摘要
目的:探讨Kisspeptin-10(KP-10)对绵羊卵泡颗粒细胞(FGCs)CCND1、CDK6、Bcl-2、THBS1、TNC和Bax基因表达的影响.方法:将分离培养的原代FGCs细胞在含不同浓度(0、10、100和1000 nmol/mL)KP-10培养基中培养,分别标记为CG组、EP-1组、EP-2组和EP-3组,采用CCK-8法检测FGCs的细胞活性,筛选出KP-10对FGCs增殖的最佳浓度与时间.采用RNA-seq技术进行转录组测序分析,应用实时荧光定量PCR(qPCR)和Western Blot方法进行验证.结果:与CG组比较,48 h时EP-2组FGCs细胞活力极显著增强(P<0.01).转录组测序显示CG组和KP组间存在202个差异表达基因,其中上调基因119个,下调基因83个.qPCR和Western Blot结果表明,KP组CCND1、CDK6、Bcl-2基因表达显著上调(P<0.05);Bax、THBS1、TNC基因表达显著下调(P<0.01;P<0.05),与转录组测序结果基本一致.Western Blot验证结果与qPCR检测结果表明,KP组BAX蛋白、THBS1蛋白、TNC蛋白表达水平极显著降低(P<0.01),CCND1蛋白、BCL-2蛋白和CDK6蛋白水平均显著升高(P<0.05或P<0.01).结论:KP-10可促进绵羊FGCs增殖及凋亡相关基因表达.KP可以降低BAX、THBS1、TNC mRNA及蛋白表达,提高CCND1、BCL-2、CDK6 mRNA及蛋白表达.
Objective:To investigate the effect of Kisspeptin-10(KP-10)on expressions of CCND1,CDK6,Bcl-2,THBS1,TNC and Bax genes in ovis aries follicular granulosa cells(FGCs).Method:Primary isolated and cultured FGCs cells were cultured in KP-10 medium with different concentrations(0,10,100 and 1000 nmol/mL)and labeled as CG group,EP-1 group,EP-2 group and EP-3 group,respectively.The cell activity of FGCs was detected by CCK-8 method.The optimal concentration and time of KP-10 for FGCs proliferation were screened,and transcriptome sequencing was performed by RNA-seq technology,and verified by real-time quantitative fluorescent PCR(qPCR)and Western Blot.Results:Compared with CG group,FGCs cell viability in EP-2 group was significantly enhanced at 48h(P<0.01).Transcriptome sequencing showed that there were 202 differentially expressed genes between CG and KP groups,among which 119 up-regulated genes and 83 down-regulated genes.The results of qPCR and Western Blot showed that the expressions of CCND1,CDK6 and Bcl-2 genes in KP group were significantly up-regulated(P<0.05).Bax gene expression was significantly down-regulated(P<0.01),THBS1,TNC gene expression was significantly down-regulated(P<0.05),which was basically consistent with the transcriptome sequencing results.Conclusion:KP-10 can induce expression of genes related to proliferation and apoptosis of FGCs in ovis aries.
作者
时小凤
袁肇方
黄一迪
巩转娣
SHI Xiao-feng;YUAN Zhao-fang;HUANG Yi-di;GONG Zhuan-di(Life Science and Eengineering College,Northwest Minzu University,Lanzhou 730030,China;Hospital,Northwest Minzu University,Lanzhou 730030,China)
出处
《西北民族大学学报（自然科学版）》
2023年第3期54-61,共8页
Journal of Northwest Minzu University(Natural Science)
基金
西北民族大学高原动物疾病创新团队建设项目(2020-88-07-3)
国家自然科学基金项目(31460684)。
