GPI-B7-1锚定肾癌细胞膜对细胞毒性T淋巴细胞杀伤自身肿瘤的促进作用

Glycosyl-phosphatidylinositol-B7-1 anchoring into renal carcinoma cell membranes enhances CTL's ability of killing carcinoma cells

目的探讨糖基磷脂酰肌醇(GPI)-B7-1锚定肾细胞癌(RCC)细胞膜能否提高细胞毒性T淋巴细胞(CTL)对自身肿瘤的杀伤能力。方法取出本实验室冻存于液氮罐中的经转基因高表达GPI-B7-1的CHO/DHFR-细胞,再从该CHO/DHFR-细胞上洗脱目的蛋白GPI-B7-1,并进行分离纯化,Western blotting法检测活性。建立肾癌细胞系CG-6327。将CTL分别与GPI-B7-1-CG-6327细胞膜、CG-6327细胞膜、GPI-B7-1-CHO/DHFR-细胞膜作用24 h,此时得到被三种不同物质活化的CTL细胞,再设单纯体外培养的CTL作为对照,分别将上述四种CTL细胞加入到已接种有CG-6327细胞的96孔板中,进行24 h杀伤试验,MTT法测定CTL的杀伤活性。结果经GPI-B7-1-CG-6327细胞膜活化的CTL细胞杀伤活性(A值)与对照组比较差异有统计学意义(P<0.05),其他几种物质活化的CTL细胞杀伤活性与对照组比较,P均>0.05。结论通过基因重组方法表达的GPI-B7-1锚定蛋白可被转移并锚定在RCC细胞膜表面提供第二刺激信号,GPI-B7-1锚定后的具有双刺激信号的RCC细胞膜对于CTL的活化有增强作用。

Objective To investigate whether GPI-B7-1 anchoring into the renal carcinoma cell membrane can improve the cytotoxic T lymphocyte（ CTL） ＇s ability of killing the cancer cells. Methods We selected the CHO / DHFR-cells that were genetically modified to highly express GPI-B7-1 in liquid nitrogen tanks,then,the target protein GPI-B7-1 was eluted from the CHO / DHFR-cells and purified. The activity was detected by Western blotting. The renal cell carcinoma cell line CG-6327 was established. We cultured the autologous CTL admixed with GPI-B7-1-CG-6327 membrane,CG-6327 membrane and GPI-B7-1-CHO / DHFR-membrane for 24 hours,and up to now,we got three kinds of CTL. Simple CTL cultured in vitro was taken as the control group,then these four kinds of CTL were added into 96-well plates which were inoculated with CG-6327 cells for the 24-hour killing experiment. The killing activity of CTL was detected by MTT. Results The killing activity（ A value） of GPI-B7-1-CG-6327 cell membrane-activated CTL was statistically different as compared with that of the control group（ P〈0. 05）. No significant difference was found in the CTL cell killing activity activated by others as compared with that of the control group,all P〉0. 05. Conclusion GPI-B7-1 anchored protein can be transferred and anchored in the cell membrane of RCC surface which provides a second stimulus,and RCC cell membrane with double stimulation signal anchored by GPI-B7-1 has a greater effect for the activation of CTL.