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急性期过敏性紫癜PBMC凋亡及其相关因素研究
中华微生物学和免疫学杂志 2000年第5期第20卷 临床免疫学
作者:李秋 杨锡强 刘恩梅 李欣 李永柏 王莉佳 张远维
单位:400014,重庆医科大学附属儿童医院
关键词:过敏性紫癜;细胞凋亡;Bcl-2;CD40L
【 摘要 】 目的 了解急性期过敏性紫癜(HSP)患儿是否存在淋巴细胞延迟凋亡及Bcl-2和CD40L对它们的影响。 方法 分别用琼脂糖凝胶电泳及流式细胞技术(FACS)分析经CD3单克隆抗体刺激培养的外周血单个核细胞(PBMC)产生的凋亡片段DNA及凋亡细胞计数;FACS检测PBMC Bcl-2的产生及CD40L表达。 结果 与正常儿童比较,急性期HSP患儿的PBMC出现延迟凋亡现象,Bcl-2产生及CD40L表达增高,且Anti-CD40L能明显纠正延迟凋亡。 结论 HSP患儿的淋巴细胞及单核细胞存在延迟凋亡,可能与Bcl-2的异常产生及CD40L过度表达有关,推测可能为HSP的发病机制之一。
Lymphocytes apoptosis and the related factors of Henoch-Schnlein's purpura in acute phase
LI Qiu, YANG Xiqiang, LIU Enmei, et al.
(Children's Hospital, Chongqing University of Medical Science, Chongqing 400014, P.R.China)
【 Abstract 】 Objective To observe whether lymphocytes apoptosis delays in children with Henoch-Schnlein's purpura (HSP) in the acute phase and investigate the effects of Bcl-2 and CD40L on it. Methods DNA fragmentation and apoptosis cell count of peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 McAb were detected by both agarose gel electrophoresis and flow cytometry. Analyses of Bcl-2 production and CD40L expression on PBMC were also conducted by flow cytometry. Results The results showed the apoptosis delay and significant increases of Bcl-2 production and CD40L expression of PBMC. This apoptosis delay condition of PBMC could be corrected when anti-CD40L McAb was added in vitro. Conclusions CD40L and Bcl-2 might involve in regulating apoptosis of PBMC of the patients. High expression of CD40L and Bcl-2 might result in delayed apoptosis of lymphcytes, which might play a very important role in setup of HSP.
【 Subject words 】 Henoch-Schnlein's purpura; Apoptosis; Bcl-2; CD40L
细胞凋亡(apoptosis)是多细胞生物体正常的细胞生理死亡过程,赖以维持自身的稳定,细胞凋亡不足或过剩均可以导致疾病。已知川崎病存在急性期外周血单个核细胞(PBMC)延迟凋亡现象,且随病情缓解PBMC凋亡恢复正常水平。也有关于系统性红斑狼疮伴淋巴细胞凋亡减少的报告〔1-3〕。本研究企图通过对过敏性紫癜(HSP)患儿外周血单个核细胞的研究,证实HSP也存在的淋巴细胞凋亡异常及其相关调控因素。
研究对象及方法
病例选择
1.HSP组:HSP急性期32例,均为本院住院患儿,男17例、女15例,平均年龄7.5岁。
2.对照组:本院门诊体检的健康同龄儿童30例作为正常对照,男14例,女16例,平均年龄6.7岁。
实验方法
1.细胞培养:采血(肝素抗凝)后,立即用淋巴细胞分离液分离PBMC,含10%小牛血清的RPMI 1640(GIBCO)调细胞浓度为106/ml。分设以下各管:①留1ml备检Bcl-2;②加CD3单克隆抗体(Anti-CD3,北京邦定公司,终浓度5μg/ml);③加Anti-CD3及Anti-CD40L(Pharmingen,终浓度5μg/ml)。从②③管各取1ml加入24孔培养板中,置37℃、5%CO2孵箱培养0、24、48及72h,备检片段DNA。
另取106细胞加佛波醇(PMA,Sigma,终浓度10ng/ml)及钙离子载体蛋白(A23187,Sigma,终浓度2μg/ml)37℃、5%CO2孵箱培养6h,备检CD40L。
2.片段DNA分析:酚、氯仿抽提及电泳法根据刘氏改良〔4〕的方法,按实验设计的时间收集细胞,经PBS洗涤后,加0.5ml低渗缓冲液,10000r/min 10min离心,取上清加等体积酚、氯仿、异戊醇抽提蛋白质。上清中加1/20体积乙酸钠,2倍体积无水乙醇,-20℃沉淀过夜并离心弃上清。用70%乙醇洗涤、干燥,将片段DNA溶于EDTA*Tris液中,加RNA酶20μg/ml,37℃水浴30min以降解RNA。加3μl溴酚蓝混匀,1%琼脂糖凝胶电泳,100V,1h,紫外灯下观察结果并照相。
3.凋亡细胞计数:按实验设计的时间收集细胞,PBS洗涤2次,用碘化丙啶(PI)染色30min后,流式细胞仪(FACS, Calibur BD公司)检测。采用DNA分析软件ModFit LT-Unititled 2.0版定量分析30000细胞中凋亡细胞百分率。
4.FACS检测Bcl-2表达:根据Yano等〔5〕提供的细胞内蛋白质检测方法改良。将新鲜分离的PBMC用PBS洗涤1次,0.25%多聚甲醛固定15min,洗涤缓冲液(含PBS/0.2%牛血清白蛋白/0.1%叠氮钠)洗涤1次;加70%甲醇1ml,4℃,1h;洗涤2次,加鼠抗人Bcl-2单抗(北京华美,终浓度5μg/ml),0~4℃,1h;洗涤2次,加羊抗鼠IgG1-FITC(1∶50)40μl(北京华美),4℃,30min,将细胞重悬于400μl PBS,FACS检测15000个细胞中Bcl-2阳性细胞率,以未加荧光抗体及Bcl-2单抗为空白对照。
5.FACS检测CD40L表达:将培养的PBMC用PBS洗涤2次,加鼠抗人CD40L-PE单抗(Pharmingen,浓度20μl/106细胞)染色(直接染色法),阴性对照用鼠抗人IgG1-PE,4℃,30min,将细胞重悬于400μl PBS,FACS检测15000个细胞中CD40L阳性细胞率。
结果
1.凋亡细胞片段DNA分析:片段DNA电泳结果(图1)可见正常对照组的PBMC在Anti-CD3刺激下,24h已有少许片段DNA出现,48h尤为明显;而HSP患儿的PBMC在48h开始出现片段DNA,当加入Anti-CD40L后,HSP的片段DNA增加,与正常对照组接近。
2.流式细胞仪(FACS)计数凋亡细胞:见表1。
3.流式细胞仪检测Bcl-2及CD40L:结果见表2、图2、图3。
图1 过敏性紫癜PBMC片段DNA电泳图
Fig 1. DNA ladder of PBMC from children in the HSP by 1% agarose gel electrophoresis in various culture conditions and times
Lane 1: Marker(EcoRⅠ/Hind Ⅲ); Lane 2,6: CD40L McAb were added in PBMC from HSP patient cultured for 24h and 48h; Lane 3,7: PBMC from normal subject cultured for 24h and 48h; Lane 4,5,8: PBMC from HSP patient cultured for 24, 0 and 48h
表1 HSP患儿PBMC凋亡百分率(
Table 1. Percentage of apoptosis positive cell of PBMC from HSP patients and controls(
0h | 24h(n) | 48h(n) | 72h(n) | |
Controls(1) | 0 |
14.71±4.96(8) |
26.23±12.05(7) |
32.79±11.69(7) |
HSP(2) | 0 | 3.13±3.21(9) | 12.26± 7.33(9) | 17.15± 4.26(8) |
HSP+CD40L McAb(3) | 0 | 12.57±8.48(6) | 18.98±11.62(6) | |
t/P* | 4.667/<0.001 | 2.685/<0.05 | 2.79/<0.015 | |
t/P** | 2.451/<0.05 | 1.161/>0.05 | ||
t/P*** | 0.471/>0.05 | 0.89/>0.05 |
*:(1) Compared with (2); **: (2) Compared with (3); ***: (1) Compared with (3)
表2 急性期HSP患儿PBMC中Bcl-2和CD40L阳性细胞率(
Table 2. Percentage of Bcl-2 and CD40L positive cells of PBMC from HSP patients and controls(
Bcl-2(n) | CD40L(n) | |
Controls |
27.96±17.92(11) |
0.54±0.58(11) |
HSP | 59.15±13.75(11) | 8.26±4.15(13) |
t | 3.273 | 5.831 |
P | <0.01 | <0.001 |
讨论 淋巴细胞凋亡是免疫系统维持自身免疫耐受的重要机制。在免疫反应过程中,淋巴细胞增殖、分化、清除外来抗原的同时,免疫系统也通过凋亡机理清除过多的和具有潜在自身免疫反应的淋巴细胞,以维持其数量和功能的平衡,凋亡过甚或不足均可产生疾病〔1〕。凋亡过多可导致免疫缺陷病。本室曾证实新生儿T淋巴细胞暂时性功能低下与淋巴细胞凋亡过甚有关〔4〕。当凋亡减少,淋巴细胞过度生长,免疫反应过度则会发生淋巴细胞增殖性疾病、恶性淋巴瘤及自身免疫性疾病等。已有资料表明,自身免疫性疾病如系统性红斑狼疮(SLE)、川崎病就存在有淋巴细胞延迟凋亡现象〔2,3,6〕。我们采用CD3单抗诱导HSP急性期患儿PBMC中淋巴细胞凋亡的研究,发现HSP患儿急性期有明显淋巴细胞延迟凋亡。
图2 HSP患儿PBMC表达CD40L的FACS图
Fig 2. Expression of CD40L by PBMC from HSP patients were assayed by flow cytometry
CD40L are shown by a continuous line,and relevant isotype controls are shown by a dotted line
A: CD40L was expressed by (8.26±4.15)% in the PBMC of the HSP patients
B: Normal controls expressed (0.54±0.58)%
图3 HSP患儿PBMC表达Bcl-2的FACS图
Fig 3. Expression of Bcl-2 by PBMC from HSP patients were assayed by flow cytometry
Bcl-2 are shown by a continuous line, and relevant isotype controls are shown by a dotted line
A: Bcl-2 was expressed by (59.15±13.75)% in the PBMC of the HSP patients
B: Normal controls expressed (27.96 ±17.92)%
细胞凋亡与增殖、分化一样,受细胞内外多种因素的正常调节,包括抗原因素,细胞表面分子如CD40-CD40配体(CD40L)、Fas-FasL和细胞因子如IL-2、IL-4、肿瘤坏死因子(TNF)等协同刺激因子及凋亡基因(如p53)和抗凋亡基因(如bcl-2家族)〔7-9〕。本研究发现HSP急性期PBMC的Bcl-2表达增强,提示HSP的淋巴细胞凋亡可能为Bcl-2依赖途径,其延迟凋亡可能与Bcl-2过度产生有关。
CD40L主要表达于活化的CD4+T细胞膜上,通过与B淋巴细胞及单核细胞膜CD40交联,可以分别活化T、B淋巴细胞及单核细胞并拮抗其凋亡〔10,11〕。本组HSP患儿淋巴细胞延迟凋亡的同时伴有淋巴细胞CD40L表达增高,体外实验Anti-CD40L有明显逆转淋巴细胞延迟凋亡的作用。
上述现象可能与免疫反应细胞过度活化。免疫反应物质异常增多,导致HSP的全身性血管炎密切相关。而CD40L与Bcl-2及淋巴细胞延迟凋亡之间的关系尚待进一步研究。
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(收稿日期:1999-01-11)