慢病毒载体介导shRNA干扰IL-6Rα基因表达削弱IL-6对猪脂肪细胞分化的影响

Lentiviral Vector-mediated shRNA Interfering Expression of IL-6Rα Attenuates the Effects of IL-6 on Differentiation of Porcine Adipocytes

为研究白细胞介素-6(IL-6)对猪脂肪细胞分化的影响及其分子机制,构建猪IL-6Rα基因RNA干扰(RNA interference,RNAi)慢病毒载体;用IL-6Rα-RNAi重组慢病毒预处理原代培养的猪前体脂肪细胞或不处理,然后用100 ng/mL IL-6处理分化第6 d的脂肪细胞48 h.通过测定甘油释放量检测脂肪细胞的脂解率;油红O染色提取法测定脂肪细胞的脂质含量;采用RT-PCR和Western印迹检测脂肪细胞分化相关基因的mRNA和蛋白表达.结果显示,IL-6显著抑制猪脂肪细胞分化,并下调PPARγ2、Perilipin A和IRS-1的mRNA及蛋白表达,同时增强ERK1/2磷酸化;IL-6Rα-RNAi预处理前体脂肪细胞则显著逆转IL-6的上述作用.总之,IL-6通过多重机制抑制猪脂肪细胞分化;而且本研究构建的IL-6Rα-RNAi重组慢病毒载体可有效阻断IL-6信号,为进一步研究IL-6的功能奠定了基础.

To explore the effect of interleukin-6 （IL-6） on differentiation of porcine adipocytes, we constructed the lentiviral RNA interference （RNAi） vector targeting the porcine IL-6Ra gene. Primary cultured porcine preadipocytes were pre-infected with or without IL-6Ra-RNAi recombinant lentivirus, and then differentiated adipocytes were incubated with or without 100 ng/mL IL-6 for 48 hours started from day 6. The glycerol release into the culture media was used as an indicator of the lipolysis, and the porcine cellular lipid accumulation was assayed with oil red O staining and extraction. The expression of adipocyte differentiation related genes were analyzed by semi-quantitative RT-PCR and Western blotting. The results showed that IL-6 significantly suppressed the differentiation of pig adipocytes with reduced PPARy/2, perilipin A and IRS-1 at mRNA and protein levels. Meanwhile, ERK1/2 phosphorylation was increased. However, when infected with IL-6Ra-RNAi, the suppression of adipocyte differentiation by IL-6 was reversed. Hence, the IL-6Ra-RNAi recombinant lentiviral vector used in this study was effectively blocked IL-6 signaling, which could be used for the functional study of IL-6 in adipocytes.