人卵巢壁层颗粒细胞和卵丘细胞体外分离及原代培养的比较

Comparison of isolation and primary culture in vitro between human ovarian mural granulosa cells and cumulus cells

目的:为建立合适卵巢功能研究的细胞模型,本研究比较了人卵巢壁层颗粒细胞(mural granulosa cells,MGCs)与卵丘细胞(cumulus cells,CCs)体外分离及原代培养的效果。方法:收集16例行辅助生殖技术治疗患者取卵日的卵泡液及其卵丘-卵母细胞复合物(223个),采用密度梯度离心法分离MGCs,机械切割法+酶解法分离CCs。应用台盼蓝染色法比较CCs与MGCs的细胞总数和存活率,通过流式细胞术检测卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)的表达来鉴定CCs与MGCs的细胞纯度,qPCR和Western blot方法检测微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、P62和Bax mRNA和蛋白的表达水平。结果:与MGCs相比,CCs的分离耗时少,细胞存活率高(P<0.05),体外培养的细胞延展性好;流式细胞术显示分离后的CCs与MGCs的FSHR表达量分别为(92.23±2.66)%和(81.33±6.57)%,差异具有显著性(P<0.05);CCs中LC3的mRNA表达水平显著低于MGCs(P<0.01),Bax的mRNA表达水平显著高于MGCs(P<0.05),CCs与MGCs的P62 mRNA表达水平没有显著差异(P>0.05),LC3、P62和Bax蛋白在CCs与MGCs中的表达差异与mRNA一致。结论:机械切割法+酶解法分离CCs方法简便,收集细胞的纯度高,细胞状态良好,可作为卵巢功能研究的细胞模型。

AIM:To establish a suitable cell model for the study of ovarian function through comparing the isolation and primary culture effect of human ovarian mural granulosa cells(MGCs)and cumulus cells(CCs).METHODS:The follicular fluid of 16 patients who underwent assisted reproductive technique and their cumulus oocyte complexes(n=223)were collected.Density gradient centrifugation was used to isolate the MGCs and the methods of mechanical cutting plus enzyme hydrolysis were used to isolate the CCs.The cell counts and survival rates were analyzed by trypan blue staining and the expression of follicle stimulating hormone receptor(FSHR)was analyzed by flow cytometry to identify the purity.The expression of microtubule-associated protein 1 light chain 3(LC3),P62 and Bax at mRNA and protein levels was determined by qPCR and Western blot,respectively.RESULTS:There had less isolation time,higher survival rate(P<0.05)and better tractility in vi⁃tro of CCs compared with MGCs.The results of flow cytometry showed that the FSHR expression of CCs and MGCs after isolation was(92.23±2.66)%and(81.33±6.57)%,respectively,with significant differences(P<0.05).The mRNA level of LC3 in CCs was significantly lower than that in MGCs(P<0.01),and the mRNA level of Bax was significantly higher than that in MGCs(P<0.05).There was no significant difference in P62 mRNA expression between CCS and MGCs(P>0.05).The difference of protein expressions of these molecules in the 2 kinds of cells were consistent with that in mRNA.CONCLUSION:Mechanical cutting method plus enzyme hydrolysis is a simple way to isolate the CCs,with high purity and good cellular state in vitro,which can be used as a cell model for ovarian function research.