Kupffer细胞Fas配体的表达及其对移植肝内淋巴细胞的杀伤作用  

Expression of Fas ligand protein in Kupffer cells and its role in inducing T cells apoptosis in rat liver graft

作　　者：涂兵[1] 梁绍勇[1] 陈勇[1] 龙飞伍[1] 彭勇[1] 刘作金[1] 徐明清[2] 严律南[2] 龚建平[1] 

机构地区：[1]重庆医科大学附属第二医院肝胆外科,重庆400010 [2]四川大学华西医院肝移植中心,成都610065

出　　处：《第三军医大学学报》2008年第14期1322-1326,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(30471696,30500473)~~

摘　　要：目的观察移植肝脏中Kupffer细胞(KCs)和T淋巴细胞(T lymphocytes,TLCs)中Fas、FasL基因和蛋白的表达,以及KCs对侵入肝脏内的淋巴细胞的细胞毒性作用。方法肝移植后0、48h分离肝移植组、氯化钆(GdCl3)组动物肝脏的KCs和TLCs,并对TLCs进一步分类;用RT-PCR法测定KCs和TLCs中Fas、FasL基因表达,用免疫组化、激光扫描共聚焦显微镜和Western blot蛋白印迹等方法测定KCs和TLCs中Fas、FasL蛋白质表达;观察不同培养条件下淋巴细胞凋亡与KCs的相互作用。结果GdCl3组分离出的KCs显著少于肝移植组,淋巴细胞数量及CD8+T细胞显著多于肝移植组(P<0.05),免疫组化和激光共聚焦显微镜显示GdCl3组FasL阳性KCs显著少于肝移植组(P<0.05),2组Fas阳性淋巴细胞无显著差异(P>0.05);RT-PCR和Western blot显示KCs中FasL mRNA及蛋白表达GdCl3组较弱(P<0.05),2组淋巴细胞中Fas、FasLmRNA无显著差异(P>0.05)。GdCl3组中分离出的KCs对TLCs杀伤率明显低于肝移植组(P<0.05),抗FasL抗体对KCs杀伤力有抑制作用。结论KCs能表达FasL蛋白,并通过Fas、FasL途径杀伤移植肝脏中的淋巴细胞,诱导移植肝脏产生免疫耐受。GdCl3能阻止KCs中FasL基因和蛋白表达,抑制肝移植中免疫耐受的产生。Objective To detect the expressions of Fas/FasL in Kupffer cells（KCs） and graft-infiltrating T lymphocytes （TLCs）, and explore the role of KCs in induced-T cells apoptosis through Fas/FasL pathway. Methods Totally 112 male SD rats were randomized into 4 groups, that is, live transplantation group （28 do- nors and 28 recipients）, and GdCI3 pretreatment group （28 donors and 28 recipients）. The donors of pretreat- ment group received intravenous injection of GdCl3at 10 mg/kg, and the donors of the other group was injected with the same volume of normal saline. Then liver transplantation was carried out with the modified two-step perfusion method. The recipients were killed immediately or 48 h after the operation. Isolated KCs and TLCs were counted and TLCs were classified using flow cytometry （FCM）. Expression of FasL protein in KCs and TLCs was evaluated with immunocytochemical staining and laser scanning confocal microscopy （I.SCM）. mRNA and protein expression of Fas and FasL in KCs and TLCs were detected with RT-PCR and Western blot. The cytotoxicity of KCs against TLCs v/a Fas/FasL system and the effect of anti-FasL antibody in this process was assessed using 3H thymidine release assays. Results Compared with the liver transplantation group, the counts of isolated KCs in GdCI3 group were lower while the number of TLCs and CD8^＋ T cell were significantly increased （ P 〈 0.05 ）, and less FasL-positive KCs and more Fas-positive TLCs were detected （P 〈 0.05 ）. Expression of FasL mRNA and its protein in KCs was decreased （ P 〈 0.05 ） , but expression of Fas mRNA and its protein in TLCs had no significant difference. An 18-hour 3H thymidine release assay showed that in 48 h after transplantation, KCs isolated from the liver transplantation group induced more TLCs than those from GdC13 group. The KCs-mediated killing of TLCs was significantly inhibited by treatment of anti-fasL antibody. Conclusion KCs-mediated TLCs killing induces liver transplantation tolerance v/a the Fas/

关 键 词：肝移植 KUPFFER细胞 Fas FAS配体 免疫耐受 淋巴细胞 

分 类 号：R392.4[医药卫生—免疫学] R394.4[医药卫生—基础医学]