【作者】 曹水； 任秀宝； 于津浦； 张新伟； 韩颖； 张维红；

【Author】 Cao Shui Ren Xiu-Bao Yu Jin-Pu Zhang Xin-Wei Han Ying Zhang Wei-Hong Department of Immunology, Tianjin Medical University Affiliated Cancer Hospital, Tianjin 300060, China

【机构】 天津医科大学附属肿瘤医院免疫学研究室；

【摘要】 目的:研究CIK细胞中CD4+T细胞(CD4+CIK)诱导肿瘤细胞凋亡的作用,并进一步探讨其相关机制。方法: 体外大规模扩增CIK细胞,应用高强度磁珠分选系统纯化 CD4+T细胞亚群,LDH法比较CD4+CIK、CD4-CIK对raji 细胞的杀伤活性;将CD4+CIK与raji细胞共孵育,应用 AnnexinV／PI染色比较4h和24h时不同效靶比时raji细胞的凋亡;Transwell板培养观察细胞间膜与膜的接触对凋亡的影响;同时检测raji细胞表面凋亡相关分子Fas的变化;应用 FasL中和性抗体阻断Fas／FasL途径后比较raji细胞的凋亡变化;ELISA法检测共培养上清中细胞因子IL-2、IFN-γ、 TNF-α的分泌量;应用半定量PCR方法及流式细胞法比较 PBMC中CD4+T细胞亚群(CD4+PBMC)与CD4+CIK上 CD40L的表达变化;ELISA法比较两种CD4+T细胞培养上清中可溶型CD40L(soluble CD40L,sCD40L)的分泌量。结果:经过多种细胞因子的诱导后,CIK细胞中的CD 3+、 CD3+CD8+、CD3+CD4+、CD3+CD56+细胞比例较PBMC明显增多(P<0．05);富集CD4+T细胞纯度达96．2％; CD4+CIK 细胞在4h时对raji细胞无明显杀伤,但可观察到早期凋亡, 24h时raji细胞凋亡率明显增加,并有部分细胞出现死亡;在 Transwell板中raji细胞凋亡率较普通96孔板培养降低明显; 共孵育过程中raji细胞表面Fas表达水平升高,且随着效靶比的升高而增加,加入FasL抗体后凋亡率明显下降;共培养上清中IL-2、IFN-γ分泌增加,TNF-α无明显变化;在 mRNA和蛋白表达水平,CD4+CIK细胞中CD40L均较PBMC 明显增加(P<0．01);其培养上清中sCD40L分泌量增加明显(P <0．05)。结论:(1)CD4+CIK可通过诱导肿瘤细胞的凋亡实现对肿瘤细胞杀伤,这一过程主要通过膜与膜的接触进行。(2) CD4+CIK主要通过Fas／FasL途径来诱导肿瘤细胞凋亡。(3) 多种细胞因子刺激后,CD4+CIK细胞表面中CD40L的表达量明显增加;培养上清中sCD40L分泌量增加。(4)CD4+CIK 可能通过CD40／CD40L交联提升肿瘤细胞表面Fas表达来诱导其凋亡。更多还原

【Abstract】 Objective: To study the apoptosis-induc-ing activity of CD4+T cells in CIKs and investigate the underlying mechanism. Methods: After large scale of amplification of CIKs in vitro, CD4+T cells subset was isolated by magnetic beads separation columns. Cytotoxicity of purified CD4+T, CD4 T cells subset in CIKs against raji cells was measured by using LDH-release assay. Apoptotic rates of raji cells cocultured with CD4+CIK after 4 hours and 24 hours were evaluated by AnnexinV staining and expression of Fas was compared by flowcytometer as well. The influence of cell-cell contact and Fas/FasL interaction on apoptotic rates of raji cells was studied by transwell plate and antibody blocking experiment .The cytokines such as IL-2, IFN- γ, TNF- β secreted in supernatant was detected by ELISA. The expression of CD40L in CD4+CIK was compared with CD4+PBMC by RT-PCR, flowcytometry and ELISA. Results: The proportions of CD3+ cells, CD3+CD8+ cells, CD3+CD4+ cells, CD3+CD56+ cells in CIKs increased significantly compared with those of PBMC. Purity of enriched CD4+T cells reached 96.2%. Few raji cells were lysed by CD4+CIK after 4h cocultured. But the apoptotic rates of raji were observed and elevated with the increasing of effector cells after 24h cocultured. Flowcytometry showed that Fas on raji cells was increased after 24h cocultured. The apoptotic rates decreased obviously after cocultured with anti-FasL blocking antibody. The cytokines ,such asIL-2, IFN- γ, in supernatant increased and there was no change about TNF- α . Comparing with PBMC, significant increase in mRNA and protein expression of CD40L in CD4+CIK was observed(P< 0.01) Conclusion: (1)CD4+CIK were the effectors capable of inducing apoptosis in tumor cells through cell-cell-contact. (2)CD4+CIK induce apoptosis of tumor cells mainly through Fas/FasL pathway.(3)Comparing with PBMC, significant increase in mRNA and protein expression of CD40L was observed in CD4+CIK(P<0.01). (4) CD4+CIK can increase the expression of Fas and induce apoptosis of tumor cell through the interaction of CD40/ CD40L.更多还原