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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。
Objective To investigate the inhibitory effect of cisplatin-resistant tumor cells on activated CD4 ~ + T cells in ovarian cancer and its mechanism, and to provide a theoretical basis for immunotherapy in patients with drug-resistant ovarian cancer. Methods The cisplatin-resistant ovarian cancer cell line SKOV3 / CDDP was established by medium-dose intermittent treatment. The drug resistance index and cross-resistance were determined by MTT assay. The apoptosis rate was detected by flow cytometry (FCM). The contents of TGF-β1 and IL-10 in tumor cell culture supernatants were detected by ELISA. CD4 + T cells were isolated from peripheral blood of normal people by immunomagnetic beads, and activated CD4 ~ + T cells by CD3 and CD28 antibodies. The expression of IL-2 in CD4 ~ + T cells was detected by ELISA. The expression of NF-κB and Snail in CD4 ~ + T cells was detected by Western blot and the expression of TGF-β1 , And the expression of P-smad2 / 3 was detected. Tumor cells were cocultured with activated CD4 ~ + T lymphocytes and the proliferation of CD4 ~ + T cells was counted. The inhibition of tumor cell adhesion to activated CD4 ~ + T cells was observed by HE staining . Results The resistance index of SKOV3 / CDDP was 32.4. The resistance to doxorubicin and paclitaxel was cross-resistant, and the apoptosis rate of SKOV3 / CDDP was significantly decreased under the action of CDDP. Compared with SKOV3 cells, the expression of TGF-β1 in the SKOV3 / CDDP cell culture supernatant increased. The SKOV3 / CDDP culture supernatant could inhibit the expression of IL-2 in activated CD4 ~ + T lymphocytes and the CD4 ~ + T lymphocytes The expression of NF-κB and Snail in CD4 ~ + T cells was decreased by Western blot. The expression of TGF-β1 and P-smad2 / 3 was increased in SKOV3 / CDDP cells (P <0.01). Conclusion The activation of TGF-β1 / P-smad signaling pathway in SKOV3 / CDDP-resistant cells increases the secretion of TGF-β1, which may inhibit the proliferation and adhesion of activated CD4 ~ + T lymphocytes by inhibiting CD4 ~ + T cells Within the NF-κB / Snail signaling pathway.