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

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。

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

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.