RNA干扰技术沉默survivin基因逆转卵巢癌细胞株SKOV3/ADM耐药性的研究  

RNA interference silencing expression of survivin gene and reversing drug resistance of ovarian cancer cell line SKOV3/ADM

作　　者：邓凯贤[1] 钟玲[1] 姜梅贤[2] 王萍玲[1] 陈莹[1] 

机构地区：[1]重庆医科大学附属第二医院妇产科,400010 [2]四川省中医药研究院附属医院急诊科

出　　处：《中华妇产科杂志》2005年第12期836-839,共4页Chinese Journal of Obstetrics and Gynecology

摘　　要：目的检测survivin基因在卵巢上皮性癌(卵巢癌)耐药细胞株SKOV3/ADM及其亲本细胞株SKOV3中的表达水平,探讨以survivin基因为靶向的RNA干扰(RNA i)能否有效沉默survivin基因在SKOV3/ADM细胞中的表达,并能否有效逆转SKOV3/ADM细胞对化疗药物的耐药性。方法半定量RT-PCR、免疫荧光法检测survivin mRNA、蛋白在SKOV3/ADM及SKOV3细胞中的表达。以survivin基因为靶向的重组质粒pshRNA-survivin及紫杉醇作用于SKOV3/ADM细胞,将SKOV3/ADM细胞分为4组,即对照组、RNA i组、紫杉醇组、RNA i+紫杉醇组,RT-PCR技术检测各组细胞的survivinmRNA表达水平,吖啶橙/溴乙锭(AO/EB)荧光染色法及末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸标记法(TUNEL)检测各组细胞的凋亡情况。结果survivin mRNA在SKOV3/ADM、SKOV3细胞中的表达水平分别为99.1%、75.3%,SKOV3/ADM、SKOV3细胞的荧光细胞数分别为(59±5)、(42±3)个,两种细胞间比较,差异均有统计学意义(P<0.05)。RNA i后SKOV3/ADM细胞中mRNA的表达水平由99.1%降低至7.9%。AO/EB荧光染色法、TUNEL法检测各组细胞凋亡数,紫杉醇组为(10.2±1.0)、(9.8±0.8)个,RNA i组为(48.5±4.9)、(49.5±4.3)个,RNA i+紫杉醇组为(71.5±6.8)、(68.3±5.7)个,RNA i+紫杉醇组与RNA i组比较,差异有统计学意义(P<0.05),RNA i+紫杉醇组与紫杉醇组比较,差异也有统计学意义(P<0.05)。结论survivin基因在SKOV3/ADM细胞中的表达显著高于SKOV3细胞。以survivin基因为靶向的RNA i可有效抑制SKOV3/ADM细胞中survivin基因的表达,有效增加了SKOV3/ADM细胞对化疗药物紫杉醇的敏感性,显著上调了SKOV3/ADM细胞的凋亡活性。Objective To examine expression of survivin gene in ovarian epithelial carcinoma drug resistant cell line SKOV3/ADM and its parental cell line SKOV3, and induction of cells apoptosis and reversal of drug resistance in SKOV3/ADM after RNA interference （RNAi） silencing survivin gene. Methods The transcription of survivin gene in cells was detected by semi-quantitative RT-PCR, the protein expression level of survivin gene was analyzed by immunofluorescence staining, SKOV3/ADM cells were treated with pshRNA-survivin and paclitaxel （Taxol）, and acridine orange （AO）/ethidium bromide （EB） staining was performed to evaluate the apoptosis of cells, Results Survivin gene mRNA expressed by 99.1% and 75.3% respectively in cell lines SKOV3/ADM and SKOV3, while fluorescent cells were 59 ± 5 and 42 ± 3 （P 〈 0. 05）, After the introduction of pshRNA-survivin into SKOV3/ADM, mRNA transcription level of survivin gene decreased distinctly from 99, 1% to 7.9% . The apoptotic cells of control group detected by AO/EB staining was 3.6 ± 0. 6, of Taxol group 10. 2 ± 1, 0, of RNAi group 48.5 ± 4. 9, of RNAi ＋ Taxol group 71, 5 ± 6.8. Apeptosis ratio between RNAi ± Taxol group and RNAi group had significant difference （ P 〈 0. 05 ）, and that between RNAi ＋ Taxol group and Taxol group also had significant difference （P 〈 0. 05 ）. Conclusions Both survivin gene mRNA and its protein are over-expressed in ovarian epithelial carcinoma cell lines SKOV3 and SKOV3/ADM, the level of survivin gene expressed in SKOV3/ADM is obviously different compared with that in its parental cell line SKOV3. RNA interference targeted against specific sequences of survivin in SKOV3/ADM cell could significantly reduce the level of survivin mRNA transcripts and protein, effectively induce the cells apoptosis and restore the sensitivity of cell to conventional chemotherapeutic agents Taxol.

关 键 词：RNA干扰 微管相关蛋白质类 细胞系 肿瘤 卵巢肿瘤 抗药性 耐药性 

分 类 号：R737.31[医药卫生—肿瘤]