葡萄球菌肠毒素激活肿瘤浸润淋巴细胞及外周血淋巴细胞体外抗人卵巢癌的对比研究

Comparative study of anti-human ovarian cancer on TIL and PBL stimulated by SEA in vitro

目的:探讨葡萄球菌肠毒素A(SEA)对卵巢癌肿瘤浸润淋巴细胞(TIL)及其外周血淋巴细胞(PBL)抗瘤活性的诱导作用。方法:取10例卵巢癌伴腹水患者实体瘤、腹水及外周血标本,分离TIL和PBL。在SEA及IL-2作用下培养,定时计数,了解其增殖情况;流式细胞仪检测其CD3、CD4、CD8表达;噻唑蓝(MTT)比色法测定其对K562及自体肿瘤细胞的细胞毒活性;酶联免疫吸附试验(ELISA)测定培养上清液中TNF-a和IFN-γ浓度。结果:10例中8例成功分离实体瘤TIL、腹水TIL及PBL。(1)SEA刺激的实体瘤TIL、腹水TIL及PBL增殖速率明显较IL-2诱导组快(P<0.05),但增殖高峰后出现下降趋势,IL-2组未出现此现象;(2)CD3+CD4+及CD3+CD8+T表达率均明显上升,其中SEA诱导组比IL-2组增加比例明显(P<0.05),以SEA作用的CD3+CD8+T比例增加最快;(3)TIL对自体肿瘤细胞的杀伤活性明显高于对K562细胞的杀伤活性(P<0.05),PBL对自体肿瘤细胞的杀伤活性则明显低于对K562细胞的杀伤活性(P<0.05),SEA激活组比IL-2组杀伤率高(P<0.05);(4)各效应细胞分泌的TNF-a、IFN-γ分别在培养的第2天和第4天达到高峰,高峰后迅速下降,SEA诱导组在前10天明显高于IL-2诱导组(P<0.05)。结论:SEA可高效、迅速诱导卵巢癌TIL的抗瘤活性。

Objective：To investigate the anti-tumor activity of tumor-infiltrating lymphocytes（TIL） and peripheral blood lymphocytes（PBL） of ovarian cancer stimulated by staphylococcal enterotoxin A（SEA） in vitro.Methods：TIL were isolated from tumor tissues and ascites,and PBL were isolated from the peripheral blood of 10 ovarian cancer patients with ascites.The isolated cells were then cultivated in the presence of SEA and IL-2.The proliferation of TIL and PBL were observed in vitro.Flow cytometer was used to detect the expression of CD3,CD4 and CD8 on the surface of TIL.The cytotoxic activity to K562 cells and autogenous tumor cells was evaluated by MTT colorimetric method and the concentration of TNF-A alpha-γ in cultivating supernatant was measured by Enzyme-linked immunosorbent test（ELISA）.Results：TIL of ovarian solid carcinoma and TIL,PBL of ascites were successfully separated from 8 patients of 10 patients.（1） SEA had great stimulatory effect,which was higher than the group of IL-2（P0.05）,on the TIL of ovarian solid carcinoma,the TIL of ascites and PBL.After the peak of proliferation,it showed a downward trend,which was not seen in the group of IL-2.（2） The expression rate of CD3＋CD4＋T-cell and CD3＋CD8＋T-cell was increased significantly.There was significant difference in the increasing rate of SEA group and IL-2 group（P0.05）,the rate of CD3＋CD8＋T which induced by SEA proliferated more quickly.（3） The killing activity of TIL on autologous tumor cells was much higher than that on K562 cells（P0.05）.However,the killing activity of PBL on autologous tumor cells was much lower than that on K562 cells（P0.05）.Each effector cells were stimulated by SEA had higher killing activity than that stimulated by IL-2（P0.05）.（4） TNF-alpha or IFN-gamma secreted by each effector cells reached peak on the second and the fouth day,and then the peak decreased rapidly.The level of TNF-alpha and IFN-gamma in the SEA group was higher than those in the IL-2 group（P0.05）.Conclusion： SEA can induce the antitumor acitivity of TIL efficiently and quickly.