摘要
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 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 RTPCR 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 C13 K 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 CI3K 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 CI3K 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 ( IC50 ) to cisplatin of CI3K cells transfected with PTEN [ (7.2 ± 0. 3 ) μmol/L] was obviously lower than those of empty-vector transfected cells and untransfected cells [ ( 12.7 ± 0. 4), ( 13.0 ± 0. 3 ) Ixmol/L; P 〈 0. 05 ]. (4) The apoptosis ratio of C 13 K 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 muhidrug-resistant human ovarian cancer cell line C13K by decreasing the expression of p-AKT.
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2007年第9期612-616,共5页
Chinese Journal of Obstetrics and Gynecology
基金
国家自然科学基金(30571950)
国家重点基础研究发展计划(2002CB513107)
关键词
卵巢肿瘤
PTEN磷酸水解酶类
顺铂
抗药性
肿瘤
Ovarian neoplasms
PTEN phosphohydrolase
Cisplatin
Drug resistance, neoplasm