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目的通过检测 PTEN 基因在卵巢上皮性癌(卵巢癌)顺铂敏感细胞株 OV2008及OV2008配对的顺铂耐药细胞株 C13K 中的表达,探讨转染 PTEN 基因能否逆转 C13K 细胞对顺铂的耐药及其相关机制。方法半定量 RT-PCR 技术和蛋白印迹法检测 OV2008和 C13K 细胞中 PTENmRNA 和蛋白的表达。将野生型 PTEN 基因真核表达质粒在脂质体介导下转染 C13K 细胞,同时以转染空载体和未转染的 C13K 细胞作为对照,分别应用 RT-PCR 技术检测各组细胞 PTEN mRNA 表达的变化,应用蛋白印迹法检测各组细胞 PTEN、蛋白激酶 B(AKT)及磷酸化 AKT(p-AKT)蛋白表达的变化;四甲基偶氮唑蓝(MTT)比色法观察转染 VFEN 基因后 C13K 细胞对顺铂敏感性的变化,流式细胞仪分析顺铂作用后的细胞凋亡情况。结果 (1)PTEN mRNA 在 OV2008和 C13K 细胞中的表达水平分别为1.02±0.05和0.45±0.03,而 OV2008、C13K 细胞中 PTEN 蛋白的表达水平分别为1.02±0.07、0.55±0.03,两种细胞 PTEN mRNA 和蛋白的表达水平分别比较,差异均有统计学意义(P<0.05)。(2)PTEN 基因转染48 h 后,C13K 细胞中 PTEN mRNA、蛋白的表达水平分别为2.04±0.10和0.94±0.04,分别与转染空载体和未转染的 C13K 细胞比较,差异均有统计学意义(P<0.01);p-AKT 蛋白的表达水平(0.94±0.07)较转染空载体(1.66±0.10)和未转染(1.68±0.14)的 C13K 细胞显著降低(P<0.05)。(3)转染 PTEN 基因的 C13K 细胞对顺铂的半数抑制浓度(IC_(50))为(7.2±0.3)μmol/L,明显高于转染空载体和未转染的 C13K 细胞[分别为(12.7±0.4)、(13.0±0.3)μmo]/L,P<0.05]。(4)顺铂作用24 h 后,转染 PTEN 基因、转染空载体和未转染的 C13K 细胞的凋亡率分别为(41.7±0.9)%、(18.6±0.7)%和(15.3±0.8)%,前者明显高于后两者(P<0.01)。结论 PTEN 基因在 OV2008细胞中的表达明显高于 C13K 细胞。转染野生型 PTEN 基因能有效提高C13K 细胞内 PTEN 基因的表达,并通过降低 C13K 细胞中 AKT 磷酸化的水平恢复 C13K 细胞对顺铂的敏感性。
Objective To detect the expression of PTEN gene in cisplatin-resistant cell line C13K, which is a cisplatin-sensitive ovarian epithelial ovarian cancer (OV2008) and OV2008-matched cell line, and to investigate whether transfection of PTEN can reverse the resistance of C13K cells to cisplatin Medicine and its related mechanisms. Methods Semi-quantitative RT-PCR and Western blotting were used to detect the expression of PTEN mRNA and protein in OV2008 and C13K cells. The eukaryotic expression plasmid of wild-type PTEN gene was transfected into C13K cells by lipofectamine. Meanwhile, the transfected empty vector and untransfected C13K cells were used as control. RT-PCR was used to detect the expression of PTEN mRNA The protein expression of PTEN, AKT and p-AKT in each group was detected by Western blotting. The expression of PTEN, p-AKT and p-AKT in each group were detected by MTT assay C13K cells after cisplatin gene sensitivity, flow cytometry analysis of apoptosis after cisplatin. Results (1) The expression levels of PTEN mRNA in OV2008 and C13K cells were 1.02 ± 0.05 and 0.45 ± 0.03 respectively, while the expression levels of PTEN protein in OV2008 and C13K cells were 1.02 ± 0.07 and 0.55 ± 0.03, respectively mRNA and protein expression levels were compared, the differences were statistically significant (P <0.05). (2) The expression of PTEN mRNA and protein in C13K cells after PTEN gene transfection for 48 h were 2.04 ± 0.10 and 0.94 ± 0.04, respectively, which were statistically different from those of transfected empty vector and untransfected C13K cells (P <0.01). The expression level of p-AKT protein (0.94 ± 0.07) was significantly lower than that of C13K cells transfected with empty vector (1.66 ± 0.10) and untransfected cells (1.68 ± 0.14) (P <0.05). (3) The half-inhibitory concentration (IC50) of C13K cells transfected with PTEN gene was (7.2 ± 0.3) μmol / L, which was significantly higher than that of untransfected and untransfected C13K cells (12.7 ± 0.4), (13.0 ± 0.3) μmo] / L, P <0.05]. (4) After treated with cisplatin for 24 h, the apoptosis rates of transfected PTEN gene transfected empty vector and untransfected C13K cells were (41.7 ± 0.9)%, (18.6 ± 0.7)% and (15.3 ± 0.8) )%, The former was significantly higher than the latter two (P <0.01). Conclusion The expression of PTEN gene in OV2008 cells was significantly higher than that of C13K cells. Transfection of wild-type PTEN gene can effectively improve the expression of PTEN gene in C13K cells and restore the sensitivity of C13K cells to cisplatin by reducing the level of AKT phosphorylation in C13K cells.