促黄体素(LH)对猪卵巢颗粒细胞细胞周期及类固醇激素分泌的影响

摘要

To investigate the effect of luteinizing hormone ( LH) on growth and steroid secretion of porcine granulosa cells, dominant follicles from porcine ovaries collected from a local slaughterhouse were selected based on the diameter ( ＞6 mm) . Granulosa cells ( GCs) obtained from porcine dominant follicles were cultured in the medium supplemented with different doses of LH ( 0 IU/ml, 2 IU/ml and 4 IU/ml) . Cell cycle and steroid secretion were analyzed by flow cytometry ,RIA and real-time fluorescence quantitative PCR, respectively. The results showed that the high dose of LH (4 IU/ml) significantly decreased the percentage of GCs from 92. 79％ to 86. 12％ at G0/G1 stage, increased the percentage of GCs from 1. 97％ to 8.43％ at S stage, and significantly improved the expression of cyclin-dependent kinase-4 ( CDK4) and Cyclin D2. The high dose of LH (4 IU/ml) significantly lowered the concentrations of estradiol ( E2) and progesterone ( P4 ) in the medium, and down-regulated the expression of P450 aromatase ( P450arom) as well as cholesterol side-chain cleavage enzyme P450 ( P450scc). The results indicate that LH regulates the growth and steroid secretion of porcine granulosa cells.%为揭示促黄体素对猪卵巢颗粒细胞生长及类固醇分泌的影响,从当地屠宰场采集猪卵巢,选择表面直径大于6 mm的卵泡(优势卵泡)作为试验材料,将从其中获得的颗粒细胞进行体外培养,并在培养基中添加不同浓度(0 IU/ml、2 IU/ml和4 IU/ml)的促黄体素(LH),通过流式细胞仪、放射性免疫和定量PCR分别检测细胞周期的情况、类固醇激素的分泌和相关基因mRNA的表达.结果显示:高剂量的LH(4 IU/ml)可使G0/G1期的细胞数量由92.79%减少到86.12%;S期细胞数量由1.97%增加到8.43%,并能显著提高细胞周期依赖性蛋白激酶-4(CDK4)和细胞周期素-D2(Cyclin D2)的表达水平;高剂量的LH(4 IU/ml)能显著减少培养基中E2和P4的浓度,并能降低芳香化酶(P450arom)和胆固醇侧链裂解酶(P450scc)的表达.结果表明LH对猪卵巢颗粒细胞生长和类固醇激素分泌有调控作用.