不同分离方法对人卵巢颗粒细胞体外培养的影响

Effects of different isolation methods on the culture of human ovarian granulosa cell in vitro

目的比较人卵巢颗粒细胞不同分离方法。方法取体外受精-胚胎移植(IVF-ET)采卵时的卵泡液,用裂解法、密度梯度离心法、酶解法分离纯化并原代培养人卵巢颗粒细胞。比较不同的分离方法获得的卵巢颗粒细胞数量、形态及其对早期培养的影响。结果裂解法得到的颗粒细胞数较密度梯度离心法、酶解法多(P<0.005,P<0.001);比较3种分离方法获得的卵巢颗粒细胞的活性和孕激素分泌量差异无统计学意义;接种24 h后,酶解法得到的卵巢颗粒细胞存活量最多(P<0.05),裂解法得到的存活细胞最少(P<0.05);裂解法获得的卵巢颗粒细胞继续培养48、72及96 h均较培养24 h有明显细胞增殖(P<0.05),且与其他组存活的细胞量差异无统计学意义。结论裂解法得到的颗粒细胞数量多,细胞生长较好,操作简单,是较理想的分离人卵巢颗粒细胞的方法。

【Objective】 To compare the different isolation methods for the human granulosa cell.【Methods】 Follicular fluid was harvested from women undergoing IVF procedures.Human granulosa cells were obtained from follicular fluid by lysis treatment,density gradient centrifugation or enzyme hydrolysis method.Then they were cultured in the medium and the quantity,morphology and effects on early development were assessed.【Results】 Lysis treatment yielded the largest amount of granulosa cells compared to that of density gradient centrifugation and enzyme hydrolysis（P ＆lt;0.005,P ＆lt;0.001,respectively）.There were no difference in the cells viability and progesterone production by either method.Cultured after 24 h,the enzyme hydrolysis group obtained relatively largest amount of survived GCs（P ＆lt;0.05）;and the lysis group obtained lest amount.Then the cells by lysis treatment were more significant proliferation cultured after 48 h,72 h,96 h than that cultured after 24 h（P ＆lt;0.05）,and was no difference compared with other methods.【Conclusion】 Lysis treatment obtained the relatively largest amount of GCs and the GCs were grew better.This method that needs less protocol than others was an ideal isolation method for human ovarian granulosa cell.