L-半胱氨酸对绵羊卵巢颗粒细胞增殖、凋亡和类固醇激素分泌的影响

Effect of L-cysteine on Proliferation,Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells

旨在探讨L-半胱氨酸(L-Cys)对体外培养绵羊卵巢颗粒细胞(GCs)增殖、凋亡和类固醇分泌的影响。本研究采集1~1.5岁龄体重相近饲养条件相同的30只健康性成熟小尾寒羊母羊新鲜卵巢,从优势卵泡(2~8 mm)中收集GCs,随机分为5组,每组6个重复,各组添加L-Cys浓度分别为0、100、300、500和700μmol·L^(-1),培养48 h后用CCK-8试剂盒进行细胞增殖活力测定,Annexin V-FITC/PI流式细胞仪检测GCs的凋亡情况,采用ELISA检测GCs培养液上清中孕酮和雌二醇的分泌水平并采用实时荧光定量PCR和Western blot分析L-Cys对绵羊颗粒细胞增殖相关基因(PCNA、CCND2、CCNB 1)、凋亡相关基因(BAX、Caspase-3、Bcl-2)和类固醇分泌相关基因的(STAR、3β-HSD、CYP 11A1、CYP 19A1)mRNA和蛋白表达的影响。结果显示,100、300和500μmol·L^(-1)组的L-Cys均可显著促进绵羊GCs的增殖活力(P<0.05),并显著抑制其凋亡率(P<0.05)。添加300μmol·L^(-1)的L-Cys显著抑制了孕酮(P 4,P<0.05)的分泌。进一步研究发现,300μmol·L^(-1)和500μmol·L^(-1)组的L-Cys可以上调增殖相关基因(PCNA、CCND2、CCNB 1)mRNA表达水平,其中300μmol·L^(-1)组的增殖蛋白(PCNA、CCND2、CCNB1)表达量显著上升(P<0.05);100μmol·L^(-1)和300μmol·L^(-1)组显著下调凋亡相关基因(BAX、Caspase-3)mRNA(P<0.05)及其蛋白表达水平(P<0.05)。添加300μmol·L^(-1)L-Cys显著降低了类固醇相关基因(STAR、3β-HSD)mRNA及蛋白表达水平(P<0.05),添加100μmol·L^(-1)和300μmol·L^(-1)L-Cys显著升高了类固醇相关基因(CYP 11A1、CYP 19A1)mRNA(P<0.05)及其蛋白表达水平(P<0.05)。综上,L-Cys通过上调基因PCNA、CCNB1、CCND2、Bcl-2 mRNA及其蛋白表达、下调基因BAX、Caspase-3 mRNA及其蛋白表达,促进绵羊GCs增殖,抑制其凋亡;L-Cys通过下调基因STAR、3β-HSD mRNA及其蛋白表达以及上调基因CYP 11A1 mRNA及其蛋白表达抑制P 4的分泌。

This study was conducted to investigate the effects of L-cysteine(L-Cys)on proliferation,apoptosis and the secretion of steroid hormones(progesterone and estrogen,P 4 and E 2)in ovine ovarian granulosa cells(GCs).Thirty sexually mature ewes aged between 1-1.5 years with the similar body weight and the same feeding condition were selected,and the fresh ovaries were collected.Then,GCs were collected from the dominant follicles(2-8 mm),and the GCs were randomly divided into 5 groups with 6 replicates per group.GCs were treated with 0,100,300,500 and 700μmol·L^(-1)L-Cys for 48 h.The cell proliferation,apoptosis assay and the secretion of steroid hormones were measured using the CCK-8 kit,Annexin V-FITC/PI flow cytometry and ELISA,respectively.Real-time quantitative PCR and Western blot were used to analyze the mRNA and protein expression of the cell proliferation related genes(PCNA,CCND2,CCNB 1),apoptosis-related genes(BAX,Caspase-3,Bcl-2)and steroid-secretion-related genes(STAR,3β-HSD,CYP 11A1,CYP 19A1).The results showed that 100,300 and 500μmol·L^(-1)L-Cys significantly promoted the cell viability(P<0.05)and significantly inhibited the apoptotic rate(P<0.05)of GCs.The addition of 300μmol·L^(-1)L-Cys significantly inhibited the secretion of P 4(P<0.05).The mRNA expression of proliferation-related genes(PCNA,CCND2,CCNB 1)were upregulated in the 300μmol·L^(-1)and 500μmol·L^(-1)L-Cys groups,while the protein expression of proliferation-related proteins(PCNA,CCND2,CCNB1)were increased in the 300μmol·L^(-1)L-Cys group(P<0.05).The mRNA and protein expressions of apoptosis-related genes(BAX,Caspase-3)were significantly downregulated in 100μmol·L^(-1)and 300μmol·L^(-1)L-Cys groups(P<0.05).The mRNA and protein expressions of the steroid-related genes(STAR,3β-HSD)were significantly decreased in 300μmol·L^(-1)L-Cys group(P<0.05),but the mRNA and protein expressions of the steroid-related genes(CYP 11A1,CYP 19A1)were significantly increased in 100μmol·L^(-1)and 300μmol·L^(-1)L-Cys groups(P<0.05).In summary,L-Cys promoted cell proliferation and inhibited the apoptosis of ovine ovarian GCs by upregulating the mRNA and protein expressions of PCNA,CCNB1,CCND2,Bcl-2 and downregulating the mRNA and protein expressions of BAX,Caspase-3;L-Cys inhibited secretion of P 4 by downregulating the mRNA and protein expressions of STAR,3β-HSD and upregulating the mRNA and protein expressions of CYP 11A1.