优选第九章淋巴细胞

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第9章B淋巴细胞ppt课件

未成熟B细胞结合骨髓中的自身抗原---细胞发生凋亡---克隆清除
未成熟B细胞结合骨髓中的自身抗原---mIgM 表达下调---进入外周淋巴器官，对抗原刺激不产生应答---无能
未成熟B细胞结合骨髓中的自身抗原---细胞中 RAG基因重新活化---轻链VJ再次重排，合成新的轻链，替代自身反应性轻链---成为一个新的B细胞---受体编辑。若受体编辑不成功，则该细胞发生凋亡或无能。
B
V 细
胞
甲1
B
V 细
胞
乙1
B
细V
胞
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D J1 1
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3、体细胞高频突变(somatic 造成的多样性 hypermutation)
被抗原激活后的B细胞在外周免疫器官生发中心分裂增殖时，IgV区基因大约每复制1000个 bp就有1对发生突变（碱基配对错误所致，而其他体细胞每次分裂中DNA分子的突变率约为1010bp），故称为体细胞高频突变。
（isotype exclusion）：
是一个B细胞克隆只表达一种 BCR，只分泌一种抗体的机制。
等位排斥——是指B细胞中位于一对染色体上的轻链或重链基因，其中只有一条染色体上的基因得到表达，先重排成功的轻链或重链基因抑制了另一条染色体上的基因的重排。
同种型排斥——是指κ轻链和λ轻链之间的排斥，κ链基因表达成功即抑制λ链基因的表达。在人Ig中κ链和λ链之比约为 2:1，而在小鼠中约为20:1。
• 可分泌CKs调节免疫应答
第一节 B细胞的分化发育
非抗原依赖期（骨髓）抗原依赖期（外周淋巴器官）
B细胞在中枢免疫器官中的分化发育过程中产生的主要事件是
–功能性BCR的表达

免疫学第九章淋巴细胞

• 细胞种类的鉴定依据：
不同免疫细胞及同类细胞的不同分化阶段可表达不同的种类和数量的表面膜蛋白或分化抗原(CD)。
分化抗原是鉴定免疫细胞种类及分化状态的标志。
免疫细胞均由骨髓的多能干细胞发育而来。
再生
多能干细胞
髓样干细胞
淋巴干细胞
B细胞
红细胞血小板肥大细胞中性粒细胞巨噬细胞树突状细胞浆细胞 T细胞 NK细胞
二、T淋巴细胞
（一）T细胞的分化发育 • T细胞来源于骨髓→在胸腺（thymus）内
发育成熟→移行至外周淋巴组织→执行特异性细胞免疫应答，参与对TD抗原的体液免疫应答。
在胸腺中完成。分三个时期：
1）双阴性期：T细胞不表达CD4分子，也无CD8分子，
2）双阳性期：T细胞先表达前TCR（β链和α链前体），并诱导CD8和CD4分子。
3）单阳性期：分化为带CD4或CD8分子的T细胞，同时表达TCR及CD3，随血流进入外周免疫器官或外周血。
CD4-CD8-TCR-
干 CD4-CD8-TCR细胞
双阴性 T细胞
CD4-,CD8γδTCR
CD4+CD8+ TCR+
双阳性 T细胞
外周
CD8+ TCR+
识别MHC Ⅰ类分子
CD8+ TCR+
阳性选择的意义：使成就的T细胞具有识别、结合 MHC的能力，在识别抗原时显示MHC约束性。
• 阴性选择：阳性选择的T细胞的TCR与 MHC-自身抗原肽显示高亲和力的被清除，只有显示低或中等亲和力的及无识别能力的被留下。
• 意义：清除自身反应性T细胞。不会和自身抗原起反应。
（二）、T细胞受体

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

B淋巴细胞复习题

第九章 B 淋巴细胞一、选择题A 型题1、BCR复合物的组成成分为（）A. mIg ,CD3B. IgM, CD79a/CD79bC. IgD, CD79a/CD79bD. mlg, lg a和lg BE. mIg, IgA 和IgG2、成熟B 细胞表达的mlg 主要为（）A. mlgMB. mlgDC. mlgGD. mlgM 和mlgGE. mlgM 和mlgD3、传递B 细胞活化信号1 的信号转导分子为（）A. CD79a 和CD79bB. CD19 和CD21C. CD3 和CD4D. CD4 和CD8E. CD40 和CD40L4、BCR与抗原结合后不能直接传递抗原刺激信号，原因是（）A. mlg 与抗原结合的亲和力不高B. mlg 的L 链胞内部分很短C. mlgM 的H 链胞内部分很短D. mlgD 的H 链胞内部分很短E. mlgM 和mlgD 的H 链胞内部分均很短5、关于BCR的叙述，下列哪项是错误的？（）A. 其化学本质是mlgB. 能有效地摄取可溶性抗原C. 识别抗原有MHC限制性D. 与抗原结合后产生B 细胞活化信号1E. B细胞活化信号1经lg a和lg B传至胞内6、 B 细胞的表面受体不包括（）A. BCRB. HlV 受体C. EB 病毒受体D. CR1 和CR2E. Fc Y R n7、下列哪种组合是 B 细胞活化的第二信号？（）A. CD80（B 细胞）——CD28（T 细胞）B. CD86（B 细胞）——CD28（T 细胞）C. CD40L（B 细胞）—— CD40（活化的T细胞）D. CD40（B 细胞）——CD40L（活化的T 细胞）E. B7（B 细胞）——CD28（T 细胞）8、下列哪种组合可抑制T 细胞的活化？（）A. CD80（B 细胞）——CD28（T 细胞）B. CD86（B 细胞）——CD28（T 细胞）C. B7（B 细胞）——CTLA-4（活化的T 细胞）D. CD40（B 细胞）——CD40L（活化的T 细胞）E. CD40L（B 细胞）—— CD40（活化的T细胞）9、关于B1 细胞，叙述错误的是（）A. 细胞表面表达CD5 和mIgMB. 其BCR/所产生的抗体与抗原结合的特异性高C. 产生于个体发育的早期D. 倾向于定位在肠道和腹膜腔E. 倾向于产生抗细菌多糖抗原的抗体10、关于B2 细胞，叙述正确的是（）A. 产生于胎儿期B. 可与多种不同的抗原表位结合，表现为多反应性C. 对蛋白质抗原的应答能力强D. 主要产生低亲和力的IgME. 可产生致病性自身抗体而诱发自身免疫病11、B1 细胞的主要功能不包括（）A. 产生抗细菌多糖抗原的抗体而抗微生物感染B. 产生抗病原体蛋白质的抗体而抗微生物感染C. 产生多反应性自身抗体而清除变性的自身抗原D. 产生致病性自身抗体而诱发自身免疫病E. 在肠道抗病原体的粘膜免疫中起重要作用12、哺乳动物B 细胞发育成熟的场所为（）A. 骨髓B. 胸腺C. 淋巴结D. 脾脏E. 粘膜伴随淋巴组织13 、关于B 细胞，下列哪种说法不正确？（）a）骨髓中的淋巴细胞主要为B细胞b）B细胞表面表达的mlg，是B细胞的抗原受体c）B细胞的抗原受体库能对众多的、无限的非己抗原产生应答d）某个B细胞产生的抗体所结合的抗原与该B细胞BCR结合的抗原相同e）B1 细胞和B2 细胞产生的抗体均有高度的特异性14、B细胞活化的辅助受体是指（）a）C D79a/CD79bb）C D40 ——CD40Lc）C D80 ——CD28d）C D86 ——CD28e) CD19 —CD21—CD81—Leu 1315、关于B1 和B2 细胞，错误的是( )a) B1 细胞初次产生于胎儿期b) B2 细胞出生后才产生c) B2 细胞的BCR 与抗原结合的特异性低，具有多反应性d) B2 细胞主要对蛋白质抗原产生应答e) B1 细胞主要对多糖抗原产生应答16 、抗体的调理作用是指( )a) 抗体与病毒表面抗原结合后，阻止病毒与靶细胞结合b) 抗体与胞内菌结合后，阻止胞内菌与靶细胞结合c) 抗体与细菌毒素结合后，阻止细菌毒素发挥作用d) 与病原体结合的抗体，其Fc 段与吞噬细胞的Fc 受体结合，促进病原体被吞噬e) 抗体与病原体表面抗原结合，直接导致病原体死亡17 、抗体的中和作用是指( )a) 抗体与病原体结合后，阻止病原体与靶细胞的结合b) 抗体与病原体结合后，直接导致病原体死亡c) 抗体与病原体结合后，促进吞噬细胞对病原体的吞噬清除d) 形成病原体- 抗体- 补体复合物，促进吞噬细胞对病原体的吞噬清除e) 抗体与可溶性抗原结合，从而阻止可溶性抗原与BCR结合18、B细胞作为专职性APC,正确的表述是( )A. B细胞通过BCR结合颗粒性抗原B. B 细胞的抗原提呈作用在自然免疫应答中十分重要C. B 细胞组成性地表达协同刺激分子D. 只有活化的B 细胞才是抗原提呈细胞E. 正常情况下，B细胞也能把可溶性自身抗原提呈给19、B 细胞的BCR 结合的抗原为( )A. TD 抗原B. TI 抗原C. 颗粒性抗原D. 可溶性抗原E. 外源性抗原和内源性抗原20 、正常情况下，B 细胞不能把可溶性自身抗原提呈给A. B 细胞不能有效地摄取可溶性抗原B. B 细胞不能有效地加工、处理可溶性抗原C. 未活化的B 细胞不能表达协同刺激分子D. 未活化的B 细胞不产生细胞因子E. B 细胞只能提呈颗粒性抗原X 型题1、BCR复合物的组成成分包括( )A. MIgMB. MIgGC. Ig aD. Ig 3E. IgG2、传递B 细胞活化信号1 的信号转导分子为( ) T 细胞T 细胞而诱导自身免疫应答的原因是( )A. CD3B. CD19C. CD21D. CD79aE. CD79b3、B1 细胞的主要功能为( )A. 产生抗细菌抗体而抗微生物感染B. 产生特异性高、亲和力强的抗体，阻止病原体与靶细胞的结合C. 产生特异性高、亲和力强的抗体，促进吞噬细胞对病原体的吞噬清除D. 产生多反应性自身抗体，清除变性的自身抗原E. 产生致病性自身抗体，诱导自身免疫病4、B 细胞的主要功能包括( )A. 产生抗体介导体液免疫应答B. 提呈颗粒性抗原给T 细胞C. 提呈可溶性抗原给T 细胞D. 分泌细胞因子参与免疫调节E. 组成性地表达协同刺激分子，提呈自身抗原，参与自身免疫应答二、填空题1、B 细胞表面的抗原受体为 ____ ，它的化学结构为_________ ，其作用是_______ 。

第九章淋巴细胞的分化成熟和受体

㈢协同刺激受体—CD40
CD40为跨膜糖蛋白，属于肿瘤坏死因子受体家族。主要表达于成熟和不成熟B细胞、单核细胞和树突状细胞等APC表面。当BCR被抗原交联后，Igα和Igβ 向细胞内发出激活第一信号，诱导B细胞表达CD40，CD40与Th2表面的CD40L结合后，向细胞内发出第二信号，导致B细胞激活、增殖并分化。
二、 B细胞表面的膜分子
IL-5
补体 (C3b,C4b )
CD28 B7-2
IgM
FcmR IL-2
CD40 CD40-L
CD32
IgG
CD23 IgE
CD21 IL-4 补体 (C3bi )
Antigen
㈠B细胞抗原识别受体-BCR复合体
㈡B细胞协同受体-CD19、CD21 和CD81
BCR有协同受体，其包括CD19、CD21和CD81 3 种膜分子，它们在B细胞膜上与BCR直接接触，其能够促进通过BCR-Igα-Igβ产生的活化信号。
♦ＴＣＲαβ基因重组
ＴＣＲ基因片段以固定的次序发生重组，从而形成具有功能的ＴＣＲα和β基因，使得Ｖ、Ｄ、Ｊ和Ｃ片段互相靠近。
TCR的V、D和J基因的重组是由重组酶参与的。 TCRβ链先发生重组，先是D基因与J基因片段重组，然后V基因片段重组到DJ形成VDJ基因，接着α链开始基因重组，α链的第一次基因重组清除了染色体上所有δ链的DJ和C基因片段（δ链基因位于Vα 和Jα之间），灭活了δ链基因，大量存在的Vα和Jα 基因片段使α链位点的重组可进行几个循环，因此不同的重组经常发生。
但其多样性与Ig基因不同，TCR分子无同型转换，TCR基因也无体细胞突变。
二者之间不同还表现在，虽然 TCRV 区基因的数量远较 IgV基因的少，但TCR分子多样性潜能，推测起来却大于Ig 分子，这是因为TCR基因形成功能性重组，就不会进一步导致蛋白受体功能和亲和性改变，另外，N区的插入多于BCR，在BCR中只有重链有N－核苷酸的插入，而TCR的αβ 链基因中都有N区的插入，也就是在α链的V-J片段和β链的V-D、 D-J之间都有N区的插入。

医学免疫学课件第九章TB淋巴细胞

◆ CD152：细胞毒T细胞抗原，CTLA-4。表达于活化的T 细胞，与B7结合可抑制活化的T细胞增殖——负调控作用。
◆ CD25：IL-2R的α亚单位，活化T细胞表面的标志。
（三）T细胞表面的其它膜分子
1. MHC分子所有T细胞均表达MHC-Ⅰ类分子活化T细胞表达MHC-Ⅱ类分子
2. 细胞因子受体（cytokine receptor，CKR） IL-1R、IL-2R、IL-4R、IL-6R及IL-7R等。静止T细胞表达低亲和力IL-2Rβγ链；活化T细胞表达高亲和力IL-2Rαβγ链
◆ 来源于造血干细胞，在胸腺中发育成熟，在外周淋巴器官识别抗原提呈细胞所提呈的特异性抗原而被激活，分化为效应性和记忆性T细胞。
（一）T细胞的分化发育
胸腺是T细胞分化、发育、成熟的场所。胸腺微环境：胸腺基质细胞（TSC）、细胞外基质、局部活性物质
T细胞发育过程中的细胞类别
◆ 双阴性细胞（double negative cell，DN）：CD4-CD8◆ 双阳性细胞（double positive cell，DP）：CD4+CD8+ ◆ 单阳性细胞（single positive cell，SP）：CD8+ 或CD4+
TCR-CD3复合体
ITAM：免疫受体酪氨酸活化基序
1. TCRαβ
α链及β链分子结构与免疫球蛋白类似，属于IgSF。α链（相当于Ig轻链）和β链（相当于Ig 重链）的可变区分别由V-J及V-D-J基因片段重排后所编码，形成特异性各不相同的TCR分子。
针对抗原产生特异性应答。
2. TCRγδ
◆ CD8：细胞毒性T细胞（CTL）的标志，与MHC-I 结合，提供CTL活化的第一信号。

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。

免疫学试题库 (9)

第九章B淋巴细胞一、单项选择1. 关于B细胞受体（BCR）的结构，下述哪项是错误的？A. 膜表面Ig主要为五聚体IgMB. 含有Igα和IgβC. 可直接识别抗原D. 识别抗原有特异性E. 属于免疫球蛋白超家族2. 以下哪一个分子与BCR交联后传入抑制性信号：A．TCR B．FcμR C．FcγRⅡ-B 即CD32 D．CD19 E．CD213. 以下哪种CD分子是特异性表达于B细胞表面的：A. CD28B. CD19C. CD3D. CD8E. CD44. 鉴别B-1细胞和B-2细胞的主要表面标志是：A. CD5B. CD21C. CD19D. CD81E. CD79a5. 不成熟B细胞表达的mIg主要是：A. mIgAB. mIgMC. mIgDD. mIgEE. mIgG6. 以下哪种CD分子不表达于B细胞：A. CD154B. CD21C. CD19D. CD81E. CD79a7. B细胞摄取抗原的方式有：A. 通过BCR特异性结合抗原B. 非特异性吞饮C. 非特异性吞噬D. A和B两种方式E. 上述A、B和C三种方式8. 组成BCR复合体的成分是：A. mIg、CD4和CD8B. mIg、CD80和CD86C. mIg、CD79а和CD79βD. CD81、CD21和CD19E. mIg、CD40和CD40L9. B细胞活化的第二信号是：A. TCR与pMHC复合物结合B. CD40与CD40L结合C. BCR与抗原结合D. CD8与抗原结合E. CD19与抗原结合10. 下列哪种分子不属于B细胞表面的协同刺激分子：A. BCRB. CD40C. CD80（B7.1）D. CD86（B7.2）E. ICAM-111. B细胞活化的辅助受体是：A. BCR复合体B. CD19/CD21/CD81C. 协同刺激分子D. C3d受体E. BCR识别特异性抗原12. 关于生发中心的描述，下列哪种说法是错误的：A. B细胞在生发中心分化成熟B. 生发中心可以分成明区和暗区两部分C. 生发中心中只有B细胞D. 生发中心的滤泡树突状细胞（FDC）可以富集并向B细胞提供抗原刺激E. 生发中心绝大多数B细胞会发生凋亡13. 关于浆细胞的描述，下列哪种说法是错误的：A. 浆细胞又称抗体形成细胞B. 浆细胞是B细胞分化的终末细胞C. 浆细胞能合成并分泌特异性抗体D. 浆细胞中含有大量粗面内质网E. 浆细胞表面仍表达BCR，可以与抗原起反应14. 关于记忆性B细胞的描述，下列哪种说法是错误的：A. 可以产生大量抗体B. 再次遇到相同抗原后可迅速活化C. 离开生发中心，进入血液参与再循环D. 寿命较长，属长寿细胞E. 不产生抗体15. B淋巴细胞分化成何种细胞可以分泌抗体？A. NK细胞B. 浆细胞C. 巨噬细胞D. 单核细胞E. 淋巴母细胞16. 下列哪种细胞不属于B细胞在中枢免疫器官（骨髓）的分化发育的阶段:A. 祖B细胞B. 前B细胞C. 未成熟B细胞D. 活化的B细胞E. 造血干细胞17. 关于B细胞的分化，下列哪种说法不正确：A. B细胞的分化分为抗原非依赖期和抗原依赖期两个阶段B. 抗原非依赖期是指B细胞在骨髓的分化阶段，此阶段B细胞分化不受抗原影响C. 抗原依赖期是指B细胞在外周淋巴器官的分化阶段，此阶段B细胞接受抗原刺激而活化、增殖D. B细胞的分化始终不需要抗原的刺激E. 未成熟B细胞膜表面可表达IgM18. 下列关于B细胞表面受体复合物哪种说法不正确：A. 是B细胞表面的主要分子之一B. 由mIg和Igα/Igβ异源二聚体组成C. mIg负责识别和结合抗原D. Igα/Igβ负责传递抗原刺激信号E. 对B细胞的功能没有作用19. B细胞具有识别抗原的能力，因其表面有：A. E受体B. mIgC. sIgAD. C3受体E. Fc受体20. 人类B细胞分化成熟的场所是：A．胸腺B．骨髓C．法氏囊D．淋巴结E．脾21. 与mIg共同组成BCR复合物的是：A．CD19和CD21 B．CD28和CD152 C．CD79a和CD79bD．CD80和CD86 E．CD40和CD40L22．表达pre-B受体的细胞是：A．祖B细胞B．大前B细胞C．小前B细胞D．未成熟B细胞E．成熟B细胞23. B细胞上的EB病毒受体是：A．CD5 B．CD19 C．CD22D．CD21 E．CD3524. 恒定地表达于成熟B细胞上的协同刺激分子是：A．CD40 B．CD40L C．CD152D．CD28 E．CD525. B-1细胞接受碳水化合物刺激后，产生的抗体主要是：A．高亲和力IgG类抗体B．高亲和力IgM类抗体C．低亲和力IgG类抗体D．低亲和力IgM类抗体E．低亲和力IgA类抗体26. 编码BCR重链的基因位于：A．第14号染色体B．第6号染色体C．第22号染色体D．第2号染色体E．第7号染色体27．B-1细胞识别的抗原主要是：A．颗粒性抗原B．蛋白质抗原C．脂类抗原D．碳水化合物类抗原E．胸腺依赖性抗原28．B细胞作为APC可藉其表面的BCR结合：A．可溶性抗原B．病毒抗原C．颗粒性抗原D．各种抗原E．细菌抗原29．可刺激成熟B细胞增殖和（或）分化的细胞因子是：A．IL-12 B．IL-8 C．TGF-βD．G-CSF E．IL-430．具有多反应性的细胞是：A．B-1细胞B．B-2细胞C．大前B细胞D．小前B细胞E．祖B细胞31．B细胞发育中阴性选择的后果是：A．表达mIgM B．细胞发育受阻滞C．受体编辑D．自身耐受E．免疫无能二、不定项选择1. B-1细胞与传统意义上的B细胞（B-2细胞）有很大不同，其主要特性和抗体应答主要特点是：A.具有自我更新的能力B.接受抗原刺激后，48h之内即可产生以IgM为主的抗体C.增殖分化过程中，可发生Ig类别转换D.具有免疫记忆，可产生再次免疫应答E.产生免疫应答需要有Th细胞辅助2. 成熟B细胞表达的mIg主要为：A. mIgAB. mIgMC. mIgDD. mIgGE. mIgE3. 关于B细胞，正确的描述有：A. 产生抗体B. 在骨髓中发育成熟C. 自胸腺发育成熟D. 具有细胞毒作用E. 活化的B细胞也可以递呈抗原4. B细胞在生发中心分化成熟中的事件有：A. 克隆增殖B. 体细胞高频突变C. Ig亲和力成熟D. 通过CD40/CD40L与T细胞相互作用E. 抗体类别转换三、名词解释1. BCR复合物四、简答与论述1. 试述B淋巴细胞的功能。

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（三）BCR重排顺序
H
重链可变区基因重排示意图
BCR胚系基因重排发生具有明显的程序化形式，首先是重链进行基因重排，随后才是轻链重排。
经过胚系基因的重排，B细胞的DNA序列与其他体细胞有很大不同，这是存在于B细胞和T 细胞中非常独特的现象。
（四）等位排斥和同型排斥
等位排斥 (allelic exclusion)：
CD19/CD21/CD81
多分子活化共受体，增强B细胞对抗原刺激的敏感性。 CD19 ：胞质区可传递活化信号； CD21 (CR2)：可结合补体C3d； CD81：连接CD19和CD21，稳定复合物结构。
B 细胞双重抗原识别模式图
三、协同刺激分子（co-stimulatory molecules ）
优选第九章淋巴细胞
第一节 B细胞的分化发育
（一） BCR的基因结构、重排及其多样性
BCR是表达于B细胞表面的免疫球蛋白，即膜型免疫球蛋白（membrane immunoglobulin, mIg）。B细胞通过BCR识别抗原，接受抗原刺激，启动体液免疫应答。
编码BCR重链或轻链的基因是以数个分隔开的基因片段的形式成簇存在的。在B细胞的分化过程中，只有经过基因重排，才能形成有功能的BCR。
在B细胞中的一对同源染色体上，只有一条染色体上的H或L
基因可得到有效表达。
抑制另一条染色体H基因重排
14号染色体有效
H基因重排
启动2号染色体链基因重排
失败
另一条染色体H基因重排
同型排斥 (isotype exclusion)：
Ig 和两型中择一表达；先表达链基因，如链基因重排
失败，才开始链基因表达。
人VH基因片段为40个，根据同源性分为7个家族，编码H链的
信号序列和V区1-98aa，包括CDR1和CDR2
D（diversity）：
人D基因片段为25个，编码V区中大部分CDR3
JH（joining）：人JH基因片段为6个，编码CDR3其余部分和第4个骨架，15-17aa
（二）重链C区基因
V

第二节 B淋巴细胞的表面分子及其作用
B细胞的膜表面分子
➢ B细胞抗原受体复合物 ➢ B细胞共受体 ➢ 协同刺激分子 ➢ 其他表面分子
一. B细胞抗原受体(BCR)复合物
mIg Ig(CD79a) Ig(CD79b)
BCR复合物结构模式图
BCR复合体介导的胞内信号转导模式图
二、B细胞共受体（coreceptor）
CD40：组成性表达，与活化T细胞CD40L结合，传递B 细胞活化第二信号； CD80/CD86：活化B细胞表达，与T细胞上CD28和 CTLA-4结合，提供T细胞活化的第二信号；其他黏附分子：ICAM-1（CD54），LFA-1等。
ICAM-1 LFA-1
协同刺激分子
B淋巴细胞重要的表面分子示意图
C
CDR1 CDR2 CDR3
重链
重链基因
VDJ
C
CH：人CH基因片段含多个外显子，在大多数情况下C区中每个结构域和铰链区是由单独外显子编码。
（三）轻链的结构
V
C
CDR1 CDR2 CDR3
轻链
轻轻基因
V
J
C
V：人V基因片段为40个，根据同源性可分7个家族，编码195aa，包括CDR1、CDR2和部分CDR3
四、其他表面分子
CD20：调节B细胞增殖与分化，B细胞特异性标志 CD22：胞浆含ITIM基序，负调节CD19/CD20/CD81 CD32：负反馈调节B细胞活化及抗体的分泌
第三节 B细胞的亚群
一、B-1细胞表面标记：CD5＋分布：腹腔、胸腔、肠壁固有层功能：参与固有免疫
J：人J基因片段为5个，编码96-108aa, 包括部分CDR3和第4 个骨架区
C：人C基因片段为1个，编码109-214aa
（四）λ轻链的结构
V
C
CDR1 CDR2 CDR3
轻链
轻轻基因
V
J
C
人Vλ约有30个，根据同源性可分10个家族；Jλ和Cλ各4个，并成对排列
BCR的基因重排
（一）重组信号序列(recombination signal sequence, RSS) 重排过程主要由一组重组酶来识别V、（D）、J基因片段两侧的重组信号序列，通过切断、修复DNA而实现的
➢ RSS 结构：回文结构七聚体富含A/T的九聚体分布：V 片段3、J片段 5和D片段的两侧
➢ RSS与重链VDJ基因重排：D+JDJ V+DJVDJ ➢ RSS与轻链VJ基因重排：V+JVJ
（二）V (D) J重组酶 (recombinase) RAG-1/RAG-2复合物（重组激活酶复合物）性质：内切酶表达：T/B淋巴细胞发育早期阶段作用：识别RSS，切断七聚体一侧末端脱氧核苷酸转移酶（TdT）表达：B细胞前体作用：通过非模板的方式将数个核苷酸加到DNA断端其它：内切酶、DNA外切酶、DNA合成酶等
小鼠：=95:5，人：= 65:35
BCR多样性产生的机制
胚系中众多V、D、J基因片段（组合多样性，combinatorial diversity）人重链：VH(40) D(25) JH(6) = 6000 人轻链：V(40) J(5) = 200 人轻链：V(30) J(4) = 120 轻、重链不同配对 6103 2.0102 + 6103 1.2102 = 1.92106 V（D）J连接的多样性（junctional diversity）体细胞高频突变（somatic hypermutation）
（二）B细胞的发育和分化
B细胞的发育阶段
B细胞中枢免疫耐受的形成
克隆清除：
未成熟B细胞表面表达的mIgM与骨髓中的自身抗原结合，会导致该细胞的凋亡。
受体编辑：
mIgM对自身抗原的识别导致RAG基因重新活化，发生轻链VJ的再次重排，合成新的轻链。
失能：
mIgM与自身抗原的结合引起mIgM表达下调，进入外周淋巴器官后对抗原刺激不应答。
免疫球蛋白的基本结构
人BCR染色体定位和功能性基因片段
肽链 BCR H
染色体定位 14q32.3 2p1112 22q11.2
编码可变区编码恒定区
V DJ
C
40 25 6
9
40 5
1
30 4
4
BCR的基因结构
（一）重链V区基因
V
C
CDR1 CDR2 CDR3
重链
重链基因
VDJ
C
VH（variable）: