黄素化颗粒细胞分离提纯方法的比较研究  

Comparison of different isolation and purification methods of human ovarian granulosa cells

作　　者：周小丹 罗金[1] 漆倩荣[1] 谢青贞[1] 钟方圆 梅忆媛 张祎明 ZHOU Xiao-dan;LUO Jin;QI Qian-rong;XIE Qing-zhen;ZHONG Fang-yuan;MEI Yi-yuan;ZHANG Yi-ming(Center for Reproductive Medicine,Renmin Hospital of Wuhan University,Hubei Clinic Research Center for Assisted Reproductive Technology & Embryonic Development,Wuhan 430060)

机构地区：[1]武汉大学人民医院生殖医学中心湖北省辅助生殖与胚胎发育医学临床研究中心

出　　处：《生殖医学杂志》2019年第6期661-666,共6页Journal of Reproductive Medicine

基　　金：国家自然科学基金面上项目(81471456)

摘　　要：目的 比较不同人卵巢颗粒细胞分离提纯方法的效果。方法 收集体外受精-胚胎移植(IVF-ET)患者取卵后的卵泡液,分别采用密度梯度离心法、红细胞裂解法、密度梯度离心法+红细胞裂解法分离、提取黄素化颗粒细胞,比较3种方法获取的细胞数量、存活率、纯度以及生长、贴壁情况。结果 密度梯度离心法获取的颗粒细胞数量最低,但体外培养生长状态良好且细胞贴壁状态最佳;红细胞裂解法获取的颗粒细胞数量均多于其他两种方法(P<0.05),但细胞存活率低,体外培养生长状态欠佳、细胞贴壁差;卵泡刺激素受体(FSHR)细胞免疫组化及荧光鉴定显示3种方法获取的颗粒细胞纯度均>90%。结论 密度梯度离心法获取的细胞数目少,但生长状态及贴壁情况最佳,是分离提纯颗粒细胞较为理想的选择方法。Objective : To compare different methods for isolation and purification of human granulosa cells (GCs). Methods : Follicular fluid was harvested after ovary extraction from the women undergoing IVF-ET. The density gradient centrifugation,red cell lysis treatment,and density gradient centrifugation plus red cell lysis treatment were respectively used for separating human GCs. The quantity,viability,purity,growth behavior and adherence of GCs retrieval were compared among the three methods. Results : Density gradient centrifugation obtained the least amount of GCs,yet accompanied with good growth behavior and adherence in vitro culture. Red cell lysis treatment led to the most amount of GCs ( P <0.05),but the worst growth status and adherence. Purity of GCs was over 90% in all methods,which confirmed by FSHR immunohistochemical and immunofluorescence methods. Conclusions : The density gradient centrifugation can obtain the least amount of GCs,but with the best growth behavior and adherence,which is an ideal isolation method for human ovarian GCs.

关 键 词：颗粒细胞 细胞分离 原代培养 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]