慢病毒载体介导的RNA干扰Akt2表达抑制猪前体脂肪细胞分化

丝氨酸/苏氨酸蛋白激酶2(serine/threonine protein kinase2,Akt 2)是胰岛素信 号通路的关键基因, 与细胞生存和癌症的发生密切相关. 但Akt2在前体脂肪细胞分化中 的作用仍然不是十分清楚. 本研究构建了慢病毒干扰载体pLentiH1-Akt2-shRNA 1, 2, 3及scrambled, 经酶切和测序鉴定均正确; 这4种干扰载体分别转染HEK293T细胞后, 均 获得有感染性的病毒颗粒并感染猪前体脂肪细胞. 转染HEK293T细胞48 h 后real-time PCR分析和转染72 h 后Western 印迹分析表明, Akt2-shRNA2 和shRNA3 介导的Akt2 mRNA和蛋白表达被显著下调 (P <0.05), 其中pLentiH1-Akt2-shRNA3 介导的对Akt2 mRNA和蛋白的干扰效率均达到70%以上 (P <0.01). 进一步研究发现, 猪前体脂肪细胞 中Akt2被有效干扰后, 细胞中脂滴明显减少并变小, 且成脂标志基因PPARγ和aP2蛋白 水平被显著下调. 本研究结果表明, Akt2 knockdown可显著抑制猪前体脂肪细胞分化.

Knockdown of Akt2 by Lentivirus Vector-Mediated RNA Interference Suppresses Differentiation of Porcine Preadipocytes

he serine/threonine kinase 2 (Akt2,or protein kinase B,PKB), is a key component of insulin signaling. Akt2 is closely related to cell survival and human cancer. Akt2 is a critical component to insulin signaling, but the role in adipocyte differentiation remains unclear. In this study, by enzyme restriction digestion and DNA sequencing, we successfully constructed pLentiH1-Akt2 shRNA 1, 2, 3 and scrambled recombinant lentivirus vectors. Virus particles were obtained by transfecting HEK 293T cells using four recombinant vectors and infected porcine preadipocytes, respectively. The results of real-time PCR at 48 hours and Western blotting at 72 hours showed that levels of Akt2 mRNA and protein were remarkably decreased in porcine preadipocytes infected with Akt2 shRNA2 and shRNA 3 recombinant lentivirus (P <0.05). Especially, Akt2 mRNA and protein expressions were suppressed more than 70% by Akt2 shRNA3 (P <0.01). The further study showed that size and number of lipid droplets were reduced and the protein levels of PPARγ and aP2 were decreased significantly after Akt2 were knocked down efficiently in porcine preadipocytes. The above results show that Akt2 knockdown by RNAi significantly represses differentiation of porcine preadipocytes.