牦牛IGF-Ⅱ干扰载体的构建及其转染对睾丸支持细胞的影响

Construction of IGF-Ⅱ shRNA Knockdown Vector and Its Effect on Sertoli Cells in Yak

旨在构建干扰胰岛素样生长因子(insulin-like growth factor-Ⅱ, IGF-Ⅱ)的重组质粒载体,研究IGF-Ⅱ对牦牛睾丸支持细胞的影响。本研究设计并合成针对牦牛IGF-Ⅱ的shRNA寡核苷酸,并将其克隆至pLKO.1质粒载体上,转染牦牛睾丸支持细胞后经嘌呤霉素筛选获得稳定的细胞株,并采用实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹法检测IGF-Ⅱ基因和蛋白的表达,采用细胞生长分析仪分析支持细胞的增殖凋亡情况。结果显示,干扰牦牛IGF-Ⅱ表达的pLKO.1质粒载体构建成功,其可在睾丸支持细胞中稳定表达,且能有效抑制IGF-Ⅱ基因的表达。与对照组相比,pLKO.1-shRNA2的干扰效率最显著,IGF-Ⅱ的表达量下调至19.1%(P<0.05),且pLKO.1-shRNA2可使内源性IGF-Ⅱ蛋白的表达量减少76.3%(P<0.05)。pLKO.1-shRNA2质粒转染及筛选得到的稳定细胞株培养48 h后的细胞分裂增殖活性显著低于对照组(P<0.05)。qRT-PCR结果显示,干扰内源性IGF-Ⅱ后,睾丸支持细胞中的IGF-Ⅰ和IGF-ⅡR基因表达出现了显著的上调(P<0.05),IGF-IR与Bcl-2的表达出现了显著的下调(P<0.05)。综上表明,牦牛IGF-Ⅱ干扰质粒构建成功,其转染至牦牛睾丸支持细胞有效抑制了IGF-Ⅱ的表达,改变了IGF-IR与Bcl-2的表达模式,潜在影响了牦牛支持细胞的分裂增殖,具体作用机制有待进一步研究。

The aim of this study was to construct shRNA recombinant vector interfering insulin-like growth factor-Ⅱ(IGF-Ⅱ), and study the effects of IGF-Ⅱ on the yak sertoli cells. The shRNA oligonucleotides targeting yak IGF-Ⅱ were designed and synthesized and it was cloned into pLKO.1 plasmid vector, after transfected into yak sertoli cells, a stable cell line was obtained by puromycin selection. The relative expressions of IGF-Ⅱ mRNA and protein were identified by qRT-PCR and Western blotting, respectively. The proliferation and apoptosis of sertoli cells were analyzed by proliferation measurement system. The results showed that the pLKO.1-shRNA vector interfering IGF-Ⅱ was successfully constructed, which was stably expressed in sertoli cells and could interfere the expression of yak IGF-Ⅱ effectively. Compared with the control group, the interference efficiency of pLKO.1-shRNA2 was the most significant, IGF-Ⅱ expression down-regulated to 19.1%(P<0.05), and could reduce the expression of endogenous IGF-Ⅱ protein by 76.3%(P<0.05). The stable cell line obtained by transfection and screening of pLKO.1-shRNA2 plasmid had significant lower activity of cell division and proliferation than that of control group after 48 h of culture(P<0.05). The results of qRT-PCR showed that the expressions of IGF-Ⅰ and IGF-ⅡR in sertoli cells were significantly up-regulated(P<0.05), and the expressions of IGF-IR and Bcl-2 were significantly down-regulated(P<0.05) after interfering IGF-Ⅱ. In conclusion, the interference plasmids of IGF-Ⅱ were successfully constructed. After interference plasmid was transfected into yak sertoli cells, it could effectively inhibit the expression of IGF-Ⅱ, change the expression pattern of IGF-IR and Bcl-2, and potentially affect the division and proliferation of yak sertoli cells, however, the specific regulation mechanism requires further studied.