Wnt10b对山羊前体脂肪细胞分化相关基因表达的影响

摘要

旨在利用RNA干扰技术,明确Wnt10b基因对山羊前体脂肪细胞分化的影响.设计合成山羊Wnt10bsiRNA序列,利用胶原酶消化法获得山羊皮下和肌内前体脂肪细胞.利用有效的siRNA序列干扰山羊皮下和肌内前体脂肪细胞Wnt10b基因表达后,检测其对细胞分化及脂肪细胞分化标志基因表达的影响.结果表明,筛选获得有效的山羊Wnt10b siRNA序列,转染山羊皮下和肌内前体脂肪细胞后,Wnt10b被显著干扰,其mRNA表达量分别下调63％ (P＜0.01)和67％ (P＜0.01);油红O染色结果显示,干扰Wnt10b基因可明显抑制山羊皮下和肌内脂肪细胞的脂滴积聚;且干扰Wnt10b基因后,在皮下前体脂肪细胞分化过程中C/EBPβ、PPARγ、SREBP1和Pref1基因出现极显著下调(P＜0.01),LPL出现显著下调(P＜0.05);而在肌内前体脂肪细胞分化过程中,AP2和LPL基因出现极显著下调(P＜0.01),C/EBPβ出现显著下调(P＜0.05),Pref1出现极显著上调(P＜0.01).本试验发现,Wnt10b基因可能通过调控C/EBPβ、PPARγ、SREBP1和LPL的表达来促进山羊皮下前体脂肪细胞分化,通过调控AP2、LPL、C/EBPβ和Pref1的表达来促进山羊肌内前体脂肪细胞分化.%The aim of this study was to investigate the effect of Wnt10b gene on the precursor adipocytes differentiation of goat by RNA interference.The goat Wnt10b siRNA sequence was designed and compounded,and the goat subcutaneous and intramuscular precursor adipocytes were obtained by collagenase digestion.The effect of Wnt10b gene expression on the cell differentiation and adipocyte differentiation marker genes expression were detected by using the effective siRNA sequence to interfere the expression of Wnt 10b gene in goat subcutaneous and intramuscu1ar precursor adipocytes.The resu1ts showed that an effective goat Wnt10b siRNA sequence was obtained,and the expression of Wnt10b was down-regulated by 63％ (P＜0.01) and 67％ (P＜0.01),respectively after transfection in goat subcutaneous and intramuscular precursor adipocytes.Results of Oil red O staining showed that the accumulation of lipid droplets were inhibited significantly by Wnt10b RNAi in goat subcutaneous and intramuscular adipocytes.The C/EBPβ,PPARγ,SREBP1 and Pref1 genes were extremely significantly down-regulated in the subcutaneous precursor adipocyte differentiation after Wnt 10b RNAi(P＜0.01),LPL was significantly down-regulated(P＜0.05).The AP2 and LPL were extremely significantly down-regulated (P＜0.01),C/EBPβ was significantly down-regulated (P＜0.05),and Pref1 was extremely significantly up-regulated (P＜0.01) in intramuscular precursor adipocytes differentiation.In this study,it was found that Wnt 10b gene could promote the differentiation of goat subcutaneous precursor adipocytes by regulating the expression of C/EBPβ,PPARγ,SREBP1 and LPL,and promote the differentiation of goat intramuscular precursor adipocytes by regulating the expression of AP2,LPL,C/EBPβ and Pref1.