转Wee1基因靶细胞抗CTL介导的细胞毒效应

目的 :研究Wee1转基因细胞抗CTL介导的细胞毒效应及抗穿孔素抗体对抗细胞毒效应的影响。方法 :体外混合NIT及淋巴细胞培养获得CTL ,并测定淋巴细胞增殖指数 (MTT法 ) ;采用脂质体将重组体Wee1Hu GFP导入哺乳动物细胞NIT ;以转染重组体前后的NIT为靶细胞 ,检测CTL介导的胞毒效应 (LDH法 ) ,同时测定抗穿孔素抗体对此效应的影响。结果 :混合细胞培养可诱导CTL的增殖 ;转染Wee1基因及加入抗穿孔素抗体NIT组发生的细胞溶解的数量最少。结论 :转Wee1基因靶细胞在一定程度上可抵抗CTL介导的细胞毒作用 ,穿孔素抗体可协同增强此抗胞毒作用。     

Weel基因修饰细胞抗CTL诱导的凋亡效应

目的 研究Weel转基因细胞抗CTL介导的凋亡效应及抗穿孔素抗体的对此抗凋亡效应的影响。方法 采用DNA转染技术将重组体Weel Hu／GFP导入哺乳动物细胞NIT(鼠胰岛细胞瘤细胞)；以转染重组体前、后的NIT为靶细胞，采用ELISA凋亡试剂盒检测CTL介导的凋亡效应。结果 转染Weel及加入抗穿孔素抗体的NIT组发生凋亡的细胞数最少。结论 Weel基因转染靶细胞在一定程度上可抵抗CTL介导的细胞凋亡，而穿孔素抗体可协同增强此抗凋亡效应。     

转Weel基因靶细胞抗CTL介导的细胞毒效应

目的：研究Weel转基因细胞抗CTL介导的细胞毒效应及抗穿孔素抗体对抗细胞毒效应的影响。方法：体外混合NTT及淋巴细胞培养获得CTL，并测定淋巴细胞增殖指数(MTT法)；采用脂质体将重组体Weel Hu／GFP导人哺乳动物细胞NIT；以转染重组体前后的NIT为靶细胞，检测CTL介导的胞毒效应(LDH法)，同时测定抗穿孔素抗体对此效应的影响。结果：混合细胞培养可诱导CTL的增殖；转染Weel基因及加入抗穿孔素抗体NIT组发生的细胞溶解的数量最少。结论：转Weel基因靶细胞在一定程度上可抵抗CTL介导的细胞毒作用，穿孔素抗体可协同增强此抗胞毒作用。     

Wee1-GFP在CTL介导的细胞毒效应中的作用研究

目的: 研究Wee1-GFP融合蛋白对胞毒T细胞介导的胞毒效应的影响.方法: 采用脂质体将融合基因Wee1-GFP导入鼠胰岛瘤细胞(NIT-1)并检测其表达.以转染pWee1-GFP和空载体的NIT-1为靶细胞, 采用ELISA凋亡试剂盒和乳酸脱氢酶法检测胞毒T细胞介导的细胞毒效应.结果: Western blot检出Wee1-GFP融合蛋白的表达.转染Wee1-GFP的靶细胞发生凋亡及损伤的细胞数比转染空载体的靶细胞少.结论: Wee1-GFP基因转染靶细胞具有一定的抵抗CTL介导的胞毒效应的能力.     

FADDdel-GFP真核表达载体的构建及其抗细胞毒效应初步观察

目的构建融合蛋白FADDdel-GFP真核表达载体pFADDdel-GFP，用该融合基因转染小鼠胰岛瘤细胞(NIT)，从而抑制死亡受体介导的细胞内凋亡信号传导，以探讨IDDM的发病机制及防治措施。方法采用基因重组技术将FADDdel定向连接于真核表达载体pEGFP-N1，用脂质体将重组子pFADDdel-GFP导入哺乳动物细胞NIT并检测其表达，采用FACS和MTT法检测特异性T细胞对转染重组子NIT引起的细胞毒效应。结果转染重组子pFADDdel-GFP的NIT可见绿色荧光蛋白表达；pFADDdel-GFP转染的NIT对特异性T细胞介导的细胞毒有明显抵抗。结论成功构建pFADDdel-GFP融合基因并获稳定表达；pFADDdel-GFP转染NIT可有效抑制死亡受体介导的细胞内凋亡信号传导。     

融合蛋白FADDdel-GFP对死亡受体介导的胰岛细胞凋亡的影响

目的研究融合蛋白FADDdel-GFP对死亡受体介导的细胞凋亡信号的生物学效应,以探讨通过基因修饰靶细胞对1型糖尿病的影响。方法用脂质体将融合基因pFADDdel-GFP导入胰岛细胞株NIT,通过细胞RT-PCR和荧光显微镜检测其表达,采用FACS检测抗-Fas抗体诱导的胰岛细胞株的细胞毒效应。结果转染重组子pFADDdel-GFP的NITf-g细胞可见绿色荧光蛋白表达;NITf-g细胞对抗-Fas诱导的细胞损伤率为10.16%,细胞内的Caspase-3的活性为12.33%,均明显低于对照组（P〈0.05）。结论成功建立了稳定表达FADDdel-GFP的胰岛细胞株;FADDdel-GFP可有效抑制死亡受体介导的细胞内凋亡信号传导。     

融合基因FADDdel-GFP修饰在胰岛细胞自杀效应中的作用

目的 研究FADDdel-GFP在Fas/FasL介导的胰岛细胞自杀效应中的作用.方法 采用DNA转染技术将重组体pFADDdel-GFP导入哺乳动物细胞NIT(鼠胰岛细胞瘤细胞);采用FACS检测细胞因子诱导后NIT细胞的Fas和FasL表达情况及细胞自杀效应;采用active caspase3检测试剂盒检测细胞因子作用前后NIT细胞的caspase-3活性变化.结果 NIT细胞表面无Fas和FasL表达,细胞因子可促其表达增加;细胞因子作用后,FADDdel-GFP修饰的NIT细胞的细胞损伤百分率和细胞内caspase3活性明显低于对照组.结论 FADDdel-GFP修饰的NIT细胞具有一定的抵抗Fas/FasL途径介导细胞自杀效应的能力.     

HBV S基因修饰树突状细胞疫苗诱导特异性CTL反应的探讨

目的分析腺病毒载体介导HBVS基因修饰树突状细胞（DCs）能否诱导抗HBV特异性CTL反应。方法制备携带HBVS基因的重组腺病毒，分别转染外周血诱导培养的DCs，观察腺病毒转染DCs效率和DCs中HBV抗原的表达；混合淋巴细胞反应测定HBV抗原基因修饰DCs刺激同种异体T淋巴细胞增殖能力；乳酸脱氢酶释放法检测特异性CTL细胞对HepG2 22．1．5靶细胞的杀伤能力。结果腺病毒载体能够高效介导HBVS基因在DCs中表达，且DC细胞形态完整；HBVS基因修饰DCs具有刺激同种异体T细胞增殖能力，同时能诱导抗HBV特异性CTL反应。结论HBVS基因修饰DCs疫苗具有增强抗HBV特异性CTL效应的能力，可能发展为一种新型抗病毒疫苗。     

颗粒酶A介导的凋亡机制研究现状

目的:CTL细胞杀伤靶细胞有两个重要方式:即分泌型和非分泌型杀伤。前者指的是如穿孔素/(颗)粒酶类介质使靶细胞裂解,后者主要指通过FAS-FASL结合等一类途径诱导凋亡。(颗)粒酶A是(颗)粒酶家族中含量较多、作用方式较独特、且相当重要的一种物质。穿孔素/(颗)粒酶A/SET复合物介导的凋亡途径是一条重要的途径,它通过引起单股DNA链缺刻和DNA修复障碍导致细胞凋亡。其在杀伤肿瘤细胞、病毒感染细胞及异体移植物细胞有重要意义,近年来对穿孔素/(颗)粒酶A/SET复合物介导的细胞凋亡途径研究比较多,本文拟在这方面作一些综述。     

穿孔素介导的细胞凋亡机制在抗流感病毒感染免疫防御中作用的研究

目的：穿孔素介导的细胞凋亡机制在流感病毒初次感染中作用的研究。方法：用流感病毒A／PR／8／34经鼻感染穿孔素基因敲除鼠和同源对照C57BL／6小鼠，采用PFU方法测定肺内流感病毒增殖状况；免疫组织化学染色方法观察小鼠病毒感染后感染细胞的凋亡情况；利用乳酸脱氢酶释放法检测感染鼠脾淋巴细胞NK活性及CTL杀伤活性。结果：穿孔素基因缺乏导致流感病毒在小鼠肺内大量增殖；小鼠清除感染病毒所需时间延长；病毒感染细胞发生凋亡的时间亦因穿孔素的缺乏而延迟；感染小鼠脾淋巴细胞NK活性及CTL杀伤活性均显著降低。结论：穿孔素依赖的细胞介导的细胞毒效应在控制流感病毒初次感染，快速清除感染病毒方面起重要作用。     

FADDdel-GFP modified mouse insulinoma cells counteract the cytotoxicity of reactive T cells

IDDM results from pancreatic beta cell destruction by islet-reactive T cells,a process that involves beta cellapoptosis.Fas-FasL pathway plays a major role in pancreatic β cell death.Fas-associated death domain protein(FADD),the component of the tumor necrosis factor receptor type 1(TNFR1) and Fas signaling complexes,isinvolved in TNFR1-and Fas-induced apoptosis.Inhibiting the function of FADD will lead to blockingdownstream apoptosis signal,which protects pancreatic β cells from destruction by Fas-FasL pathway.In thisstudy we constructed eukaryotic expressing vector of fusional protein FADDdel-GFP named pFADDdel-GFP.After pFADDdel-GFP was transfected into NIT,the expression of FADDdel-GFP in NIT was detected byfluorescence microscopy and the resistance of NIT transfected with pFADDdel-GFP to cytotoxicity mediated byspecial T cells was detected by FACS and MTT.The results showed that NIT modified by pFADDdel-GFPobviously resisted cytotoxicity mediated by special T cells.Therefore,it may be useful in the prevention ortreatment of IDDM by intervening Fas-FasL pathway.Cellular & Molecular Immunology.2004;1(5):383-386.     

Two Distinct Mechanisms of Cytotoxity Mediated by Herpes Simplex Virus-Specific CD4+Human Cytotoxic T Cell Clones

The present study was undertaken to elucidate the mechanisms responsible for the cytotoxicity of herpes simplex virus (HSV)-specific CDA4⁺human cytotoxic T lymphocyte (CTL) clones, focusing on perforin and membrane-bound lymphotoxin (LT) (tumor necrosis factor-β). Two HSV-specific CDA4⁺CTL clones, which expressed both perforin and membrane-bound LT, exerted HSV-specific cytotoxicity and cytotoxicity against LT-sensitive L929 cells. These CDA4⁺CTL clones lysed HSV-infected cells directly in an HLA class II-restricted manner and did not exhibit "bystander killing." The culture supernatants of these clones stimulated with HSV antigen showed no cytotoxicity against HSV-infected cells or L929 cells, suggesting that adhesion to target cells is essentail to their antigen-specific and antigen-nonspecific cytotoxicities. The cytotoxicities of these clones against HSV-infected autologous cells were inhibited by an anti-CD3 monoclonal antibody but not by an anti-LT antibody. Conversely, their cytotoxicities against L929 cells appeared to be partially inhibited by the anti-LT antibody but not by the anti-CD3 monoclonal antibody. Furthermore, target cell DNA fragmentation induced by these CDA4⁺CTL clones was apparently observed in L929 cells but only faintly detected in HSV-infected autologous cells. L929 cell DNA fragmentation was also inhibited by adding the anti-LT antibody to CDA4⁺CTL cultures. these data suggest that some CDA4⁺CTL possess at least two cytolytic mediators, i.e., perforin and membrane-bound LT simultaneously, and can exert both antigen-specific and antigen-nonspecific cytotoxicity via two distinct mechanisms, necrosis and apoptosis.     

CD44抗体诱导急性髓性白血病细胞增殖抑制作用的研究

目的　探讨抗CD4 4抗体对白血病细胞增殖的影响 ,以及对T淋巴细胞介导的白血病细胞的细胞毒作用的影响 ;阐明抗CD4 4抗体的抗肿瘤效应机制。方法　利用3 H TdR掺入法检测肿瘤细胞的DNA合成 ;利用PI染色 流式细胞仪法分析肿瘤细胞的细胞周期变化 ;利用PKH2 PI双染色 流式细胞仪法检测T淋巴细胞所介导的细胞毒作用。结果　抗CD4 4抗体可明显地抑制高表达CD4 4分子的急性髓性白血病MML 1细胞的增殖 ,并且利用抗GAM抗体交联抗CD4 4抗体后 ,可直接诱导Fas敏感的MML 1细胞的凋亡改变。另外 ,抗CD4 4抗体单独即可抑制IL 2激活的T淋巴细胞介导的细胞毒作用 ;协同抗CD2抗体后 ,其对IL 2激活的T淋巴细胞介导的细胞毒作用的抑制更加明显。结论　CD4 4分子的表达可能与肿瘤的发生发展有一定的关系。抗CD4 4抗体对MML 1细胞的增殖抑制作用 ,可能与启动了Fas系统所介导的凋亡信号有关。另外 ,抗CD4 4抗体对T淋巴细胞所介导的白血病细胞的细胞毒作用的抑制 ,可能与其部分地阻断了CD4 4分子的表达有关     

The differential contribution of granzyme A and granzyme B in cytotoxic T lymphocyte-mediated apoptosis is determined by the quality of target cells

Complementary approaches with purified molecules or transfected cytolytic effector cells have suggested that both, granzyme A (gzmA) and granzyme B (gzmB), similarly contribute to CTL-mediatedand perforin (perf)-dependent apoptotic nuclear damage (DNA fragmentation) in target cells. Studies employing gzmA or gzmB single-knockout mice on the other hand indicated that gzmB is the prominent CTL effector molecule for the rapid induction of DNA fragmentation, with gzmA playing only a minor part. We have now taken ex vivo-derived virus-specific or in vitro generated alloreactive CTL from mice deficient in either gzmA or gzmB and a panel of three target cells to reinvestigate this unresolved issue. We show that rapid CTL-mediated DNA fragmentation of L1210.3 target cells is solely dependent on gzmB, whereas the DNA fragmentation of EL4.F15 target cells by the same CTL population is mainly induced by gzmA and only marginally by gzmB. Moreover, CTL-induced apoptosis of a third target cell, MC57G, was partially dependent on both gzmA and gzmB activities. The differential contribution of the two gzms to apoptosis was further verified by their distinct sensitivity tocaspase inhibitors. The data suggest that both, gzmA and gzmB, have a similar potential to induce rapid perf-mediated apoptosis but that their individual contribution to the underlying intracellular processes is dictated by the quality of the target cell.     

Regulation of Fas(Apo-1/CD95)- and perforinmediated lytic pathways of primary cytotoxic T lymphocytes by the protooncogene bcl-2

Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl-2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl-2 protein in the murine mastocytoma line P815 or in concanavalin A-activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl-2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl-2 and suggest that these two pathways converge at a step prior to Bcl-2 inhibition.