【摘要】 探讨用密度梯度离心法快速、有效地分离大鼠卵泡膜细胞.选取23~25 d雌性大鼠卵巢,用Percoll密度梯度离心法将卵泡膜细胞分离纯化,3β-羟基类固醇脱氢酶(3β-hydroxysteroid dehydrogenase,3β-HSD)组织化学染色用于卵泡膜细胞纯度检测.分别用0.1 U/mL和1.0 U/mL卵泡刺激素(Follicle-stimulating hormone,FSH)及黄体生成素(luteinizing hormone,LH)处理细胞,无血清培养48 h后,酶联免疫法检测培养液中雄烯二酮和雌二醇的水平.分离所得细胞中,3β-HSD染色阳性细胞与总细胞数之比大于90%;LH组的雄烯二酮水平显著高于对照组和FSH组(P<0.05),LH组中1.0 U/mL组的雄烯二酮水平又高于0.1 U/mL组.各组均未检测到雌二醇及孕酮.3β-HSD组织化学染色可快速有效地检验所分离的卵泡膜细胞的纯度,分离所得的卵泡膜细胞可对LH产生反应,且其中几乎没有混杂颗粒细胞.更多还原

【Abstract】 To explore a rapid,effective method to separate rat ovary theca cells by density gradient centrifu-gation.The ovaries of 23~25-days-old were collected.The theca cells were separated by Percoll density gra-dient centrifugation procedure.The purity of theca cell was examined by 3β-hydroxysteroid dehydrogenase(3β-HSD) staining.The separated cells were cultured for 48 h under serum-free cell culture condition with 0.1 U/mL or 1.0 U/mL FSH or LH,then the concentration of androstenediol and estradiol of the culture medium were measured by enzyme-linked immunosorbent assay.In the separated cells,the ratio of 3β-HSD positive cells to the total cells was more than 90%.The level of androstenediol in LH cultured media was significantly higher than that of the FSH cultured and control media(P<0.05);In LH cultured media,the level of androstenediol in 1.0 U/mL LH was significantly higher than that of in 0.1 U/mL LH.The level of androstenediol was no difference between FSH cultured media and control media(P>0.05).3β-HSD staining could be applied as a rapid and effective method to examine the purity of the separated theca cells.The procedure described by this study can separate rat ovary theca cells which is reacted with LH stimulation and barely mixed with contaminated graunlosa cells.更多还原