浅层皮质、副皮质区和皮质淋巴窦组成。
➢髓窦（medullary sinus）髓质淋巴窦，与
皮质淋巴窦的结构相同，但较宽大，腔内的巨噬细胞较多，故有较强的滤过作用。
（3）淋巴结内的淋巴通路：输入淋巴管被膜下淋巴窦
输出淋巴管
髓窦
皮质窄通道
淋巴流经一个淋巴结约需数小时，含抗原愈多则流速愈慢。
2．淋巴细胞再循环（recirculation of lymphocyte）周围淋巴器官和淋巴组织内的淋巴细胞可
血窦外侧有较多
的巨噬细胞，其
突起可通过内皮
间隙伸向窦腔。
2．脾的血液通路脾动脉分支为小梁动脉，小梁动脉分支进入动脉周围淋巴鞘内，称为中央动脉。中央动脉沿途发出一些小分支形成毛细血管供应白髓，其末端膨大形成边缘窦。中央动脉主干在穿出白髓进入脾索时分支形成一些直行的微动脉，形似笔毛，故称笔毛微动脉。
髓质位于中央、色浅、相互连续。胸腺基质细胞：胸腺上皮细胞＋巨噬c
胸腺细胞：淋巴干细胞 T细胞

第9章-免疫细胞之二：T淋巴细胞和B淋巴细胞

（5）CD28/CTLA-4
CD28有两条相同的链组成，糖蛋白，表达于T细胞、浆细胞及5％的CD3＋胸腺细胞表面。 CTLA-4（CD152）主要表达于激活的T细胞表面、 CTL表面。CD28／CTLA-4的配体为B7。
B7包括B7-1（CD80）、B7-2（CD86），为跨膜蛋白。主要表达于B细胞、激活的单核细胞、树突状细胞、激活的T细胞和NK细胞表面。
自治区精品课程
B细胞的分化发育过程
阴性选择
自治区精品课程
生发中心
*** T淋巴细胞
T细胞（T lymphocte）是机体内功能活跃的细胞群体，介导细胞免疫应答，并辅助B细胞对TD抗原的体液免疫应答。来源于骨髓造血干细胞，在胸腺内分化、发育，成熟的T细胞定居于外周淋巴组织。在识别由APC提呈的特异性抗原后被激活、分化为效应细胞和记忆细胞，参与适应性免疫应答和免疫记忆的维持。
自治区精品课程
**成熟B细胞特征
细胞表面同时表达mIgM和mIgD，并与Igα和Igβ 结合形成复合物。表达由CD19、CD21和CD81组成共受体。表达CR1、CKR和丝裂原受体等。
自治区精品课程
B细胞在骨髓中的发育
自治区精品课程
2. 外周发育
成熟的B细胞（Bn）迁移、定居与外周免疫器官。若接受抗原刺激，B细胞即增殖、分化为浆细胞并产生抗体，此即为B细胞的外周发育，亦称为B细胞发育的抗原依赖期。
自治区精品课程
Selection processes in the thymus (C)
清除自身反应性T细胞
自治区精品课程
T细胞个体发育及谱系
自治区精品课程
T细胞在胸腺中的选择过程

第九章 B细胞(2012)

二、抗原识别受体多样性产生的机制
1、组合造成的多样性
2、连接造成的多样性
3、体细胞高频突变造成的多样性
二、抗原识别受体多样性产生的机制
1、组合造成的多样性
理论上Ig V区基因片段的组合加上轻重链组合后的多样性约为1.9106。
2、连接造成的多样性：
各基因片段连接的不精确性：插入、缺失、或核苷酸替换等，增加了BCR和Ig的多样性。
主要事件：功能性BCR的表达自身免疫耐受的形成
一、BCR的基因结构及其重排
BCR—膜型免疫球蛋白（mIg)
免疫球蛋白肽链由两部分构成，即可变区（V）和恒定区（C），由两个基因编码。两个基因编码一条肽链。重链V区基因是由三种胚系基因片段V、D、J拼接而成。轻链V区基因是由V、J两个基因片段拼接成。 V区基因的下游是编码C区的基因。 V基因是几个基因片段在B细胞发育过程中发生重排。
（1）T细胞激活信号2 （2）抑制T细胞活化 CD152只表达于活化T细胞，含ITIM。
3、其它黏附分子： ICAM（CD54)、LFA－1
（四）其他表面分子
CD20 ：B细胞特异性标志，是治疗性单抗
识别的靶分子。
CD22：B细胞的抑制性受体 CD32：FcRⅡ，负反馈调节B细胞活化及抗体的分泌
提高B细胞对Ag刺激的敏感性。
B细胞辅助受体 CD19胞浆区有9个 Tyrosine残基，可募集信号分子。抗原刺激诱导补体活化，产生C3d 。C3d两端结合抗原和CD21。 CD21与CD19交联，接近BCR,由CD19分子增强 BCR对抗原敏感性。
CD21：EB病毒受体
（三）协同刺激分子
BCR的胚系基因

淋巴细胞

Carcinogenesis vol.33no.3pp.661–669,2012doi:10.1093/carcin/bgr320Advance Access publication January4,2012Hypoxia-inducible factor-1a inhibition by a pyrrolopyrazine metabolite of oltipraz as a consequence of microRNAs199a-5p and20a inductionSeul Gi Kang1,2,y,Woo Hyung Lee1,2,y,Young Hun Lee3, Yong Sup Lee3and Sang Geon Kim1,2,Ã1Department of Pharmacy,College of Pharmacy and2Research Institute of Pharmaceutical Sciences,Seoul National University,Seoul151-742,South Korea and3Department of Pharmaceutical Science,College of Pharmacy, Kyung Hee University,Hoegi-Dong,Seoul130-701,South KoreaÃTo whom correspondence should be addressed.College of Pharmacy,Seoul National University,Sillim-dong,Kwanak-gu,Seoul151-742,South Korea. Tel:þ822-880-7840;Fax:þ822-872-1795;Email:sgk@snu.ac.krOltipraz,a cancer chemopreventive agent,has antitumor and anti-angiogenic effects.In animal models and clinical studies,a consider-able amount of oltipraz is metabolized to pyrrolopyrazines, including M2,7-methyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine; M3,7-methyl-8-(methylsulﬁnyl)-6-(methylthio)pyrrolo[1,2-a]pyra-zine and M4,7-methyl-6,8-bis(methylsulﬁnyl)pyrrolo[1,2-a]pyra-zine.In view of the role of hypoxia-inducible factor-1(HIF-1)a in tumor growth and angiogenesis,this study investigated whether pyrrolopyrazine metabolites of oltipraz inhibit HIF-1a induction, and if so,what the molecular basis is.M2treatment inhibited the induction of HIF-1a by a variety of stimuli including insulin, hypoxia,CoCl2and hydrogen peroxide in HCT116cells,whereas M3or M4failed to do so.Consistently,M2prevented HIF-1a target gene induction.Moreover,it inhibited cancer cell invasion and migration.M2caused no change in the expression of HIF-1a transcript but increased the levels of precursor forms of micro-RNAs(miRNAs)199a-5p and20a,but not those of primary forms, indicating facilitation of the maturation process of the miRNAs by M2.Increased levels of the miRNAs contributed to HIF-1a re-pression,as shown by the results of experiments using mimics. Consistently,M2treatment inhibited de novo synthesis of HIF-1a, as supported by decreased incorporation of[35S]-methionine into HIF-1a with no changes in its ubiquitination or degradation. 7-Ethyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine,a synthetic analog of M2,had a similar inhibitory effect.In conclusion,M2 with pyrrolopyrazine structure and its7-ethyl congenor have the ability to prevent the induction of HIF-1a,which may result from the inhibition of HIF-1a de novo synthesis,as mediated by the induction of miR-199a-5p and miR-20a.IntroductionAn activated complex of hypoxia-inducible factor-1(HIF-1)a facil-itates to induce the expression of the genes implicated in the adapta-tion of cancer cells to tumor microenvironments where the utilization of oxygen and nutrients are signiﬁcantly restricted(1–3),which plays a crucial role in cancer cell proliferation,angiogenesis and invasion/ migration(4,5).Moreover,a considerable amount of HIF-1a is de-tected in aggressive and/or malignant cancers(6,7).A series of com-pounds(e.g.rapamycin,YC-1,resveratrol,radicicol and17-AAG) have been unveiled as the inhibitors of HIF-1a(4,5,8,9).Since they may possess toxic and/or adverse effects(5,9),recent pharmacolog-ical interventions in the activity of HIF-1a are limited.Thus,advances are required in the development of tailor-made inhibitors of HIF-1a.Oltipraz[4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione]is a can-cer chemopreventive agent(10–12)and exerts antitumor and anti-neoangiogenic effects in tumor xenograft animal models(13,14).It has an inhibitory effect on the growth of pulmonary adenomas and forestomach cancers(15,16).In mammals,oltipraz is metabolized into oxidized forms via main pathways:ﬁrst,oxidative desulfuration of the thione to yield4-methyl-5-(pyrazin-2-yl)-3H-1,2-dithiol-3-one (M1)with no further metabolism and second,desulfuration,methyl-ation and molecular rearrangement to produce7-methyl-6,8-bis(me-thylthio)pyrrolo[1,2-a]pyrazine(M2,a pyrrolopyrazine structure), which may be further metabolized to other forms(M3and M4) (17,18).In our clinical trials,oltipraz underwent prompt and substan-tial molecular conversion into mostly M2after oral administration in human(19).At a high dose(i.e.90mg,q.d.),extended residence time of oltipraz in the body may have caused increased formation of M2(19).Ourﬁndings indicated that oltipraz or its synthetic derivatives have the ability to inhibit the induction of HIF-1a in human colon cancer cells by not only facilitating its degradation but also decreasing its de novo synthesis(14).It is noteworthy that oltipraz and M2share sim-ilar pharmacokinetic proﬁles,suggesting that the M2metabolite is highly likely to contribute to the beneﬁcial effect of oltipraz and serve as the bioactive metabolite.In addition,both M1and M2induce the expression of GSTA2gene,prevent mitochondrial injury by activating adenosine monophosphate-activated protein kinase and protect hep-atocytes from reactive oxygen species(20,21).M2treatment effec-tively activated NF-E2-related factor2,suggesting that it may be pharmacologically active(20).Some NF-E2-related factor2activa-tors have the ability to inhibit the expression of HIF-1a.For example, sulforaphane and resveratrol inhibit HIF-1a activity by reducing its stability(22,23).Nonetheless,it remains to be established whether M2has an effect on HIF-1a.In view of the possibility that M2inhibits HIF-1a and has antican-cer potential,this study examined the effects of M2and its synthetic congenors on the expression and activity of HIF-1a.We also explored whether M2suppresses HIF-1a-dependent gene transactivation and,if so,the underlying basis.Here,we identiﬁed the induction of speciﬁc microRNAs(miRNAs)by M2treatment and their inhibitory effect on the translation of HIF-1a.Moreover,new derivatives of M2that have pyrrolopyrazine structure were synthesized with the aim of discover-ing other candidates that similarly inhibit HIF-1a.Now,we report the inhibitory efﬁcacy of M2and its7-ethyl substitute on the induction of HIF-1a.Ourﬁndings indicate that these agents increase the levels of mature miR-199a-5p and miR-20a for HIF-1a repression,which is a unique and novel mechanism for the inhibition of HIF-1a activity by pharmacological means.Materials and methodsCells and cell culture conditionsHCT116and HT29cells,human colon cancer cell lines,were obtained from ATCC(Rockville,MD).The cells were maintained in growth medium con-taining Dulbecco’s modiﬁed Eagle’s medium,10%fetal bovine serum and5% penicillin–streptomycin at37°C in a humidiﬁed atmosphere containing5% CO2.For all experiments,cells were grown to80–90%conﬂuency and were deprived of serum for16h before drug treatment.To create hypoxic conditions, they were transferred to a hypoxic chamber(Forma Scientiﬁc,Marietta,OH), where the cells were maintained at37°C in an atmosphere containing5%CO2, 1%O2and94%N2.MaterialsInsulin,H2O2,MG132and CoCl2were purchased from Sigma–Aldrich (St Louis,MO).Antibodies speciﬁcally directed against HIF-1a and HIF-1b were obtained from BD Biosciences Pharmingen(San Jose,CA).Anti-ubiquitin antibody was supplied from Sigma–Aldrich.Antibodies recognizingAbbreviations:HIF-1,hypoxia-inducible factor-1;miRNA,microRNA;mRNA,messenger RNA;S6K1,p70ribosomal S6kinase1;UTR,untranslatedregion.y These authors contributed equally to this work.ÓThe Author2012.Published by Oxford University Press.All rights reserved.For Permissions,please email:journals.permissions@661 at Shanghai Jiao Tong University on March 27, 2012 / Downloaded fromp70ribosomal S6kinase1(p70S6K1),p-p70S6K1,lamin A/C and HSP70 were obtained from Cell Signaling Technology(Beverly,MA).Chemical synthesis of M2and congeners1)Synthesis of6,8-bis(methylthio)-7-phenylpyrrolo[1,2-a]pyrazine(N3a)at Shanghai Jiao Tong University on March 27, 2012/Downloaded fromChromosomal DNA was precipitated with trichloroacetic acid and extracted with a solution containing0.5%SDS and0.5N NaOH.The radioactivity was measured using a liquid scintillation counter.In vitro cell invasion/migration assaysAn in vitro cell invasion/migration assay was performed using a24-well TranswellÒas described previously.The lower side of theﬁlter was covered with type I collagen,whereas its upper side was coated with Matrigel(Collaborative Research,Lexington,KY).The lower compartment was occupied with serum-free media with0.1%bovine serum albumin.HCT116cells were located in the upper compartment of the TranswellÒplate,maintained with10%serum for24h with vehicle or M2,ﬁxed with methanol and then were stained with he-matoxylin for10min,brieﬂy followed by eosin staining.The invasive phenotype was determined by quantifying the cells that migrated to the lower side of theﬁlter with microscopy(magniﬁcation,Â200). Thirteen visualﬁelds were counted for eachﬁlter,and each sample was assayed in triplicate.An in vitro cell migration assay was per-formed using a24-well TranswellÒunit with polycarbonateﬁlters. Experimental procedures were the same as for the cell invasion assay but that theﬁlter was not coated with Matrigel.Data analysisOne-way analysis of variance procedures were used to assess signiﬁcant differences among treatment groups.For each signiﬁcant treatment effect, the Newman–Keuls test was utilized to compare multiple group means.ResultsInhibition of HIF-1a and its target gene inductionInsulin treatment induces HIF-1a through both de novo synthesis and protein stabilization(26,27).First,we investigated whether the oxi-dized metabolites of oltipraz inhibit HIF-1a induction by insulin in HCT116cells.Previously,oltipraz treatment at10or30l M sup-pressed the induction of HIF-1a(14).Treatment of the cells with M2inhibited HIF-1a induction,whereas M3,M4or M1failed to do so(Figure1B).M2at30l M completely inhibited the induction of HIF-1a,beginning from1h at least up to6h,and exhibited a dose–response effect(Figure1C).The inhibitory effect of M2on HIF-1a was con-ﬁrmed in another cell line(HT29).In addition,M2had an inhibitory effect on HIF-1a induction by other stimuli including hypoxia,CoCl2 or H2O2in HCT116cells(Figure1D).HIF-1a exists as a full length with826amino acids(28).The expression of a shorter form of HIF-1a is observed presumably because alternative splicing produces the smaller form of HIF-1a(735aa)with a weak activity(28).HIF-1a forms a heterodimer with its binding subunit HIF-1b, which undergoes nuclear translocation for target gene induction (3,29).Nuclear HIF-1a content was assessed in HCT116cells treated with M2in the presence or absence of insulin.M2treatment almost completely prevented the ability of insulin to elevate nuclear HIF-1a content in either HCT116or HT29cells(Figure2A).Consistently, M2abrogated increases in HIF-1a target gene transcript levels(i.e. vascular endothelial growth factor and glucose transporter1) (Figure2B).Moreover,the results of hypoxia-response element re-porter gene assays conﬁrmed the inhibitory effect of M2on HIF-1a-dependent gene transcription(Figure2C).Fig.1.M2inhibition of HIF-1a induction by insulin.(A)The chemical structures of oltipraz and its pyrrolopyrazine metabolites.(B)The effects of oltipraz’s metabolites on the induction of HIF-1a by insulin.HCT116cells were treated with each metabolite or oltipraz(30l M each)for1h and continuously incubated in a medium containing100nM insulin for6h.(C)The time course and concentration–response effects of M2.HCT116or HT29cells were treated with30l M M2for 1–6h(left).They were also exposed to the indicated concentration of M2for6h(middle).The band intensity of HIF-1a relative to HIF-1b was quantiﬁed by scanning densitometry of the immunoblots(right).Value represents mean±SE fromﬁve independent experiments(treatment mean signiﬁcantly different from vehicle-treated control,ÃÃP,0.01,or insulin,##P,0.01).(D)The effects of M2on the induction of HIF-1a by a variety of stimuli.HCT116cells were treated with30l M M2for 1h and were continuously incubated under the condition of hypoxia(1%oxygen)or in a medium containing100l M CoCl2or0.5mM H2O2for the indicated times.HIF-1a inhibition by pyrrolopyrazines663 at Shanghai Jiao Tong University on March 27, 2012 / Downloaded fromInduction of miR-199a-5p and miR-20a/bDespite the repression of HIF-1a protein,M2had no effect on the level of HIF-1a transcript,indicating that posttranscriptional mecha-nism might be involved in this process (Figure 3A).Since miRNAs play a role in the posttranscriptional regulation of HIF-1a ,we exam-ined the effect of M2on the expression of miRNAs that potentially interact with the 3#-untranslated region (UTR)of HIF-1A messenger RNA (mRNA).In an effort to identify miRNAs responsible for the inhibition of HIF-1a expression by M2,we searched the TargetScanDatabase and discovered that four sequence motifs of the 3#-UTR of HIF-1A match seed sequences of 10candidate miRNAs (i.e.miR-199a-5p,miR-20a/b,miR-93,miR-138,miR-18a/b,miR-106a/b and miR-519c).Among the candidate miRNAs,M2treatment signiﬁ-cantly elevated the miRNA levels of miR-199a-5p,miR-20a and miR-20b in HCT116cells (Figure 3B).This effect of M2was also observed in HT29cells.Consistently,transfection of HCT116cells with each miRNA mimics or a mixture of the mimics almost com-pletely abrogated HIF-1a induction (Figure 3C).The mixture was also active in HT29cells (data not shown).These results support the contention that M2inhibits HIF-1a induction and which may result from the upregulation of miR-199a-5p and miR-20a/b that pair to the complementary-binding sites within the 3#-UTR of HIF-1A mRNA.To determine how M2elevates the levels of miR-199a-5p and miR-20a,their precursor levels were assessed by real-time PCR assays (Figure 3D).Interestingly,M2treatment increased the levels of pre-miR-199a and pre-miR-20a,but not their primary precursors,suggest-ing that the miRNAs induction may be mediated by the maturation process of the primary miRNAs.Since miR-20a is transcribed within the miR-17-92cluster,other mature miRNAs expressed from the cluster (miR-17,miR-18a,miR-19a,miR-19b-1and miR-92a-1)were also monitored (Figure 3E).M2treatment had no effects on the levels of the miRNAs,being consistent with our contention that M2affects the maturation process of miR-199a and miR-20a but not their general transcription.In addition,M4,another pyrrolopyrazine metabolite,did not change miR-20a/b levels but decreased miR-199a-5p level (Figure 3F).Taken together,our results demonstrate that M2has the ability to facilitate the maturation of primary precursors to miR-199a-5p and miR-20a that block the translation of HIF-1A mRNA.Inhibition of HIF-1a de novo synthesis35S-Methioninepulse labeling experiments were performed to verifythe inhibitory effect of M2on the synthesis of HIF-1a protein.As expected,M2treatment abolished the de novo synthesis of HIF-1a in HCT116cells (Figure 4A).Since HIF-1a is continuously degraded by the 26S proteasome complex after multiple ubiquitination (3),treat-ment with MG132(a proteasome inhibitor)promoted HIF-1a accu-mulation in cells treated with insulin (Figure 4B,upper).This effect was almost entirely abolished by simultaneous M2treatment in HCT116or HT29cells.Moreover,M2diminished the accumulation of ubiquitinated HIF-1a by MG132(Figure 4B,lower).These results are in line with the notion that the inhibition of HIF-1a de novo synthesis by M2restrained the ubiquitination and proteosomal degra-tion of HIF-1a because of a decrease in its supply.In a continuing effort to ﬁnd the molecular mechanism of HIF-1a inhibition by M2,we next investigated whether M2alters the stability of HIF-1a .Cycloheximide,a general inhibitor of protein synthesis,was used to prevent HIF-1a de novo synthesis.HCT116cells treated with cycloheximide in combination with insulin displayed a gradual decrease in the level of HIF-1a as a function of time (Figure 4C).Simultaneous treatment of the cells with M2did not change the rate of HIF-1a degradation.These results demonstrate that HIF-1a repres-sion by M2may stem from the inhibition of HIF-1a de novo synthesis but not the change in the protein stability.Inhibition of cell proliferation and invasion/migrationHIF-1a activation promotes neovascularization,which features a key pro-cess in blood and nutrient supply (1–3).It is expected that HIF-1a sup-pression by M2inhibits cell proliferation.Having identiﬁed the ability of M2to suppress HIF-1a ,we examined its effect on cell proliferation and invasion/migration.M2signiﬁcantly decreased the rate of DNA synthe-sis,as indicated by the result of [3H]-thymidine incorporation assay (Figure 5A).Moreover,M2treatment attenuated serum-induced inva-sion/migration of HCT116cells (Figure 5B).Similarly,transfection with a mixture of miR-199a-5p and miR-20a mimics signiﬁcantly decreased the cell invasion and migration (Figure 5C).Our observations show that the inhibition of HIF-1a by M2may contribute to decreasing the pro-liferation and invasion/migration of the cancercell.Fig.2.M2inhibition of HIF-1a target gene induction.(A )Inhibition of HIF-1a nuclear accumulation by M2.The levels of nuclear HIF-1a weremeasured in the lysates of HCT116or HT29cells treated as described in Figure 1B.Immunoblottings for lamin A and HSP70conﬁrmed the purities of nuclear (NF)and cytoplasmic fractions (CF),respectively.(B )Real-time PCR assays of HIF-1a target gene transcripts.After serum deprivation (16h),HCT116cells were treated with 30l M M2for 1h and were continuously incubated with 100nM insulin for 12h.b -Actin was used as a normalizing reference.Value represents mean ±SE from ﬁve independent experiments (treatment mean signiﬁcantly different from vehicle-treated control,ÃÃP ,0.01,or insulin,##P ,0.01).(C )Hypoxia-response element (HRE)reporter gene assay.HRE-A549cells that had been stably transfected with the HRE-luciferase construct were incubated with 100nM insulin for 24h following M2treatment for 1h.Luciferase activity was measured on the lysates.Value represents mean ±SE from ﬁve independent experiments (treatment mean signiﬁcantly different from vehicle-treated control,ÃÃP ,0.01,or insulin,#P ,0.05;##P ,0.01).S.G.Kang et al.664at Shanghai Jiao Tong University on March 27, 2012/Downloaded fromHIF-1a repression by 7-ethyl-6,8-bis(methylthio)pyrrolo[1,2-a ]pyrazine Based on the novel pharmacologic effect of M2,pyrrolopyrazine analogs were synthesized with the aim of identifying other candidates that inhibit HIF-1a (Figure 6A).7-Ethyl-6,8-bis(methylthio)pyrro-lo[1,2-a ]pyrazine (N3b)had an inhibitory effect on HIF-1a induction,whereas 6,8-bis(methylthio)-7-phenylpyrrolo[1,2-a ]pyrazine (N3a)failed to do so (Figure 6B).Thus,7-ethyl derivative (N3b),but not 7-phenyl derivative (N3a),of pyrrolopyrazine was active in inhibiting HIF-1a .N3b treatment also induced the expression of miR-199a-5p and miR-20a,as did M2(Figure 6C).Overall,our results demonstrate that M2with pyrrolopyrazine structure and its 7-ethyl congenor have the ability to prevent the induction of HIF-1a ,and which may result from the inhibition of HIF-1a de novo synthesis,as mediated by the induction of miR-199a-5p and 20a.DiscussionHIF-1a is closely involved in angiogenesis and tumor growth (1–3).In previous studies,oltipraz inhibits the expression of vascular en-dothelial growth factor at the molecular level and has the ability to inhibit tumor growth and angiogenesis in tumor-bearing animal models (e.g.angiosarcoma and colon cancer)(13,14,30).Oltipraz treatment inhibits the activity of HIF-1a and its target gene trans-activation,thereby repressing angiogenesis and tumor growth (14).This effect appears to result from not only an increase in HIF-1a ubiquitination but also its accelerated degradation.In addition,olti-praz decreased the ability of insulin to produce H 2O 2,reactive oxy-gen species that inhibit prolyl hydroxylase domain protein-mediated hydroxylations of two proline residues of HIF-1a and consequent degradation of HIF-1a by the ubiquitin-proteasome system (14).Thus,the inhibition of HIF-1a by oltipraz results from destabiliza-tion of HIF-1a .Our results and others showed that the formation of M2occurs quickly after oltipraz administration in human or animals (19)and that M2exerts similar or possibly more potent beneﬁcial effects than its parent compound in several experimental models.Therefore,it was anticipated that M2inhibits the activity of HIF-1a in cancer cells.Here,we demonstrated for the ﬁrst time that M2and its 7-ethyl congener,but not M3and M4,have an inhibitory effect on the in-duction of HIF-1a .Moreover,our results demonstrated that M2treat-ment diminished cancer cell invasion and migration.In this study,we found no accumulation of ubiquitinated HIF-1a after M2treatment:Fig.3.The induction of mature miR-199a-5p and miR-20a/b by M2.(A )The effect of M2on HIF-1a mRNA level.HIF-1a mRNA levels were measured in HCT116cells treated as described in Figure 1B.Actinomycin D (an inhibitor of gene transcription)was used as a positive control.Value represents mean ±SE from ﬁve independent experiments (treatment mean signiﬁcantly different from vehicle-treated control,ÃÃP ,0.01).(B )The effect of M2on the expression of miR-199a-5p and miR-20a/b.The levels of miRNAs were determined by real-time PCR assays in cells treated with 30l M M2for 3h after serum deprivation.Each miRNA level was normalized to that of U6snRNA.Value represents mean ±SE from three to ﬁve independent experiments (treatment mean signiﬁcantly different from vehicle-treated control,ÃÃP ,0.01).(C )HIF-1a repression by the mimics of miR-199a-5p or miR-20a.HIF-1a was immunoblotted on the lysates of HCT116cells treated with the mimics of 100nM miR-199a-5p,miR-20a or combination of both (50nM each)for 3h.Equal loading of proteins was veriﬁed by immunoblottings for HIF-1b .(D )Real-time PCR assays.The levels of primary precursor or precursor miRNAs were measured as described in the legend to panel B.(E )The effect of M2on the levels of miRNAs in the miR-17-92cluster.(F )The effect of M4on miR-199a-5p and miR-20a/b levels.For the panels D,E and F,HCT116cells were treated with M2(or M4)as described above.Value represents mean ±SE from four to ﬁve independent experiments (treatment mean signiﬁcantly different from vehicle-treated control,ÃP ,0.05,ÃÃP ,0.01).N.S.,Not signiﬁcant.HIF-1a inhibition by pyrrolopyrazines665at Shanghai Jiao Tong University on March 27, 2012/Downloaded fromM2failed to change the half-life time of HIF-1a .Thus,the basis of HIF-1a inhibition by M2seems to differ from that of oltipraz (14).This is in line with our previous observation that M2,unlike oltipraz,failed to protect hepatocytes from oxidative stress elicited by arach-idonic acid and iron (21).So,it is probably that M2has a unique molecular basis for HIF-1a repression.Pyrrolopyrazine thione,an intermediate metabolite of oltipraz,causes the reduction of the heme iron via the interaction with cytochrome c (31–33).The membrane-bound cytochrome c then scavenges superoxide radicals and dimin-ishes excessive H 2O 2production (31–33).However,M2did not have this effect (21).Thus,the lack of accumulation in ubiquiti-nated HIF-1a by M2might result from decreased de novo synthesis.NF-j B heterodimer complex consisting of p50and p65transactivates the HIF-1A gene (34),which may be antagonized by certain chemicals (e.g.YC-1)(35).Unlike YC-1,M2had no effect on p50/p65expression or NF-j B reporter activity (data not shown),excluding the possibility that M2inhibits HIF-1a by repressing NF-j B activity.The lack of change in HIF-1A mRNA content by M2suggested that it inhibits HIF-1a at the posttranscriptional level.This hypothesis is strengthened by our observation showing a decreased translational rate of HIF-1a in cells treated with insulin that elevates HIF-1a level through a translational mechanism (26,27).Consistently,M2treatment signiﬁcantly restrained the synthesis rate of HIF-1a .Since 5#-terminal oligo pyrimidine sequences play a role in p70S6K1-dependent translation of HIF-1A mRNA (36),the inhibition of p70S6K1by olti-praz might contribute to inhibiting HIF-1a (14).Unlike oltipraz,M2failed to change p70S6K1phosphorylation (supplementary Figure S1is available at Carcinogenesis Online;the capital S denotes that the ﬁgure can be found in the supplemental material),implying that a dif-ferent mechanism exists for the translational regulation of HIF-1a by M2.The miRNAs are small non-coding RNAs that decrease the expres-sion of target proteins via the inhibition of mRNA translation with enzymatic and regulatory functions (37,38).The inhibitory action of miRNA results from imprecise base pairing of miRNAs withtheFig.4.Inhibition of HIF-1a de novo synthesis by M2.(A )[35S]-Methionine incorporation assay.HCT116cells that had been cultured in a medium free of methionine,cysteine and glutamine were pulse labeled with 100mCi/ml [35S]-methionine for 1h and were incubated in a medium containing methionine,cysteine and glutamine for 1.5h.[35S]-Methionine incorporation to HIF-1a was assessed in the lysates.Value represents mean ±SE from ﬁve independent experiments (treatment mean signiﬁcantly different from vehicle-treated control,ÃÃP ,0.01,or 35S labeling alone,##P ,0.01).(B )The effect of M2on the ubiquitination and degradation of HIF-1a .HCT116or HT29cells transfected with the plasmid encoding for His-tagged ubiquitin (His-Ubi)were treated with MG132(10l M,3h)and were treated with insulin (100nM,3h)or insulin plus M2.Immunoblotting for ubiquitin was done on HIF-1a immunoprecipitates.HIF-1b wasimmunoblotted on the lysates.Value represents mean ±SE from ﬁve independent experiments (treatment mean signiﬁcantly different from vehicle-treated control,ÃÃP ,0.01,or insulin alone,##P ,0.01).(C )The effect of M2on HIF-1a stability.HCT116cells were incubated with insulin or insulin þM2for the indicated times after cycloheximide (CHX,20l g/ml)treatment (upper).HIF-1a or HIF-1b was immunoblotted on the lysates (lower).Value represents mean ±SE from ﬁve independent experiments (treatment mean signiﬁcantly different from zero-time control,ÃÃP ,0.01,or insulin þCHX at respective time,##P ,0.01).S.G.Kang et al.666at Shanghai Jiao Tong University on March 27, 2012/Downloaded from3#-UTR of complementary mRNAs (37,38).It is known that a cluster of miR-17-92and miR-519c participate in hypoxia-induced HIF-1a accumulation (39,40).Under a normoxic condition,miR-17-92tran-scribed from the same cluster may target complementary HIF-1A mRNA for the regulation of its translation,playing an inhibitory role in cancer proliferation in an oxygen-independent manner (39).In rat hepatic sinusoidal endothelial cells,downregulation of miR-199by ethanol increased the levels of HIF-1A and endothelin receptor 1tran-scripts (41).Certain phytochemicals (i.e.curcumin,epigallocatechin-3-gallate and isoﬂavone)also affect the expression of miRNAs,leading to alterations in cellular signaling and biological behavior (42).However,the modulation of miRNAs responsible for HIF-1a translation by chemical means had never been explored.Here,we demonstrate for the ﬁrst time that M2and its 7-ethyl congenor induce the expression of miR-199a-5p and miR-20a responsible for the pre-vention of HIF-1a induction.Oltipraz and its major metabolite of M1,however,failed to increase the levels of miR-199a-5p andmiR-20aFig.5.Inhibition of cancer cell proliferation and invasion/migration by M2.(A )Inhibition of DNA synthesis by M2.HCT116cells were treated with 30l M M2for 24h and continuously exposed to 1l Ci/ml[methyl-3H]-thymidine for 8h.Cell proliferation was determined by measuring radioactivity.Value represents mean ±SE from ﬁveindependent experiments (treatment mean signiﬁcantly different from vehicle-treated control,ÃÃP ,0.01,or serum alone,##P ,0.01).(B )Repression of cell invasion/migration by M2.Invaded/migratedHCT116cells were counted under light microscope (magniﬁcation,Â200).Value represents mean ±SE from four independent experiments (treatment mean signiﬁcantly different from vehicle-treated control,ÃP ,0.05,ÃÃP ,0.01,or serum alone,#P ,0.05).(C )The effect of a mixture of miR-199a-5p and miR-20a mimics on cell invasion/migration.HCT116cells were treated with miR-199a-5p and miR-20a mixture (50nM each).Value represents mean ±SE from four independent experiments (treatment mean signiﬁcantly different from vehicle-treated control,ÃÃP ,0.01,or serum alone,#P ,0.05,ÃP ,0.01).Fig.6.The effects of synthetic analogs of M2on the induction of HIF-1a .(A )The chemical structures of pyrrolopyrazine derivatives (N3a and N3b).(B )HIF-1a repression by N3b.HCT116cells were treated with each pyrrolopyrazine derivative (30l M each)for 1h and were continuously incubated in a medium containing 100nM insulin for 6h.Value represents mean ±SE from ﬁve independent experiments [treatment mean signiﬁcantly different from insulin treatment alone (100%),ÃÃP ,0.01].(C )The effect of N3b treatment on miR-199a-5p and miR-20a expression.miR-199a-5p and miR-20a levels were measured by real-time PCR assays in HCT116cells treated with 30l M N3b for 3h after serum deprivation.Each miRNA level was normalized to that of U6snRNA.Value represents mean ±SE from ﬁve independent experiments (treatment mean signiﬁcantly different from vehicle-treated control,ÃP ,0.05,ÃÃP ,0.01).HIF-1a inhibition by pyrrolopyrazines667at Shanghai Jiao Tong University on March 27, 2012/Downloaded from。

(人卫5版医学免疫学)第九章 B淋巴细胞

第九章 B淋巴细胞
精选ppt
1
第一节 B细胞的分化发育
精选ppt
2
（一）BCR的基因结构及其重排
编码BCR的基因在胚系阶段以分隔的、数量众多的基因片段的形式存在，在B细胞的分化发育过程中，这些基因片段发生重排和组合，从而产生数量巨大、能识别特异性抗原的BCR。
精选ppt
3
BCR的胚系基因结构
• CD22：抑制性受体，负调节B细胞共受体。 • CD32（FcγRⅡ）：FcγRⅡB亚型负反馈调
节B细胞活化及抗体的分泌。
精选ppt
34
第三节 B淋巴细胞的亚群
精选ppt
35
CD5+B-1细胞
• 少数；
• 主要定居于腹膜腔、胸膜腔和肠道固有层中；
• 无需Th细胞的辅助，主要产生低亲和力的IgM，能与多种不同的抗原表位结合，表现为多反应性；
40
25
精选ppt
5
(40)
(25)
(4)
精选ppt
6
BCR的基因重排及其机制
➢BCR胚系基因中被分隔开的基因片段通过基因重排，形成VDJ（重链）或VJ（轻链）连接后，再与C基因片段连接，进一步加工、组装成有功能的BCR。
➢Ig首先是重链发生基因重排，随后是轻链重排。
➢IgV区基因的重排主要是通过重组酶的作用来实现的。
（二）提呈可溶性抗原活化B细胞
精选ppt
39
此课件下载可自行编辑修改，此课件供参考！部分内容来源于网络，如有侵权请与我联系删除！感谢你的观看！
精选ppt
24
结构：mIg-Igα/Igβ(CD79a/CD79b)复合物功能：
• mIg：B细胞的特征性表面标志，识别结合特异性Ag。

【医学免疫学】第9章 B淋巴细胞

B2细胞： CD5-，特异性B细胞，参与适应性免疫能特异性识别抗原（单特异性），介导对蛋白质类抗原（ TD-Ag ）的应答，可产生以IgG为主的各类抗体及免疫记忆。
三、B细胞的功能
产生抗体：中和作用、激活补体、调理作用、ADCC 提呈可溶性抗原免疫调节-1趋化MΦ、DC、NK、 Th等
（2）CD80和CD86（ B7-1和 B7-2，即B7）表达在活化B细胞和其他APC表面。与CD28结合，提供T细胞活化的第二信号。与CTLA-4结合，抑制T细胞的活化。
（3）黏附分子：ICAM-I, LFA-1等
二、B细胞亚群
B1细胞： CD5+，非特异性B细胞，参与固有免疫主要分布于腹膜腔、胸膜腔和肠道固有层；主要介导对多糖抗原（ TI-Ag ）的应答，产生低亲和力的、多反应性的IgM类抗体；可产生天然抗体和多种自身抗体。
成熟B细胞同时表达mIgM+mIgD；浆细胞膜不表达mIg；记忆B细胞不表达mIgD。
Ig /Ig （CD79a和CD79b)：胞浆区含有ITAM 功能是转导BCR与抗原结合产生的活化
BCR-Ig /Ig 复合物
2.B细胞共受体 (辅助受体)
CD19/CD21/CD81非共价相联，作用：增强B细胞与抗原结合的稳定性，并与Ig /Ig 共同转导抗原刺激信号。
CD19----转导活化信号
CD21----即CR2，为C3d的受体 ----也是EB病毒的受体，与EB病毒感染B细胞有关。
iC3b可裂解为C3c、C3f、C3dg=C3d+C3g)
3、共刺激分子（1）CD40
主要表达在B细胞、单核细胞和树突状细胞等APC表面 CD40与CD40L（活化T细胞表面）结合，提供B细胞活化的第二信号。

第九章淋巴造血系统疾病

A型题1 下列细胞中诊断霍奇金淋巴瘤最重要的是：A 曲核细胞B 霍奇金细胞C 双核的R-S细胞D 免疫母细胞E 嗜酸性白细胞2霍奇金淋巴瘤中预后最差的是：A 混合细胞型B 淋巴细胞消减型C 结节硬化型D 淋巴细胞为主型中的结节状浸润E 淋巴细胞为主型中的弥漫性浸润3各型霍奇金淋巴瘤中出现多数陷窝细胞的是：A 结节硬化型B 混合细胞型C 淋巴细胞为主型D 淋巴细胞消减型中的网状细胞型E 淋巴细胞消减型中的弥漫纤维化型4下列哪一种细胞一般不见于非霍奇金淋巴瘤：A裂细胞B无裂细胞C曲折核细胞D组织细胞E镜影细胞5有Ph1染色体标志的主要是哪一种白血病：：A 急性粒细胞性白血病B 慢性粒细胞性白血病C 急性淋巴性白血病D 慢性淋巴性白血病E 单核细胞性白血病6粒细胞性白血病时，白血细胞在肝内的浸润部位是：A 肝包膜下B 集中在小叶中心C 集中在汇管区D 散在于肝小叶E 围绕在小叶下静脉周围7急性粒细胞性白血病时，在骨内、骨膜下或其他器官内，可由白血病细胞形成瘤结，外观呈某种颜色，称为：A 白色瘤B 黄色瘤D 棕色瘤E 转移瘤8 脾脏在哪种白血病时肿大得最重，可能重达5000ɡ？A 急性粒细胞性白血病B 慢性粒细胞性白血病C 急性淋巴性白血病D 慢性淋巴性白血病E 单核细胞性白血病9白血病时骨髓的病变多以何处最重？A股骨B胫骨C掌骨D指骨E椎骨10慢性粒细胞性白血病白血细胞内的硷性磷酸酶比相应白细胞的如何？A 升高B 降低C 有时升高，有时降低D 无明显改变E 虽有量的差异，但一般细胞化学检查难以反映这些差异。

11在癌症患者周围血液中找到恶性细胞，说明此患者A 已发生了血道转移B 并发了白血病C 即将发生转移D 不一定即将发生转移E 已是晚期癌症患者12关于白血病患者血内白细胞总数及其功能，以下哪些是对的？A 数目一定多于正常，但其细胞的功能低于正常B数目多或少于正常，但其细胞的功能也低于正常C数目一定多于正常，其细胞的功能也常高于正常D数目可以少于正常，其细胞的功能却高于正常E数目也可以少于正常，但其细胞的功能却高于正常13白血病时，白细胞在心、肾等器官的浸润A 常形成瘤结，原组织结构淋巴细胞为主型明显破坏B常不形成瘤结，但原组织结构明显破坏消失C常形成瘤结，但原组织结构常没有明显破坏D 常不形成瘤结，原组织结构常没有明显破坏E 是很少见到的现象14按预后的好坏，霍奇金淋巴瘤四型的排列顺序应为A 混和细胞型——淋巴细胞减少型-——结节硬化型——淋巴细胞为主型B淋巴细胞减少型——混和细胞型——结节硬化型——淋巴细胞为主型C淋巴细胞为主型——结节硬化型——淋巴细胞减少型——混和细胞型D——结节硬化型——混和细胞型——淋巴细胞减少型E 淋巴细胞减少型——淋巴细胞为主型——结节硬化型——混和细胞型15下列哪种恶性肿瘤属于T细胞型淋巴瘤？A 浆细胞样淋巴细胞型淋巴瘤B 蕈样霉菌病C 大无核裂泸泡中心细胞型淋巴瘤D非Burkitt淋巴瘤E绿色瘤16下列哪种恶性肿瘤属于B细胞型淋巴瘤？A 组织细胞性淋巴瘤B Sezary综合征C 曲折核淋巴细胞型淋巴瘤D Burkitt淋巴瘤E 恶性颗粒细胞瘤17关于恶性组织细胞增生症，下列叙述哪项正确？A是组织细胞增生症的一种类型B又称急性弥漫性组织细胞增生症C又称慢性进行性组织细胞增生症D是T细胞性淋巴瘤的一种E是组织细胞及其前身细胞进行性恶性增生引起的一种全身性疾病18与EB病毒感染有关的造血系统肿瘤是：A蕈样霉菌病B Burkitt淋巴瘤C绿色瘤D毛细胞白血病E恶性组织细胞增生症19瘤细胞间散在多数吞噬各种细胞碎屑的巨嗜细胞，形成所谓“满天星”图像的恶性肿瘤是：A脂肪肉瘤B恶性组织细胞增生症C恶性纤维组织细胞瘤D恶性巨细胞瘤E Burkitt淋巴瘤20霍奇金淋巴瘤中预后最好的组织学类型是：A淋巴细胞为主型B淋巴细胞减少型C结节硬化型D混和细胞型E 以上都不是21恶性淋巴瘤是：A单核巨噬细胞系统发生的肿瘤B淋巴结发生的肿瘤C淋巴结和结外淋巴组织发生的肿瘤D淋巴结窦组织细胞发生的肿瘤E淋巴结的B淋巴细胞发生的肿瘤22典型的镜影细胞是：A多核瘤巨细胞B细胞大，浆丰富，双色性或嗜酸性C核大，核膜厚，核呈空泡状D有大的嗜酸性核仁E双核并列，有大的嗜酸性核仁，形似镜影的R—S细胞23典型的慢性粒细胞性白血病时P h1阳性，其预后：A放射治疗效果好B放射治疗效果差C化疗效果差D化疗效果好E免疫治疗效果好24恶性淋巴瘤的发病率在各种恶性肿瘤中居第几位？A第3位B第5位C第8位D第11位E第15位25霍奇金淋巴瘤在我国发病率占全部恶性淋巴瘤的：A 5%~10%B 10%~20%C 20%~30%D 30%~40%E 40%~50%26关于恶性淋巴瘤的叙述，下列哪项是错误的？A是原发于淋巴结和结外淋巴组织的恶性肿瘤B分为霍奇金淋巴瘤、非霍奇金淋巴瘤和淋巴肉瘤三大类型C肉眼呈结节状，可有假包膜，切面呈“鱼肉样”D淋巴组织结构破坏，瘤细胞弥漫分布，瘤细胞间可有网状纤维穿插包绕E青少年多见，是儿童最常见的恶性肿瘤之一27下列哪项不属于霍奇金淋巴瘤的类型？A淋巴细胞为主型B组织细胞型C淋巴细胞减少型D结节硬化型E混合细胞型28下列哪项不属于白血病的主要类型？A急性粒细胞性白血病B急性淋巴（母）细胞性白血病C慢性单核细胞性白血病D慢性粒细胞性白血病E慢性淋巴细胞性白血病29下列哪项不属于急性粒细胞性白血病（FAB分类）A绿色瘤B毛细胞白血病C急性单核细胞性白血病D急性红白白血病E急性巨核细胞白血病30关于白血病与类白血病反应的鉴别，下列叙述哪项不正确？A引起类白血病的原因去除，血象不恢复正常B类白血病反应时一般无明显贫血和血小板减少C类白血病反应时粒细胞有严重毒性改变，胞浆内有毒性颗粒和空泡等D类白血病反应时中性粒细胞的硷性磷酸酶活性和糖原皆明显增高E类白血病反应时细胞内不见P h1染色体31在霍奇金淋巴瘤结节硬化型中不易见到的细胞是：A 陷窝细胞B嗜酸粒细胞C幼稚的淋巴细胞DR-S细胞E成纤维细胞32患者颈部淋巴结肿大时，下列哪种疾病的可能性最小？A颅内肿瘤淋巴结转移B恶性淋巴瘤C鼻咽癌淋巴结转移D甲状腺癌淋巴结转移E肺癌淋巴结转移33淋巴细胞性白血病的原发部位主要是：A 淋巴结B骨髓C胸腺D脾脏E散在的淋巴组织34大量幼稚的白细胞弥漫浸润于肝窦内，这是：A急性感染时的类白血病反应B恶性淋巴瘤转移至肝脏C淋巴细胞性白血病D粒细胞性白血病E髓外造血B型题：B细胞型淋巴瘤B T细胞型淋巴瘤C 组织细胞型淋巴瘤D霍奇金淋巴瘤E Burkitt淋巴瘤35最常见的淋巴瘤是36镜影细胞是诊断上述那种疾病的重要依据？37非霍奇金淋巴瘤中很少见的类型是38“满天星”图像是上述哪种疾病的病变特点之一？39蕈样霉菌病属于A、R—S细胞在细胞中所占比例最多B、常有较多浆细胞，嗜酸性细胞及组织细胞的浸润C、没有R—S细胞D、陷窝细胞多E、嗜酸性粒细胞，中性粒细胞和浆细胞数量很少40、淋巴细胞为主型霍奇金淋巴瘤41、混合细胞型霍奇金淋巴瘤42、结节硬化型霍奇金淋巴瘤43、淋巴细胞消减型霍奇金淋巴瘤C型题：A、存在于皮质内B、存在于髓质内C、存在于二者之内D、存在于二者之外44、正常淋巴结内淋巴滤泡45结节性淋巴瘤的结节X型题：46组织细胞增生症X（Langerhans细胞组织增生症）包括：A恶性组织细胞增生症B急性弥散性组织细胞增生症C慢性进行性组织细胞增生症D骨的嗜酸性肉芽肿E巨大淋巴结增生症47白血病的分类方法包括：A根据病情急缓和白血病细胞成熟程度B根据增生细胞的类型C根据周围血内白细胞的数量D白血病的免疫学分类E根据瘤细胞的分化程度48、属于淋巴滤泡生发中心的细胞是：A、免疫母细胞B、小核裂细胞C、大无核裂细胞D、浆细胞E、曲折核细胞49、对于诊断混合细胞型霍奇金病意义较大者有：A、多数典型的R—S细胞B、多数典型的隐窝细胞C、较多纤维组织条索形成D、大量淋巴细胞和组织细胞E、数量较多的各种炎性细胞50、对于诊断结节硬化型霍奇金病意义较大者有：A、多数典型的R—S细胞B、多数隐窝细胞C、大量淋巴细胞和组织细胞D、大量各类炎性细胞E、较多纤维组织条索形成51、属于白血病继发变化的有：A、发热B、出血C、脾肿大D、感染E、贫血52、Burkitt淋巴瘤的特征是：A、多见于儿童B、大量免疫母细胞C、大量呈结节性增生的小无核裂细胞D、大量弥漫性增生的小无核裂细胞E、出现“满天星”现象53霍奇金淋巴瘤的分期依据是：A病变位于横膈的一侧或两侧B瘤细胞的分化程度C累及几组淋巴结D累及淋巴结外器官的情况E病变范围越广，预后越差54恶性组织细胞增生症时可出现：A肝肿大B脾肿大C淋巴结肿大D骨髓巨核细胞减少E全血细胞增多55、恶性组织细胞增生症的特征：A、多见于老年B、长期发热C、反应性组织细胞增生D、病变仅累及造血系统E、组织细胞进行性恶性增生56、下列细胞中对霍奇金病具有重要诊断价值的为：A、霍奇金细胞B、R—S细胞C、镜影细胞D、隐窝细胞E、多核R—S细胞57、病人颈部淋巴结肿大常应考虑的疾病是：A、淋巴结反应性增生B、嗜酸性肉牙肿C、淋巴结结核D、淋巴结转移癌E、恶性淋巴瘤58白血病时组织内容易出血主要与下列哪些因素有关？A凝血酶原减少B骨髓内白血病细胞的浸润C制造血小板的细胞减少D巨核细胞变为瘤细胞E血管的破坏59急性白血病时周围血象的表现常是：A半数病人白细胞不增多B其中有大量的原始和幼稚的白细胞C红细胞总数多低于正常D血小板总数一般减少E早期即出现贫血60恶性组织细胞增生症的特点是：A局部组织细胞异常增生性恶性肿瘤B瘤细胞有吞嗜现象，吞嗜红细胞的现象是该病的重要特征C肿瘤细胞内非特异性酯酶、酸性磷酸酶和溶菌酶皆为阳性D较多见于儿童和青壮年，晚期可出现黄疸、贫血、白细胞和血小板减少及进行性衰竭E是一种进行性、全身性的恶性程度很高的肿瘤二名词解释1、白血病2、淋巴样肿瘤3、非霍奇金淋巴瘤4、镜影细胞5、恶性淋巴瘤6、髓样肿瘤7、绿色瘤8、类白血病反应9、组织细胞增生症10、恶性组织细胞增生症11陷窝细胞（腔隙型细胞）12霍奇金淋巴瘤13 R—S细胞三填空1、造血系统包括和。

医学免疫学第九,十章 B、T 淋巴细胞

IgV区基因的重排主要通过重组酶的作用实现的。
浙中医大微免教研室刘文洪
9
重组酶包括：①重组激活酶基因（RAC）②末端脱氧核苷酸转移酶③DNA外切酶，DNA合成酶等。
重组酶作用：可以从众多的V(D)J基因片段中个选择 1个V片段，1个D片段和一个J片段重排在一起，形成V(D)J连接，最终表达为有功能的BCR
4.体细胞高频突变：在已完成Ig基因重排的基础上，成熟B细胞在外周淋巴器官生发中心接受抗原刺激后发生。不仅能增加抗体的多样性，而且可导致抗体的亲和力成熟。
浙中医大微免教研室刘文洪
14
（三）B细胞在中枢免疫器官中的分化发育
造血干细胞→淋巴样干细胞→祖B细胞→前B细胞 →未成熟B细胞→成熟B细胞
CD32 有a和b两种亚型，其中b亚
型即FcγRIIB ，负向调控B细胞的活化及抗体的分泌。
浙中医大微免教研室刘文洪
38
Fc受体 (1) 组成：主要为FcγRⅡ-B1分子，含一个ITIM
（immuno-receptor tyrosine-based inhibitory motifs）免疫受体酪氨酸抑制基序基序。
52
第一节 T细胞的分化发育
HSC在骨髓中分化成淋巴样祖细胞。可经血液循环进入胸腺，在胸腺中完成T细胞的发育，成为成熟T细胞，再经血液循环进入外周淋巴器官，主要定居于外周淋巴器官的胸腺依赖区，接受抗原刺激发生免疫应答。
高糖类多反应性 IgM>IgG
低/无低/无
B-2 -
出生后由骨髓产生
低蛋白质单特异性,尤其在免疫后 IgG>IgM
高有
浙中医大微免教研室刘文洪
43
第四节 B细胞的功能

淋巴细胞

B-1主要生物学功能： 1）接受碳水化合物抗原刺激，产生低亲和力IgM，
参与对多种细菌（尤其体腔中）的抗感染免疫； 2）产生低亲和力IgM类的生理性自身抗体，参与
对衰老、蜕变自身细胞的清除； 3）通过产生IgM类自身抗体参与某些自身免疫病
的发生
2.B-2细胞
表面标志CD5出生后产生，定位于淋巴器官。接受蛋白质抗原刺激，产生高亲和力抗体,有免疫记忆性。
（三）协同刺激分子 CD40表达于成熟的B细胞表面 B7（CD80/CD86）表达于活化的B细胞表面
二、BL的亚群
B cell precursor
Mature B cell
B
B
Plasma cell
B2cells
B
Distinct B cell precursor
IgG CD5
B
IgM - no other isotypes
（四）协同刺激分子
TL完全的活化需要两种信号的协同作用。
存在于APC和TL表面的协同刺激分子相互作用形成协同刺激信号，以扩大适应性免疫应答的免疫效应。
1、CD28：
相应配体是B7分子。与配体B7结合后产生活化信号。
2、CTLA-4（CD152）：细胞毒性TL（相关）抗原4 胞浆区含免疫
Th 细胞的功能
2、细胞毒性TL
（ cytotoxic T lymphocyte， Tc或CTL）
特异性介导靶细胞裂解或凋亡释放穿孔素，致靶细胞裂解、死亡；释放颗粒酶，借助穿孔素的孔道进入靶细胞，靶细胞凋亡；高表达FasL，通过Fas/FasL途径导致细胞凋亡。
3、调节性TL （regulatory T cell， Tr）

第九章淋巴系统

第九章淋巴系统第一节淋巴和淋巴管课时练一、单项选择题1.下列淋巴管道中的淋巴不含有淋巴细胞的是（）A.毛细淋巴管B.淋巴管C.淋巴干D.淋巴导管2.家畜的淋巴液沿淋巴管向心流动，最后归入（）A.动脉B.静脉C.毛细血管D.毛细血管静脉端3.关于淋巴的生理意义，下列说法错误的是（）A.调节血浆和组织细胞之间的体液平衡B.起免疫、屏障、防御作用C.重新吸收组织液中的蛋白质D.参与糖类的运输4.在下列血管和淋巴管中，管壁通透性最大的是（）A.动脉B.静脉C.毛细血管D.毛细淋巴管5. 家畜的小肠绒毛内可吸收脂肪的是（）A.毛细血管B.淋巴干C.毛细淋巴管D.淋巴导管6.淋巴导管是家畜体内最大的淋巴集合管，它有（）A. 1条B. 2条C. 3条D. 5-6条7.家畜的胸导管收集的淋巴约为全身（）A. 1/2B. 2/3C. 3/4D. 4/58.关于家畜淋巴系统构成，下列叙述错误的是（）A.淋巴系统由淋巴管、淋巴器官和淋巴组织组成，与循环系统有密切联系。

B.淋巴管是静脉的辅助导管，在其通路上有许多淋巴结。

C.毛细淋巴管与毛细血管一样遍布于全身组织器官。

D.右淋巴导管是全身最长的淋巴集合管。

二、多项选择题9. 家畜体内的淋巴管是淋巴的通道，分为（）A.毛细淋巴管B.淋巴管C.淋巴干D.淋巴导管10.下列物质中，可以通过毛细淋巴管管壁的有（）A.蛋白质B.脂肪C.细菌D.糖类11.右淋巴导管短而粗，紧靠胸前口气管的右侧，其末端注入（）A.前腔静脉B.后腔静脉C.右颈静脉D.左颈静脉三、填空题12.血液到达毛细血管动脉端时，其中一部分进入组织间隙形成。

它与组织、细胞进行后，大多渗入，还有一部分则进入成为。

13.在淋巴管的通路上有许多。

淋巴只有通过它之后才含有。

14.脂肪在小肠黏膜上皮细胞吸收时，因颗粒较大，不易进入，只有通过经乳糜池→→→心脏，参与血液循环。

四、识图题15.左下图是胸导管、淋巴结分布模式图(背面)，请写出图中的1,2,3,4,5,6,7,8所示部位名称。

第9章T淋巴细胞和B淋巴细胞

T细胞占淋巴细胞总数的65%～85%,在脾脏中占30～50%，外周血中占60～70% 。
B细胞在骨髓或腔上囊中分化成熟，因而称为骨髓依赖性淋巴细胞(bone marrow-dependent lymphocyte)，简称B淋巴细胞。
血液:20％～25％淋巴结:占1／4 脾:1／2
一、淋巴细胞来源及分化二、T淋巴细胞三、B淋巴细胞
TCR特异识别由 MHC分子提呈的抗原肽
CD3分子转导T细胞活化的第一信号
CD4分子 Th的重要表面标志； CD4分子与MHC-Ⅱ类分子结合，是Th细胞
TCR识别抗原的共受体； HIV受体
CD8分子 CTL的重要表面标志； CD8分子与MHC-Ⅰ类分子结合，是CTL细
胞TCR识别抗原的共受体
共刺激信号
CD80（B7-1）和CD86（B7-2）与CD28结合提供T活化的第二信号
CD275 属于CD28家族
3）B细胞表面其它分子 MHC分子组成性表达MHC-Ⅱ类分子 CKR CK与其相应受体结合参与或调节B细胞
活化、增殖、分化
CR 例如，CR1、CR2，CR2为EB病毒受体 Fc 受体与IgG的Fc段结合丝裂原受体
丝裂原
B
母
B 细胞
淋巴母细胞
美洲商陆（PWM）对T、B细胞均有致有丝分裂作用脂多糖（LPS）是常用的小鼠B细胞丝裂原
二. B细胞亚群及功能
根据CD5表达与否，分为 B1细胞（表达CD5分子） B2细胞（不表达CD5分子）
B cell precursor
在T细胞表面与CD3以非共价键结合而形成 TCR-CD3复合物，为T细胞识别抗原和转导信号的主要单位.
TCR 主要的T细胞（90%外周血T）仅识别APC膜表面的MHC-抗原肽复合物

第9、10章淋巴细胞

2.B淋巴细胞表面的分子
(1)B细胞抗原受体 (2)Fc受体(Fc receptor,FcR)
(3)补体受体(complement receptor, CR)
(4)丝裂原受体
(5)其他表面分子
B细胞抗原受体
•细胞表面的免疫球蛋白(sIg)。
•sIg既是抗原的受体, 又是表面抗原,具有免疫球蛋白的抗原决定簇,能与抗免疫球蛋白的抗体特异性结合。
1、 T淋巴细胞表面分子及其作用
• T细胞抗原受体(TCR)
•
所有T细胞表面具有识别
和结合特异性抗原的分子结构称T细胞抗原受体(TCR)。
•
95％T细胞的TCR是由α链
和β链，分为可变区(V区) 和恒定区(C)区两个功能区。
C区与细胞膜相连,并有4～5
个氨基酸伸入胞浆,而V区则为与抗原结合部位。
巨噬细胞：既是通讯兵又是战斗家
抓住大肠杆菌
吞噬大肠杆菌
吞噬外来物
吞噬老化的红血球
单核巨噬细胞(mononuclear phagocyte)
•(1)来源及发育：
• 单核细胞在骨髓分化成熟进入血液，经血流随机分布到全身多种组织器官中,分化成熟为巨噬细胞。 • 巨噬细胞寿命较长 (数月以上),具有较强的吞噬功能。不同组织部位的巨噬细胞名称不同。
B细胞和抗体——作战用的导弹、子弹,人一生可分泌出10亿种以上的抗体）
1. B细胞的发育
人类的祖B细胞（pro-B）最早出现于妊娠第8周的胚胎中。妊娠中期以后，祖B细胞在骨髓中完成其发育成熟的全过程，前B(pre-B)、未成熟和成熟B细胞等几个阶段。细胞表面BCR的组成随着B细胞的发育阶段不同而变化
虫)等, 可以抗肿瘤免疫、抗感染免疫和移植物排斥反应、清除自身的衰老细胞

9. B细胞

BCR -complex
接受抗原特异信号
CD79a/CD79b (Ig/Ig)
ITAM
2.B细胞共受体
由CD19/CD21/CD81 组成功能是提高BCR
对抗原识别的敏感性。
BCR交联对B细胞激活是必须的
BCR交联使足够多的Igα和Igβ聚集在一起，并启动了传递信号的酶链反应
3.B细胞表面共刺激分子
3、等位基因排斥和同种型排斥。
BCR多样性
1. 组合多样性和随机性（combinational diversity）－胚系基因片段VJ和VDJ的组合
2. 连接多样性（junctional diversity）－胚系基因片段连接的不精确性－胚系基因重排时核苷酸的插入
3. 体细胞高频突变（somatic hyper-mutation）限于BCR
受体编辑
克隆清除克隆失能成熟B Nhomakorabea胞BCR基因重排
1976年，日本学者发现编码Ig可变区基因和恒定区基因是不连续的，之后重排后再组合在一起。这个过程就在B细胞分化成熟过程中。这个发现对我们理解BCR 的多样性是非常有帮助的。
Susumu Tonegawa,1987年诺贝尔生理医学奖主要贡献：免疫多样性
CR1
B淋巴细胞
丝裂原受体 BCR复合体
CD19-CD21-CD81
1.B 细胞抗原受体复合物
BCR:B细胞抗原受体，是嵌入细胞膜的膜免疫球蛋白（mIg），是B细胞的特征性标记，功能是特异性识别不同的抗原分子。
CD79a/CD79b（ Ig/Ig ）：转导BCR识别抗原的信号。
BCR-膜表面免疫球蛋白（mIg）
第九章 B淋巴细胞
基本概念

医学免疫学课件第9章 B淋巴细胞

前 B 细胞 DNA
基因重排
V1
V2
V3
Vn
J1 J2 J3 J4 J5
C
Vn J1
J2
V3
J
3
V1
V2
J4 J5
C
成熟 B 细胞 DNA
V1
V2 J4 J5
C
初级转录本 RNA 剪切
V1
V2 J4 J5
C
V2 J4
J5
C
mRNA
V2 J4 C
VL
CL
1.胚系基因
Ig 重链基因重排
V1
N-氨基酸插入
V、D、J片段连接多样性
95和96处，由9个核苷酸任意组合编码2个氨基酸。
N-氨基酸插入
温州医学院微生物学与免疫学教研室张丽芳
体细胞高频突变造成的多样性 (somatic hypermutation)
成熟的B 细胞重排的V区基因，往往在抗原的刺激下发生点突变，突变的频率非常高（每次细胞分裂，大约每1000个bp中就有一对发生突变，而其他体细胞的突变频率为10-10bp。）。
V2D2J3 J4
V2D2J3
Cm Cd
V2 D2 J3 Cm
J4 Cm Cd
J4
Cm
V2D2J3
Cd
V2 D2 J3 Cd
L
VH
Cm
L
VH
Cd
抗原识别受体多样性形成机制
• 组合造成的多样性 • 连接造成的多样性 • 体细胞高频突变造成的多样性
组合造成的多样性（combinatorial diversity）
Immature B cell recognises MULTIVALENT

医学免疫学-第九章 B细胞

2、连接多样性（junctional diversity）
• 重排过程中V、D、J片段之间的连接并不准确，可以插入、
替换或缺失核苷酸，从而产生新的序列，显著增加BCR和
Ig的多样性。 – 密码子错位，后序列不变； – 框架移位，后序列完全改变； – N序列插入：TdT 能将N-核苷酸插入待接DNA 断端。
（二）抗原识别受体多样性产生的机制
• 胚系阶段分隔的基因片段，在细胞发育过程中通过多种机制发生重排，产生数量巨大的特异性抗原受体，以识别不同的抗原。
1. 组合多样性（combinational diversity）； 2. 连接多样性（junctional diversity）； 3. 受体编辑（receptor editing）； 4. 体细胞高频突变（somatic hypermutation）。
Ig/Ig
B细胞的发育阶段
• 替代性BCR复合物 • 表达于Pro-B和Pre-B细胞，属免疫球蛋白家族。 • λ5链和Vpre-B链组成假轻链（L），组成替代BCR。 • 传递Pre-B细胞进一步分化必不可少的信号。
（四）B细胞中枢免疫耐受的形成
• 前B细胞发育至未成熟B细胞，膜表面开始表达mIgM，接受自身抗原刺激，形成免疫耐受。 ① 克隆清除（clone deletion）； ② 受体编辑； ③ 失能（anergy）。
• 在已完成基因重排的基础上，成熟B细胞在外周淋巴器官的生发中心接受抗原刺激后发生的突变，可导致抗体的亲和力成熟。 – 编码V区CDR部位的基因序列发生碱基的点突变。
（三）B细胞在中枢免疫器官中的分化发育
1. 祖B细胞（pro-B cell） 2. 前B细胞（pre-B cell） 3. 未成熟B细胞（immature B cell） 4. 成熟B细胞（mature/naїver） B cell

解剖学基础(第4版)《淋巴系统》PPT课件

第9章第2节淋巴系统电子课件中职高教版解剖学基础（第4版）
第九章淋巴系统 P145
学习目标掌握：胸导管；脾的位置。理解：淋巴系统的组成；腋淋巴结群；腹股沟浅淋巴结群。
概述： 1 . 淋巴系统的组成（1）淋巴组织
网状组织 + 大量淋巴细胞 + 巨噬细胞等构成，分布于：（1）空腔脏器的粘膜下，如淋巴滤泡、弥散淋巴组织等；（2）参与淋巴器官的构成，如淋巴小结。
管。成群分布于人体一定部位。功能：产生淋巴细胞，滤过淋巴，参与免疫。
1 .淋巴结的微细结构
被膜：致密结缔组织。
小梁：被膜伸入实质形成，构成支架。实质：分皮质和髓质。
（1）皮质由浅层皮质、副皮质区和皮质淋巴窦构成。 1）浅层皮质（B细胞区）含大量淋巴小结，主要由B细胞构成。淋巴小结在抗原刺激下可形成生发中心。
淋巴结内的淋巴通路：
淋巴输入淋巴管
皮质淋巴窦
皮质淋巴组织
髓质淋巴组织
髓窦
输出淋巴管
淋巴细胞、抗体↑ 淋巴
抗原↓
淋巴结功能：产生淋巴细胞，滤过淋巴，参与免疫。
2. 全身主要淋巴结群
（1）头颈部淋巴结群
下颌下淋巴结：下颌下腺周围及内部，收
集面部和口腔的淋巴，注入颈外侧深淋巴结。
颈外侧浅淋巴结: 沿颈外V排列。
3 .功能分泌胸腺激素，产生、培育、
输送T细胞。
第九章淋巴系统 P145
学习目标掌握：胸导管；脾的位置。理解：淋巴系统的组成；腋淋巴结群；腹股沟浅淋巴结群。
一、单项选择题
练习题
1．淋巴器官不包括
A．脑垂体
B．淋巴结
C．扁桃体
D．脾
2．脾的以下说法，错误的是 A．位于右季肋区 C．长轴与第10肋一致

9第9章淋巴细胞

组织学与胚胎学-第9章 免疫系统

第9章-免疫细胞之二：T淋巴细胞和B淋巴细胞

第九章 B细胞(2012)

淋巴细胞

(人卫5版医学免疫学)第九章 B淋巴细胞

【医学免疫学】第9章 B淋巴细胞

第九章 淋巴造血系统疾病

医学免疫学第九,十章 B、T 淋巴细胞

淋巴细胞

第九章 淋巴系统

第9章T淋巴细胞和B淋巴细胞

第9、10章 淋巴细胞

9. B细胞

医学免疫学课件 第9章 B淋巴细胞

医学免疫学-第九章 B细胞

解剖学基础(第4版)《淋巴系统》PPT课件

组织学与胚胎学-第9章免疫系统

第九章淋巴造血系统疾病

第九章淋巴系统

第9、10章淋巴细胞

医学免疫学课件第9章 B淋巴细胞