稳转hTERT绵羊睾丸细胞系的建立

Establishment of Stable Expression hTERT Testicular Primary Cell Line

羊睾丸原代细胞的体外培养是探究牛羊病毒致病机理的重要材料,实际科研工作中常受困于此类细胞传代性差、性质不稳定等缺点。外源性导入人端粒酶逆转录酶基因(hTERT)可以促使端粒酶活性的表达,延长细胞体外培养寿命是一种有效建立细胞永生化的方法。本试验中,通过RT-PCR获得hTERT。利用同源重组的技术,成功的构建了hTERT的真核表达质粒和慢病毒质粒。利用慢病毒表达系统,建立了稳转hTERT绵羊的睾丸细胞系。间接免疫荧光和蛋白免疫印迹试验研究结果均显示,hTERT基因已成功整合进入绵羊睾丸基因组中并稳定表达。第30代次的羊睾丸细胞生长较快,性状较稳定,表明已成功构建了具有永生化特性的绵羊睾丸细胞系,为草食动物病毒的相关研究提供重要的细胞模型。

The in vitro culture of primary sheep testicular cells is an important material to explore the pathogenic mechanism of virus infection of bovine and sheep.The actual scientific research work is often trapped by the shortcomings such as poor passage and unstable properties of these cells.Exogenous introduction of human telomerase reverse transcriptase gene(human telomerase reverse transcriptase gene,hTERT)can promote the expression of telomerase activity extend the lifespan of the cells in vitro,which is an effective method to establish cell immortalization.In this study,hTERT was amplified by RT-PCR and the eukaryotic expression plasmid and lentivirus plasmid of hTERT was constructed by homologous recombination technique.Then,the stable sheep testicular cell lines were established by using the lentivirus expression system.The results of indirect immunofluorescence and Western blotting showed that the hTERT gene had been integrated into the sheep testicular genome with stable expression.The sheep testicular cells of the 30th passage grew faster and their characters were relatively stable,which indicated that an immortalized sheep testicular cell line was established which could provide an important cell model for the related research of herbivore virus.