卵巢癌耐药细胞对活化CD4^+T细胞免疫活性的抑制及其机制

Inhibitory effects of drug-resistant oophoroma cell line on immune activities of activated CD4^+T cells and the mechanism

目的探讨卵巢癌顺铂耐药肿瘤细胞对活化CD4^+T细胞的抑制作用及其机制,为卵巢癌耐药患者的免疫治疗提供理论依据。方法用中等浓度间歇作用法建立耐顺铂卵巢癌细胞SKOV3/CDDP,MTT法测定细胞耐药指数及交叉耐药性,流式细胞术(FCM)检测细胞凋亡率。ELISA检测肿瘤细胞培养上清液中TGF-β1、IL-10的含量。抽取正常人外周血用免疫磁珠分离CD4^+T细胞,用CD3和CD28抗体刺激活化CD4^+T细胞。用含20%肿瘤培养上清液培养,ELISA检测CD4^+T细胞IL-2表达量的变化,Western blot检测CD4^+T细胞内NF-κB、Snail的表达变化及肿瘤细胞中TGF-β1、P-smad2/3的表达变化;将肿瘤细胞与活化的CD4^+T淋巴细胞共培养,计数CD4^+T细胞增殖情况,HE染色观察肿瘤细胞对活化CD4^+T细胞黏附的抑制作用。结果SKOV3/CDDP的耐药指数为32.4,对阿霉素、紫杉醇产生交叉耐药性,在CDDP作用下,SKOV3/CDDP凋亡率明显降低。与SKOV3细胞相比,SKOV3/CDDP细胞培养上清TGF-β1表达量升高,SKOV3/CDDP培养上清能抑制活化的CD4^+T淋巴细胞IL-2的表达,抑制CD4^+T淋巴细胞的增殖及黏附作用;Western blot检测显示CD4^+T细胞内NF-κB、Snail的表达降低;SKOV3/CDDP细胞中TGF-β1和P-smad2/3的表达升高(P<0.01)。结论 SKOV3/CDDP耐药细胞TGF-β1/P-smad信号通路激活,细胞分泌TGF-β1增多,对活化CD4^+T淋巴细胞的增殖及黏附起抑制作用,可能是通过抑制CD4^+T细胞内的NF-κB/Snail信号通路实现的。

The study aimed to determine the effects of drug-resistant oophoroma cell line SKOV3/CDDP on activated CD4＋T lymphocytes and the mechanism. Middle doses intermittent induction method was used to establish a human CDDP-resistant ovarian cancer cell line（SKOV3/CDDP）. Then ELISA was employed to measure the concentration of TGF-β1 and IL-10 secreted by SKOV3/CDDP and SKOV3 cells; ELISA was also used to detect the concentration of IL-2 secreted by activated CD4＋T lymphocytes which was cultivated with normal culture medium, 20% serum of SKOV3/CDDP or 20% serum of SKOV3 cells. The proliferation of activated CD4＋T lymphocytes was detected by counting after co-culture with SKOV3/CDDP cells. Western blot was used to test the levels of NF-κB and Snail in CD4＋T lymphocytes and the levels of TGF-β and P-smad2/3 in SKOV3/CDDP cells.The adhesive ability of activated human CD4＋T lymphocytes was probed with SKOV3/CDDP cells using HE staining.Data showed that the drug resistant index of SKOV3/CDDP cells were 32.4,demonstrating cross resistance to other chemotherapy drugs（ADM, PTX）. Compared with SKOV3 cells, SKOV3/CDDP cells had low apoptosis rate, and secreted more TGF-β1. The supernatant of SKOV3/CDDP cells suppressed the proliferation of activated CD4＋T cells by in hibiting the secretion of IL-2. The expression of NF-κB and Snail was decreased in CD4＋T lymphocytes after stimulation with the drug-resistant cell; the expression of TGF-β1 and P-smad2/3 were higher in SKOV3/CDDP cells as compared with SKOV3. Taken together, suppression of immune activities of activated CD4＋T cells by SKOV3/CDDP cells may correlate with TGF-β1, and achieve by inhibiting the NF-κB-Snail signaling pathway in CD4＋T cells.