PTEN基因逆转卵巢上皮性癌细胞耐药的机制研究

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0V2008及0V2008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0V2008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PTENmRNA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（M1Tr）比色法观察转染PTEN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）PTENmRNA在0V2008和C13K细胞中的表达水平分别为1．02±0.05和0．45±0.03,而0V2008、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PTENmRNA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48h后,C13K细胞中PTENmRNA、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（IC50）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）μmol／L,P〈0,05]。（4）顺铂作用24h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0V2008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。

Reversal of drug resistance in human ovarian cancer cells by wild-type PTEN gene and its mechanisms

OBJECTIVE: To examine expression of PTEN gene in ovarian cancer cisplatin-sensitive cell line OV2008 cells and cisplatin-resistant cell line C13K cells, and evaluate the effect of wild-type PTEN gene on reversing cisplatin-resistance of C13K cells and underlying mechanisms. METHODS: The expression of PTEN mRNA and protein in OV2008 and C13K cells were detected by semi-quantitative RT-PCR and western blot. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine 2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, protein kinase B (AKT), phospho-AKT (p-AKT) protein were analyzed by western blot in PTEN transfected and untransfected C13K cells. Proliferation and chemosensitivity of cells to cisplatin were measured by methyl thiazolyl tetrazolium (MTT), and cell apoptosis was detected by flow cytometry after treatment with cisplatin. RESULTS: (1) The expression of PTEN mRNA and protein (1.02 +/- 0.05, 1.02 +/- 0.07) in OV2008 cells were significantly higher than those in C13K cells, which were 0.45 +/- 0.03 and 0.55 +/- 0.03 respectively (P < 0.05). (2) After transfected with PTEN gene for 48 hours, the expression of PTEN mRNA and protein in C13K cells were 2.04 +/- 0.10, 0.94 +/- 0.04 respectively. Compared with C13K cells transfected with empty vector (1.04 +/- 0.04, 0.36 +/- 0.03) and untransfected C13K cells (1.03 +/- 0.05, 0.37 +/- 0.03), the difference was significant respectively (P < 0.01). The expression of p-AKT protein (0.94 +/- 0.07) was lower than those in control groups (1.66 +/- 0.10, 1.68 +/- 0.14; P < 0.05). (3) The 50% inhibition concentration (IC(50)) to cisplatin of C13K cells transfected with PTEN (7.2 +/- 0.3) micromol/L] was obviously lower than those of empty-vector transfected cells and untransfected cells (12.7 +/- 0.4), (13.0 +/- 0.3) micromol/L; P < 0.05]. (4) The apoptosis ratio of C13K cells with wild-type PTEN transfection, empty vector transfection and untransfected were (41.7 +/- 0.9)%, (18.6 +/- 0.7)% and (15.3 +/- 0.8)% respectively (P < 0.01). CONCLUSIONS: PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in multidrug-resistant human ovarian cancer cell line C13K by decreasing the expression of p-AKT.