GDF9对绵羊卵丘细胞增殖的影响

Effect of GDF9 on Sheep Cumulus Cell Proliferation

本试验旨在探究生长分化因子9(GDF9)对卵丘细胞扩展相关基因和激素受体基因表达量及激素分泌的影响,为GDF9在绵羊卵泡发育中的作用提供依据。以绵羊卵丘细胞为研究对象,通过在低血清细胞培养液中添加不同浓度(0、50、100、200、400 ng/mL)的GDF9,培养绵羊卵丘细胞48 h后,提取细胞总RNA,利用实时荧光定量PCR技术,以β-actin为内参基因,检测卵丘细胞扩展相关基因透明质酸合酶2(HAS2)、前列腺素内过氧化物合酶2(PTGS2)、穿透素3(PTX3)及激素受体相关基因卵泡刺激素受体(FSHR)、促黄体生成素受体(LHR)和雌激素受体(E2R)的mRNA相对表达量;利用酶联免疫吸附法(ELISA)测定培养液中卵丘细胞分泌的雌二醇(E2)和孕酮(P4)含量。结果显示:在细胞培养液中添加200 ng/mL GDF9时,HAS2、PTX3、FSHR、E2R和LHR的mRNA相对表达量极显著高于对照组与其他处理组(P<0.01);PTGS2 mRNA相对表达量极显著高于对照组、50和400 ng/mL GDF9组(P<0.01),显著高于100 ng/mL GDF9组(P<0.05)。当添加400 ng/mL GDF9时,各基因mRNA相对表达量均极显著低于200 ng/mL GDF9组(P<0.01);E2分泌量极显著高于对照组与50 ng/mL GDF9组(P<0.01),显著高于100 ng/mL GDF9组,与200 ng/mL GDF9组差异不显著(P>0.05)。100、200和400 ng/mL GDF9组P4分泌量显著高于对照组(P<0.05),与50 ng/mL GDF9组没有显著差异(P>0.05),且3组之间差异不显著(P>0.05)。综上所述,GDF9能够促进绵羊卵丘细胞扩展,并参与绵羊卵丘细胞激素分泌的调控。

The purpose of this study was to investigate the effect of growth differentiation factor 9(GDF9)on the gene expression of cumulus cells expansion and hormone receptors as well as hormone secretion,in order to provide evidence for the role of GDF9 in the development of sheep cumulus cells.Sheep cumulus cells were used as the research object in this study,and were cultured for 48 h by adding different concentrations(0,50,100,200,400 ng/mL)GDF9 to low serum cell culture medium.Total RNA were extracted from the cells,usingβ-actin as the reference gene,Real-time quantitative PCR technology were used to detect the cumulus cells expansion related genes hyaluronic acid synthase gene 2(HAS2),prostaglandin lead oxide synthase 2(PTGS2),pentraxin 3(PTX3)and hormone receptor genes follicle-stimulating hormone receptor(FSHR),luteinizing hormone receptor(LHR)and estrogen receptors(E2R).Using the enzyme-linked immunosorbent assay(ELISA)method to test the content of E2 and P4.The results showed that HAS2,PTX3,FSHR,E2R and LHR mRNA relative expression of 200 ng/mL GDF9 group was extremely significantly higher than the control group and other GDF9 groups(P<0.01),PTGS2 mRNA relative expression was extremely significantly higher than the control group and 50,400 ng/mL GDF9 groups(P<0.01),and significantly higher than 100 ng/mL GDF9 group(P<0.05).When added 400 ng/mL GDF9,the relative mRNA expression of all the mentioned-above genes were all extremely significantly lower than that of the 200 ng/mL GDF9 group.Moreover,the E2 secretion level was extremely significantly higher than that of the control group and 50 ng/mL GDF9 group(P<0.01),significantly higher than that of the 100 ng/mL GDF9 group(P<0.05),while had no significant difference from the 200 ng/mL GDF9 group(P>0.05).When added 100,200 and 400 ng/mL GDF9,the concentration of P4 was significantly higher than the control group(P<0.05),and there was no significant difference from the 50 ng/mL group(P>0.05),and there was no significant difference between 100,200 and 400 ng/mL GDF9 groups(P>0.05).To sum up,GDF9 could promote the expansion of sheep cumulus cells and participated in the regulation of hormone secretion of sheep cumulus cells.