【作者】 高慧；

【导师】 王彦；

【作者基本信息】 青岛大学 ， 妇产科学， 2012， 硕士

【摘要】 目的：探讨采用脂质体2000 (lipofectamin2000)转染miRNA27a (microRNA miRNA)模拟物(mimics)及抑制物(inhabitors)对卵巢癌细胞系的顺铂(DDP)耐药性的研究。方法：本试验采用了小剂量DDP间歇诱导卵巢癌细胞系skov3建立卵巢癌顺铂耐药细胞系skov3/DDP:1.MTT检测细胞耐药系数；2.实时荧光定量PCR (Real-time PCR RT-PCR)检测skov3/DDP及skov3中miRNA27a的表达情况；2lipofectamin2000转染miRNA27a inhabitors、mimics,并用荧光显微镜检测转染效；3.MTT检测细胞增殖情况；4.实时荧光定量PCR检测多耐药基因(Multdrug Resistance Gene MDR1)基因的表达情况；5.流式细胞术检测细胞凋亡；6Western blot检测糖(P-gp)蛋白表达情况。结果：1skov3/DDP相对于skov3耐药系数为5.08；2miRNA27a在skov3/DDP中的表达明显高于skov3,约为2.80倍,有统计学意义；3skov3/DDP与其转染miRNA27a inhabitors, skov3及其转染mimics相比,MDR1的表达无统计学意义；4.转染抑制物的skov3/DDP的P-gp蛋白的表达较skov3/DDP降低了32.90%,转染mimics的skov3的P-gp蛋白的表达较skov3升高了20.71%；5.转染inhibitors及阴性对照(Negative Control NC)的Skov3/DDP在顺铂作用下存活率不同,有统计学意义；6.转染inhibitors的skov3/DDP与转染NC的skov3/DDP相比,前者凋亡率为10.37±2.58%,后者凋亡率为3.12±1.34%,有统计学意义。结论：上皮性卵巢癌耐药可能与miRNA27a的表达上调有关；miRNA27a可能是通过抑制MDR1基因的翻译来发挥作用而不通过抑制MDR1的转录发挥作用的；下调miRNA27a可以逆转卵巢癌细胞系的耐药性,增加其对顺铂的敏感性。更多还原

【Abstract】 Objective:to investigate down-regulation and up-regulationm miRNA27a and its relation with drug resistance in human ovarian cancer skov3/DDP.Methods:This test uses a small dose of DDP for intermittenting ovarian cancer cisplatin-resistant cell lines. MTT was used to know the resistant factor and drug sensitivity; real-time PCR was used to detect the expression of miRNA27a in skov3/DDP and skov3. The cells were translated miRNA27a inhibitor, negative control. miRNA27a mimics and negative control by lipofectamin2000. The expression of MDR1 gene in the cells and transltated cells was measured by real-time PCR. cell proliferation analysis was measured by MTT. cell apoptosis analysis was measured by Flow cytometry. p-glycoprotein (p-gp) was measured by western blot.Results:(1) Compared with skov3, resistance coefficient of skov3/DDP is 5.08. (2) miRNA27a was an average of 2.80 -fold higher expression level in skov3/DDP cells than that in skov3 ceils, with a significant difference between the two groups(P<0.05), (2) skov3/DDP cells transfection with inhibitors of miR-27a showed that the same levels of MDR1 mRNA without a significant difference; skov3 cells transfection with miR27a mimics also show the same level of MDR1 Mrna without a significant different; P-gp protein levels decreased 32.9% than that in the Nc group, P-gp protein levels increased 20.7% in skov3 transfected miRNA27a mimics than that in the Nc group.(3) cell proliferation in kov3/DDP cells transfection with inhibitors of miR-27a compared with skov3/DDP transfencted negative control has a significant different. (4)apoptosis rate (10.37±2.58%) was higher than that in the Nc group(3.12±1.34%), and there were significant differences between two groups(allP<0.05).Conclusion:varian epithelial cancer to drug resisntance is related to miRNA27a’s high-expression. miRNA-27a may regulate P-gp expression to increase drug resistance to cisplatin; Down-regulate miRNA-27a may reduce drug resistance to cisplatin.更多还原