大肠癌细胞功能性Fas配体的表达度与肿瘤免疫逃逸的关系

目的:表达Fas配体(fasligand,FasL)的肿瘤周围激活的Fas阳性淋巴细胞凋亡异常增加.肿瘤的发生在很大程度上是因为转化的细胞不能正常凋亡所致.观察Fas与FasL在结肠癌细胞株的表达,在体外验证结肠癌细胞SW620是否可以诱导淋巴细胞发生凋亡.方法:用免疫组织化学SABC法观察结肠癌细胞系和Jurkat T淋巴细胞系Fas与FasL的表达,为癌细胞的Fas和FasL的功能提供形态学依据.采用非放射性细胞毒分析,测定SW620结肠癌细胞与Jurkat靶细胞共培养后LDH释放值检测效应细胞的杀伤效应.结果:结肠癌SW620细胞FasL染色呈阳性反应,阳性反应物主要定位于癌细胞的胞膜及核周区,而Fas染色几乎呈阴性反应.Jurkat细胞系Fas,FasL免疫组化染色呈阳性反应,阳性反应物主要定位于癌细胞的胞膜.CytoTox96非放射性细胞毒分析显示大肠癌SW620细胞系与激活的T细胞(Jurkat T细胞)共培养,诱导对Fas介导的凋亡敏感的T淋巴细胞(Jurkat T细胞)明显凋亡,并随着SW620接种浓度增加和共培养时间的延长,Jurkat T细胞凋亡的比例显著增加.用含有乙酸肉豆佛波醇(phorbol 12-myristatel3-ac-etate,PMA)加离子霉素(ionomycin)的DMEM培养基孵育48 h的大肠癌SW620细胞系与激活的T细胞(Jurkat T细胞)共培养,Jurkat T细胞凋亡的比例也明显增加.结论:结肠癌细胞SW620表达功能性FasL,能杀伤Fas阳性Jurkat T淋巴细胞,形成免疫逃逸.

To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

基因转移FasL诱导人黑色素瘤细胞凋亡的体外研究

目的:探讨体外基因转移Fas配体(Fas-Ligand,FasL)对恶性人黑色素瘤细胞凋亡的影响.方法:用携带人FasLcDNA的缺陷型重组腺病毒载体(Ad-FL),在体外转导2株黑色素瘤细胞,并使其表达;通过流式细胞仪、RT-PCR法进行Fas/FasL表达检测,TUNEL法及荧光显微镜检测细胞凋亡状况.结果:流式细胞仪和RT-PCR检测两株黑色素瘤细胞表面均表达Fas,不表达FasL,而Ad-FL转导的两株黑色素瘤细胞均能表达FasL;Ad-FL能显著诱导两株黑色素瘤细胞在体外凋亡或抑制其生长.结论:重组腺病毒FasL在体外诱导人黑色素瘤细胞凋亡效果显著.

小鼠FasL基因真核表达载体的构建及在HEK293细胞中的表达

目的 构建含有小鼠Fas Ligand (FasL)基因的重组真核表达载体,并检测FasL蛋白在其稳定转染的HEK293细胞中的表达情况,为构建表达小鼠Fas L的树突状细胞(DC)模型,深入研究DC联合T细胞防治移植物抗宿主病(GVHD)的新方法奠定基础.方法 从小鼠脾脏中提取RNA并逆转录为cDNA,以该cDNA为模板扩增FasL基因,插入pcDNA3.1(+)载体中构建pcDNA3.1(+)-FasL重组质粒,利用脂质体将其转染HEK293细胞,用G418筛选稳定表达细胞系,Western blot确定重组蛋白的表达.结果 通过酶切及测序证实FasL序列正确插入表达载体pcDNA3.1(+),Westemblot检测证实转染重组质粒的细胞能正确表达FasL蛋白,G418筛选后能够得到稳定表达FasL的抗性细胞株即FasL-HEK293.结论 重组质粒pcDNA3.1(+)-FasL和稳定表达FasL的HEK 293细胞成功构建,为后续FasL-DC细胞的研究打下了坚实基础.

狼疮肾炎患者外周血单核细胞表达有功能的Fas

目的探讨狼疮肾炎(LN)患者单核细胞凋亡加速的机制.方法对17例LN患者进行观察及以15例正常人为对照.采用流式细胞仪检测单核细胞Fas和Fas Ligand(FasL)的表达;单核细胞与重组人FasL共孵育,碘化丙啶染色、流式细胞仪检测单核细胞凋亡,四甲基偶氮唑蓝(MTT)法观察单核细胞体外存活率.结果 LN患者单核细胞Fas的表达较正常人明显上调(P＜0.05);处于狼疮活动期的LN患者与狼疮静止期的LN患者之间Fas的表达差异无显著性意义(P＞0.05).正常人和LN患者的单核细胞表面均未检测出FasL的表达.单核细胞与重组人FasL在体外共同培养2h后,LN患者单核细胞的凋亡较正常人明显增加(P＜0.05),单核细胞与重组人FasL在体外共同培养6 h后,LN患者单核细胞的存活率较正常人明显低下(P＜0.05).结论 LN患者单核细胞表面功能性Fas表达上调是LN患者单核细胞凋亡加速的可能机制.

胃粘膜癌变过程中Fas、FasL的表达及意义

目的:通过检测胃粘膜癌变过程中凋亡相关蛋白Fas、FasL的表达,探讨其在胃癌发生发展中的作用.方法:采用免疫组化SP法检测41例慢性浅表性胃炎(CSG)、19例慢性萎缩性胃炎伴肠化生(CAGIM)、22例异型增生(Dys)和37例胃癌(GC).结果:Fas在CSG、CAGIM、Dys和GC组中的阳性表达率依次下降,FasL的阳性表达率则逐渐升高;Fas、FasL在高中分化GC组的阳性表达率均显著高于低分化GC组.结论:Fas、FasL的阳性表达率在胃癌发生过程中呈现不同的变化趋势,提示由Fas/FasL诱导的胃粘膜细胞凋亡异常与胃癌形成可能有密切关系.在胃癌患者,与癌细胞分化有关的Fas、FasL表达的一致性有助于判断肿瘤的恶性程度和预后.

CMV-hFasL转基因小鼠容易被小剂量的链尿菌素诱导至糖尿病

AIM: To investigate the role of Fas-FasL pathway in the pathogenesis of streptozotocin (STZ)-induced type I diabetes mellitus. METHODS: Low dose injections of STZ were used to induce type I diabetes mellitus in the CMV-hFasL transgenic mice. Blood glucose concentration was measured with Glucotrand Plus blood glucose test strips. Expression of hFasL was detected by RT-PCR and Western blotting. The severity of insulitis was determined by histologicalexamination. Expressions of IL- 1 β and TNF-α mRNA in the pancreas were detected by semi-quantitative RT-PCR analysis. Fas expression in apoptotic RIN-5F cells was also confirmed by RT-PCR in vitro. RESULTS: hFasL was expressed in the islets of CMV-hFasL transgenic mice. The transgenic mice were sensitive to diabetic induction than the control WT mice. IL-1 β and TNF-α expressions in the pancreas of CMV-hFasL transgenic mice were far more than that in WT mice. We also found STZ and IL-1β could both induce higher expression of Fas in RIN-5F. The combining of Fas-FasL could lead to the apoptosis of β cells in the CMV-hFasL transgenic mice. CONCLUSION: Fas-FasL interaction plays a significant role in the pathogenic mechanism of type I diabetes mellitus.