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目的:探讨细胞色素P45017α-羟化酶/17,20裂解酶(CYP17)小干扰RNA(siRNA)对CYP17基因表达和多囊卵巢综合征(PCOS)卵泡膜细胞雄激素合成的抑制效果。方法:合成4条靶向CYP17基因的特异性siRNA和非特异性siRNA。将0.4μg携带报告基因增强型绿色荧光蛋白和CYP17基因的质粒和siRNA共转染入HeLa细胞,荧光显微镜观察及流式细胞术检测绿色荧光蛋白的表达反映对CYP17表达的影响,筛选抑制效果最佳的siRNA。卵泡膜细胞取自PCOS患者和非PCOS妇女各5例,将筛选出的最佳CYP17siRNA以最佳浓度转染人卵泡膜细胞,荧光定量实时逆转录多聚酶链反应检测CYP17mRNA的表达,并且检测培养液中CYP17作用产物雄烯二酮和前体孕酮水平。结果:siRNA1221处理后,HeLa细胞中外源性CYP17的表达显著降低,抑制率达50﹪,卵泡膜细胞中CYP17mRNA水平和雄烯二酮产生降低,但与对照组比较,统计学上无显著差异。结论:RNA干扰技术可能提供一种PCOS高雄激素基因治疗的新方法。
OBJECTIVE: To investigate the inhibitory effects of cytochrome P45017α-hydroxylase / 17,20-lyase (CYP17) siRNA on CYP17 gene expression and androgen synthesis in human ovarian follicular cells of patients with polycystic ovary syndrome (PCOS). Methods: Four specific and non-specific siRNAs targeting CYP17 gene were synthesized. 0.4μg plasmid and siRNA carrying enhanced green fluorescent protein and CYP17 gene of reporter gene were co-transfected into HeLa cells. Fluorescent microscope and flow cytometry were used to detect the expression of green fluorescent protein (GFP) Good siRNA. Follicular cells were collected from 5 patients with PCOS and non-PCOS. The best CYP17siRNAs were selected and transfected into human follicular cells at the optimal concentration. The expression of CYP17mRNA was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) The CYP17-acting products, androstenedione and progesterone levels. Results: After treatment with siRNA1221, the expression of exogenous CYP17 in HeLa cells was significantly reduced and the inhibitory rate was 50%. The levels of CYP17 mRNA and androstenedione decreased in the control group, but there was no significant difference between the two groups. Conclusion: RNA interference technology may provide a new method of PCOS gene therapy for hyperandrogenism.