人卵巢颗粒细胞体外分离培养后连续传代的观察

Observation on serial passage of human ovarian granulosa cells after isolation and culture in vitro

目的观察两种方法分离的人卵巢颗粒细胞(GCs)体外培养后的连续传代情况。方法选取行辅助生殖技术治疗的不孕患者,收集促排卵后穿刺取卵获得的卵泡液(FF),分别用密度梯度离心法和红细胞裂解法分离人GCs,进行体外培养、扩增及连续传代,并用免疫荧光法对原代及传代后GCs进行鉴定。结果红细胞裂解法获得的GCs数量较密度梯度离心法显著增多(P<0.05),密度梯度离心法分离的GCs的24 h贴壁率显著高于红细胞裂解法(P<0.05)。两种方法分离的GCs均在7 d出现短梭形改变,10 d左右开始退化。两种方法分离的原代GCs在第3~5天为快速增殖期,第7天细胞增殖达到顶峰,第10天左右细胞量下降,每个时间点密度梯度离心法处理的细胞量均显著高于红细胞裂解法(P<0.05);传代后细胞1~5 d为快速增殖期,7 d左右达顶峰,10 d左右下降,各时间点两种分离方法所获GCs传代培养的细胞量均无统计学差异(P>0.05)。两种方法分离的GCs均可进行连续传代,细胞形态无明显差异,且连续传代后的GCs仍表达卵泡刺激素受体(FSHR)。结论密度梯度离心法较红细胞裂解法可获得生长情况更为良好的原代GCs;两种分离方法所获GCs均可体外连续传代。

Objective:To observe the status of serial passage of human ovarian granulosa cells(GCs)isolated by two methods.Methods:Follicular fluids were collected during transvaginal oocyte retrieval from infertility patients undergone assisted reproductive therapy.Human GCs were respectively isolated by density gradient centrifugation and red blood cell lysis method for in vitro culture,amplification and serial passage.Immunofluorescence was used to identify the primary and post-passage GCs.Results:The number of cells obtained by the red blood cell lysis method was significantly more than that of the density gradient centrifugation method(P<0.05).The 24 hours attachment rate of GCs isolated by the density gradient centrifugation method was significantly higher than that of the red blood cell lysis method(P<0.05).Both groups of GCs became short spindle in 7 days and began to degenerate at about 10 days.The primary GCs isolated by the two methods had a rapid proliferation stage on the 3^(rd)-5^(th) day,the cell proliferation reached the peak on the 7^(th) day,and the cell amount decreased on the 10^(th) day.At each time point,the number of GCs isolated by density gradient centrifugation was significantly higher than that by red blood cell lysis method(P<0.05).The cells proliferated rapidly from 1 to 5 days after passage,peaked at about 7 days and decreased at about 10 days.There was no significant difference in the number of GCs cells obtained by the two separation methods at each time point(P<0.05).GCs isolated by the two methods could be serially passaged,and there was no significant difference in cell morphology.After serial passage,GCs still expressed FSH receptor(FSHR).Conclusions:Compared with the red blood cell lysis method,the density gradient centrifugation method can obtain the primary GCs with better growth,and the GCs isolated by both methods can be serially passaged in vitro.