RNA干扰对雄激素合成关键酶17α-羟化酶/17,20裂解酶作用的实验研究  

The effect of small interfering RNAs agaist cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17) on androgen biosynthesis in theca cells obtained from women with polycystic ovarian syndrome

作　　者：杜静[1] 梁晓燕[1] 曾海涛[1] 舒益民[1] 姚书忠[1] 朱波[1] 庄广伦[1] 

机构地区：[1]中山大学附属第一医院生殖中心,广州510080

出　　处：《国际妇产科学杂志》2008年第3期219-221,共3页Journal of International Obstetrics and Gynecology

基　　金：国家自然科学基金(编号:30571956;30300372);广东省自然基金(编号:31690;21874);GZST(编号:2004J-C0111)

摘　　要：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 effects of Cytochrome P450 17α-hydroxylase/17, 20-1yase（CYP17）small interfering RNA （siRNA） on the expression of CYP17 and androgen biosynthesis in theca cells from women with polycystic ovarian syndrome （PCOS）. Methods： Four siRNAs targeting the CYP17 gene and one non-specific siRNA were synthesized. 0.4μg of plasmid, in which expressed the enhanced green fluorescent protein （EGFP）reporter gene, the CYP17 gene, and a siRNA were co-transfeeted into HeLa cells, After transfeetion the silencing EGFP was measured by fluorescence microscopy and flow cytometry. Theca cells were isolated from five women with PCOS and five women without PCOS then were cultured in the presence or absence of the most effective siRNA at the most effective concentration. After 72 hours in euhured, the media was collected to evaluate androstenedione and progesterone production. The suppressive effect of the siRNA on CYP17 mRNA was determined by real-time reverse transcriptionpolymerase chain reaction analysis. Results： Following treatment with siRNA 1221, the expression of exogenous CYP17 in HeLa cells was reduced by up to 50%. The CYP17 mRNA expression and androstenedione production of thecal cells were slightly, but not significantly,reduced compared to cell exposed to the non-specific siRNA. Conclusion.RNAi may have applications as gene therapy for reducing hyperandrogenism associated with PCOS.

关 键 词：RNA干扰 细胞色素P450 17α-羟化酶/17 20裂解酶 雄激素合成 多囊卵巢综合征 卵泡膜细胞 

分 类 号：R711.75[医药卫生—妇产科学]