HSP60对天祝白牦牛原代培养的支持细胞增殖的影响

发布时间：2018-03-18 01:36

本文选题：HSP　切入点：天祝白牦牛　出处：《核农学报》2017年08期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:HSP60 plays an important role in regulating testicular development and spermatogenesis. Sertoli cells are the only somatic cells in the testis that are directly associated with spermatogenic cells. In order to study the effect of HSP60 on the proliferation of testicular Sertoli cells of white yak, primary Sertoli cells of white yak were isolated and cultured by combined enzyme digestion. The cells were identified by immunofluorescence staining and Feulgen staining after purification by differential adherent method and Tris-HCL hypotonic buffer solution. 96.2% high purity Sertoli cells were obtained by flow cytometry. HSP60 overexpression vector pIRES2-EGFP-HSP60 was constructed and siRNA was synthesized to silence HSP60. after transient transfection of Sertoli cells, it was found that HSP60 mRNA in overexpression group was up-regulated at 244872 h. The proliferative efficiency of Sertoli cells in the over-expressed HSP60 group was significantly higher than that in the blank control group and the blank plasmid group, and the proliferation of Sertoli cells in the over-expressed HSP60 group was significantly higher than that in the blank control group and the blank plasmid group. The proliferative rate of Sertoli cells in the silencing HSP60 group was lower than that in the blank control group and the negative control group, but the difference was not significant. The results showed that the proliferation marker gene, cyclin D1) and proliferating cell nuclear antigen (PCNA), were detected by qPCR. The expression of cyclin D1 gene in over-expressed HSP60 group was significantly higher than that in blank control group and empty plasmid group at 48 h, and was significantly higher than that in blank control group and empty plasmid group at 72 h. The expression of cyclin D1 gene and PCNA gene in the silencing HSP60 group was lower than that in the blank control group and the negative control group after 72 h. The effect of HSP60 on the proliferation of testicular Sertoli cells in white yak was positively regulated. The results of this study laid a theoretical foundation for further study of the effects of HSP60 on testicular development and spermatogenesis in white yak.
【作者单位】：甘肃农业大学动物医学院;甘肃农业大学动物科技学院;
【基金】：“十二五”农村领域国家科技计划项目(2013AA02505-3)
【分类号】：S823.85

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