体外颗粒细胞培养中抗苗勒管激素与卵泡刺激素受体mRNA表达的相关性  

Correlation between anti-Mullerian hormone and follicle-stimulating hormone receptor mRNA expression in granulosa cells in vitro

作　　者：张迪[1] 桑丽英[2] 崔蓉[2] 苗竹林[2] 钟兴明[2] 韦相才[2] 

机构地区：[1]暨南大学附属第一医院妇产科,广州510632 [2]广东省计划生育科学技术研究所,广州510600

出　　处：《广东医学》2012年第7期883-886,共4页Guangdong Medical Journal

基　　金：广东省科技计划项目(编号:2009B030801270);广东省自然科学基金项目(编号:10151008010000000)

摘　　要：目的体外培养小鼠原代颗粒细胞培养液中,加入不同浓度的抗苗勒管激素(AMH),比较各组间小鼠颗粒细胞上卵泡刺激素受体(FSHR)mRNA的表达情况,探讨体外培养颗粒细胞中AMH与FSHR mRNA表达的关系。方法选取4～5周的昆明雌性小鼠,腹腔注射孕马血清促性腺激素,48～54 h后颈椎脱臼法处死小鼠,取双侧卵巢,采用机械分离法释放颗粒细胞,胰蛋白酶消化细胞,低速离心分离细胞,用含15%胎牛血清的DMEM/F12培养基置于37℃、5%CO2培养箱中培养。显微镜观察细胞形态,流式细胞术分离细胞,并用免疫荧光法对所培养的颗粒细胞进行鉴定。同时在体外培养的小鼠原代颗粒细胞培养液中加入不同浓度(5、10、20、30、40ng/mL)的AMH,测定每组培养基中AMH的终浓度,并用荧光定量PCR技术检测每组颗粒细胞中FSHR mRNA的表达。结果分离培养的颗粒细胞纯度大于80%;随AMH浓度加大,小鼠颗粒细胞中FSHR mRNA的表达逐渐降低,当AMH浓度为5和10 ng/mL时,与对照组比较差异有统计学意义(P<0.05),且5 ng/mL浓度组颗粒细胞上FSHR mRNA的表达量最高。结论用机械分离加胰酶消化加低速离心能得到纯度较高活性很好的颗粒细胞,用FSHR鉴定颗粒细胞是一种简便易行的方法,AMH浓度与颗粒细胞上FSHR mRNA表达呈一定负相关,表明AMH有可能通过抑制FSHR表达降低FSH敏感性,从而参与卵巢早衰的病理生理过程。Objective To investigate the correlation between anti-Mullerian hormone（AMH） and follicle-stimulating hormone receptor（FSHR） mRNA expression in the cultured granulosa cells in vitro.Methods Kunming female mice were peritoneally injected with pregnant mare serum gonadotropin（PMSG） and sacrificed 48 to 54 hours after injection for collection of ovarian.Granulosa cells were isolated by mechanical method,trypsin digestion and low-speed centrifugation.Granulosa cells were diluted and incubated in DMEM/F12 medium containing 15% of fetal bovine serum at 37℃ in water-saturated environment of 5% CO2.Cell morphology,flow cytometry and immunofluoroscence were used for identification of ovarian granolosa cell.The primary granolosa cells were exposed to different concentrations of AMH.Real time-PCR was applied to analyze the FSHR mRNA expression in the ovarian granulosa cells.Results Cell purity was more than 80%.AMH induced concentration-dependent down-regulation of FSHR mRNA expression,and showed significant effect at 5 and 10 ng/mL.Conclusion granulosa cells can be obtained by mechanical method combined with trypsin digestion and brachytely centrifugation.FSHR staining provides quick and convenient approach in identifications of granulosa cells.AMH down-regulates FSHR mRNA in granulosa cells,suggesting a possible mechanism of pathogenesis of premature ovarian failure.

关 键 词：颗粒细胞 抗苗勒氏管激素 卵泡刺激素受体 卵巢早衰 

分 类 号：R711.75[医药卫生—妇产科学]