CD40L在CD4^＋CIK诱导Fas抵抗乳腺癌细胞凋亡中的机制研究

Study on the proapoptotic effect of CD40L in apoptosis of Fas-resistant breast cancer cells induced by CD^4+CIKs

目的:探讨CD40/CD40L交联在诱导Fas抵抗乳腺癌细胞凋亡中的免疫学机制。方法:体外大规模扩增自体CIKs细胞,利用磁珠分选系统纯化其中CD4+T细胞亚群(CD4+CIK)。用抗Fas激活性抗体CH11诱导乳腺癌细胞系MDA-MB-231细胞凋亡,比较有无CD4+CIK培养上清预处理对MDA-MB-231细胞凋亡率的影响。MDA-MB-231细胞与CD4+CIK在不同效靶比下共孵育6小时和24小时,分别用流式细胞仪检测凋亡率和膜Fas表达,定量RT-PCR法检测胞浆内Bax、Bcl-2、FADD和c-FLIP的mRNA含量。在共培养系统中加入抗FasL、抗CD40L和抗IFN-γ中和抗体,分别比较MDA-MB-231细胞凋亡率、膜Fas表达和四种凋亡相关基因的mRNA含量。结果:MDA-MB-231细胞对CH11诱导的凋亡有抵抗,但经过CD4+CIK培养上清预处理后,敏感性显著提高。MDA-MB-231细胞与CD4+CIK共孵育24小时后,细胞凋亡率明显升高,并与膜Fas表达量呈正相关(r=0.618,P<0.01)。在共培养体系中添加抗FasL和抗CD40L中和抗体可完全清除增加的细胞凋亡,而抗IFN-γ中和抗体只能部分清除,凋亡率分别从(33.70±2.77)%降至(7.57±1.15)%、(7.80±0.26)%和(14.21±1.70)%。但MDA-MB-231细胞膜Fas表达可以等效地被抗CD40L和抗IFN-γ中和抗体封闭,分别从(25.90±2.45)%降至(6.93±1.56)%和(8.73±4.70)%。定量RT-PCR结果显示MDA-MB-231细胞与CD4+CIK共孵育6小时时胞浆中c-FLIP表达下降与凋亡率显著相关(r=0.898,P<0.05),而抗CD40L中和抗体可诱导c-FLIP表达升高。结论:CD40/CD40L交联和IFN-γ刺激均参与了CD4+CIK诱导的MDA-MB-231细胞凋亡,但其Fas抵抗性的逆转主要是通过CD40/CD40L交联抑制c-FLIP的mRNA合成而实现的。

Objective：To study the proapoptotic effect of CD40/CD40L cross-linking induced by CD4^＋CIKs on Fas-resistant breast cancer cells and the underlying mechanisms.Methods：After large scale of amplification of CIKs in vitro,the subset of CD4^＋CIK cells was isolated by magnetic bead separation columns.The typical Fas-mediated apoptosis of MDA-MB-231 cells induced by anti-Fas agonist CH11 was measured at pretreatmunt with or without supernatants of CD4^＋CIKs.Apoptotic rates of MDA-MB-231 cells cocultured with CD4^＋CIK for 24 hours at different ratio of E/T were evaluated by AnnexinV staining and expression of Fas was compared by flowcytometry as well.Then the antibodies against FasL,CD40L,IFNγ were added in the coculture,respectively.Apoptotic rates and the expression of Fas on MDA-MB-231 cells were detected.The mRNA expression of Bax、Bcl-2、FADD、c-FLIP in MDA-MB-231 cells after cocultured with CD4^＋CIK for ether 6 h or 24 h was analyzed by real-time quantitative RT-PCR method,and the antibody blocking experiment was studied.Results：The susceptibility to Fas-mediated apoptosis of insensitive MDA-MB-231 cells increased after being pretreated with supernatants of CD4^＋CIK.After co-culturing with CD4^＋CIK,the apoptotic rates of MDA-MB-231 cells increased,which significantly correlated with the enhanced expression of membrane Fas（r=0.618,P〈0.01）.The elevated apoptotic rates were blocked fully by either anti-FasL or anti-CD40L,and partly by anti-IFN-γ antibodies,which decreased from（33.70±2.77）% to（7.57±1.15）%,（7.80±0.26）% and（14.21±1.70）%.However the Fas expression on MDA-MB-231 cells inhibited equally by either anti-CD40L or anti-IFN-γ,which dropped from（25.90±2.45）% to（6.93±1.56）% and（8.73±4.70）%.The antibody blocking experiment showed that anti-CD40L rather than anti-IFN-γ antibody induced significant increase of c-FLIP during 6 h after coculturing with CD4^＋CIKs,which was significantly negatively correlated with the apoptosis induced in MDA-MB-231 cells（r=0.898,P〈0.05）.Conclusion：Both CD40L and IFN-γ participate the Fas-dependent apoptosis of MDA-MB-231 cells induced by CD4^＋CIK.However,reversion of Fas-resistance is mediated mainly through CD40/CD40L ligation rather than IFN-γ stimulation by inhibiting synthesis of c-FLIP.