20β-羟基黄体酮的制备及孕激素样作用研究  

Study on preparation of 20β-hydroxyprogesterone and determination of pro-gesterone activity in vitro

作　　者：闫淑静 陈春丽[1] 谢湘云[1] 高晓黎[1,2] YAN Shujing;CHEN Chunli;XIE Xiangyun;GAO Xiaoli(College of Pharmacy,Xinjiang Medical University,Urumqi 830017,China;Xinjiang Key Laboratory of Active Components and Release Technology of Natural Medicines,Urumqi 830017,China)

机构地区：[1]新疆医科大学药学院,乌鲁木齐830017 [2]新疆天然药物活性组分与释药技术重点实验室,乌鲁木齐830017

出　　处：《新疆医科大学学报》2022年第6期666-671,共6页Journal of Xinjiang Medical University

基　　金：新疆维吾尔自治区重大科技专项项目(2107A01002)。

摘　　要：目的制备20β-羟基黄体酮(20β-Hydroxyprogesterone,20β-DHP),以大鼠子宫内膜基质细胞(Rat endometrial stromal cells,RESC)为研究载体,研究其孕激素样作用。方法化学合成20-羟基黄体酮(20-Hydroxyprogesterone,20-DHP),经半制备高效液相色谱分离制备20β-DHP,并对其进行含量测定。从雌性SD大鼠体内原代分离培养RESC,采用免疫荧光法对其进行鉴定、纯度检测,绘制生长曲线。RESC培养后分为空白组、阳性对照(黄体酮,Progesterone,P4)组、20β-DHP组、20α-DHP组,采用MTT法观察20β-DHP及其同分异构体20α-DHP对RESC增殖的影响,采用RT-qPCR法检测3种药物对孕酮受体(Progesterone receptor,PR)和增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)基因表达的影响。结果分离得20β-DHP单体,纯度为96.27%。原代分离培养的RESC的免疫荧光结果显示标志蛋白Vimentin阳性,纯度在90%以上,活力良好。MTT结果显示20β-DHP及其同分异构体20α-DHP均可促进RESC增殖,且呈剂量依赖性,与P4作用类似;RT-qPCR结果显示20β-DHP及其同分异构体20α-DHP均可上调PCNA基因表达,下调PR基因表达(P<0.05),20β-DHP仅在12 h内对PR表达具有下调作用。结论20β-DHP及其同分异构体20α-DHP对RESC具有类似P4的孕激素作用,但20β-DHP对PR的下调作用受时间影响。Objective 20β-Hydroxyprogesterone(20β-DHP)was prepared,and rat endometrial stromal cell(RESC)was used as research carrier to explore its progesterone-like effect.Methods 20-Hydroxyprogesterone(20-DHP)was chemically synthesized;20β-DHP was separated and prepared by semi-preparative with high performance liquid chromatography,and its content was determined.Primary RESCs were isolated and cultured from female SD rats,iden⁃tified and purified by immunofluorescence method,and the growth curves were drawn.Primary cultured RESCs were divided into the blank group,the positive control progesterone(P4)group,the 20β-DHP group,20α-DHP group,the effect of 20β-DHP and its isomer 20α-DHP on proliferation of RESC was observed by MTT method,and the effects of three drugs on progesterone receptor(PR)and proliferating cell nuclear antigen(PCNA)gene expression were detected by RT-qPCR.Results 20β-DHP monomer was isolated with purity of 96.27%.The immunofluorescence results of pri⁃mary isolated and cultured RESCs showed that marker protein Vimentin was positive with a purity of more than 90%and good viability.MTT results showed that both 20β-DHP and 20α-DHP can promote proliferation of RESCs in a dose-dependent manner and similar to effect of P4;RT-qPCR results showed that both 20β-DHP and 20α-DHP could up-regulate PCNA gene expression(P<0.05)and down-regulate PR gene expression(P<0.05),but 20β-DHP down-regulated PR expression only within 12h.Conclusion 20β-DHP and 20α-DHP had a progestogenic effect similar to P4 with RESC,but down-regulation of PR by 20β-DHP was affected by time.

关 键 词：20β-羟基黄体酮 同分异构体 大鼠子宫内膜基质细胞 孕酮受体 孕激素样作用 

分 类 号：R96[医药卫生—药理学]