摘要
目的:建立有效分离、提纯及培养人卵丘颗粒细胞的方法,并与人壁层颗粒细胞的体外培养相比较。方法:收集卵胞浆内单精子显微注射取卵时的卵泡液和直接机械分离卵母细胞所得的卵丘颗粒细胞复合物,直接将卵丘颗粒细胞复合物接种于培养皿中培养。用密度梯度离心法分离卵泡液中的人壁层颗粒细胞。用免疫荧光法检测卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)的表达;用CCK-8法检测细胞生长曲线;用ELISA检测这2种颗粒细胞分泌雌激素的能力。结果:直接法所得人卵丘颗粒细胞培养24 h后可贴壁,体外培养生长状态与人壁层颗粒细胞相似;免疫荧光检测显示两者均表达FSHR;CCK-8实验结果表明,两者体外培养生长曲线相似;ELISA结果显示两者分泌雌激素能力相当。结论:利用机械切割获得人卵子周围卵丘颗粒细胞复合物直接培养的方法操作简单,获得的人卵丘颗粒细胞具有与人壁层颗粒细胞相似的生长状态、生长曲线以及雌激素分泌能力,可作为人颗粒细胞亚群体外培养方法的补充。
AIM: To establish an effective method for purification and culture of human cumulus cells( CCs)in vitro,and to compare the characteristics between CCs and mural granulosa cells( MGCs). METHODS: Follicular fluid and cumulus complex from the patients undergoing intracytoplasmic sperm injection were collected. CCs were mechanically cut from cumulus complex and then directly inoculated on a Petri dish,and MGCs were obtained from follicular fluid through density gradient centrifugation. The expression of follicle stimulating hormone receptor( FSHR) was determined by immunofluorescence. The cell growth curves were measured by CCK-8 assay. The secretion of estrogen was detected by ELISA. RESULTS: After incubated for 24 h,the adherence of CCs was observed. CCs and MGCs had similar growth characteristics and FSHR expression. The similar cell growth curves were observed by CCK-8 assay and the results of ELISA showed that they had comparable secretion of estrogen. CONCLUSION: Direct culture of CCs mechanically cutting from cumulus complex is an effective method. CCs had similar growth characteristics,growth curves and secretion of estrogen to MGCs cultured in vitro and could be a substitutive source of granulosa cell subsets.
引文
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