HSP60对天祝白牦牛原代培养的支持细胞增殖的影响

HSP60对动物的睾丸发育、精子发生起着重要的调控作用。支持细胞是睾丸内唯一与生精细胞直接发生联系的体细胞,对精子发生、成熟等过程有直接影响。为研究HSP60对白牦牛睾丸支持细胞增殖的调控作用,本试验通过组合酶消化法分离培养白牦牛原代支持细胞,通过差速贴壁法和Tris-HCL低渗缓冲液进行纯化处理,运用免疫荧光染色和Feulgen染色鉴定细胞,经流式细胞仪检测得到96.2%高纯度支持细胞。构建HSP60过表达载体p IRES2-EGFP-HSP60,合成靶向siRNA沉默HSP60,瞬时转染支持细胞后,经qPCR检测发现过表达组HSP60 mRNA在24、48、72 h等时间点均上调,沉默组HSP60 mRNA在24、48、72 h等时间点均下调。MTS试验检测细胞的增殖情况,过表达HSP60组中的支持细胞增殖效率各时间点均显著高于空白对照组和空质粒组;沉默HSP60组中支持细胞的增值率各时间点均低于空白对照组和阴性对照组,但差异不显著。qPCR检测细胞增殖标志基因细胞周期蛋白D1(cyclin D1)和增殖细胞核抗原(PCNA),结果表明,过表达HSP60组的cyclin D1基因在48 h时的表达显著高于空白对照组和空质粒组,PCNA基因的表达在24、48、72 h时均显著高于空白对照组和空质粒组;沉默HSP60组cyclin D1基因和PCNA基因的表达在24、48、72 h时均低于空白对照组和阴性对照组,但差异不显著。综上,HSP60在白牦牛睾丸支持细胞增殖调控中的作用是正向调控。本试验结果为进一步研究HSP60对白牦牛睾丸发育和精子发生的影响奠定了理论基础。

Effect of HSP60 on Proliferation of Primary Cultured Tianzhu White Yak Sertoli Cells

HSP60 plays an important role in regulating the development of testis and spermatogenesis.Sertoli cell is the only somatic cell in testis that have direct influence on germ cells,which would affect spermatogenesis and sperm mature.In order to study the effect of HSP60 on proliferation of white yak sertoli cells,the sertoli cells were isolated and purified by using enzymatic digestion,differential adhesion and Tris-HCL buffer,and identified by immunofluorescence staining and Feulgen staining.The results showed that the purity of sertoli cells were 96.2％ by flow cytometry.The over-expression vector pIRES2-EGFP-HSP60 and siRNA-HSP60 was synthesized.After transient transfection,the expression of HSP60 mRNA was up-regulated in over-expression groups and down-regulated in silent groups at 24、48、72h.The cell proliferation detected by MTS showed that the over-expression groups were significantly higher than the negative control (NC) at 24、48、72h.The silence groups were lower than NC at 24、48、72h,but the difference was not significant.qPCR showed that in over-expression groups the expression of cyclin D1 gene at 48h was significantly higher than NC and PCNA gene at24、48、72h were significantly higher than NC.In silence groups,the expression of cyclin D1 gene and PCNA gene at 24、48、72h were lower than NC,but the difference were not significant.This study suggested that the regulation of HSP60 gene in white yak sertoli cell of cell proliferation was positive.This study provides the theoretical foundation for the effect of HSP60 on white yak testis development and spermatogenesis.