过表达Fad24对猪DFAT细胞在Dex成脂诱导条件下对胞内受体及辅助分子表达的影响

Effect of overexpression of Fad24 on intracellular Dex receptor and auxiliary molecular expression in porcine DFAT cells under lipid-forming induction conditions

旨在探讨过表达Fad24对猪去分化脂肪细胞(DFAT)在成脂诱导条件下对胞内地塞米松(Dex)受体GR及其高亲和性辅助分子Hsp90和FKBPs表达的影响。用过表达载体pcDNA-Fad24和空载体pcDNA瞬时转染猪DFAT细胞,并在高浓度Dex(1μmol/L)成脂诱导液中诱导成脂分化,然后根据形态学观察检测诱导分化情况,用Real-time PCR和Western blot技术检测对GR及Hsp90和FKBPs表达的影响。结果显示,pcDNA空载体组在诱导5 d时可诱导出成脂细胞,而pcDNA-Fad24过表达组完全抑制成脂分化,细胞积聚形成多个小结节,诱导7 d后形成典型的成骨样结节,茜素红染色证实结节表面有矿化物质沉积。Real-time PCR检测证实,过表达组GRαmRNA的表达量在成脂诱导48 h显著上调(P<0.01),而GRβmRNA的表达量在成脂诱导12 h显著上调(P<0.01);GR的高亲和性辅助分子Hsp90 mRNA的表达量在成脂诱导48 h显著上调(P<0.01);FKBP 51 mRNA的表达量在成脂诱导24 h显著上调(P<0.01),而FKBP 52 mRNA的表达量在成脂诱导48 h显著上调(P<0.01)。Western blot检测证实,上述各因子的蛋白质表达谱与mRNA表达谱基本一致。过表达Fad24对猪DFAT细胞由成脂分化向成骨分化转化作用,与成脂诱导48 h时调控GRα及其Hsp90和FKBP 52表达的上调,以及GRβ和FKBP 51表达下调相关。

The aim of this experiment was to investigate the effect of overexpression of Fad24 on the expression of intracellular dexamethasone(Dex)receptor GR and its high-affinity helper molecules Hsp90 and FKBPs in porcine dedifferentiated adipocytes(DFAT)under lipogenic induction conditions.Porcine DFAT cells were transiently transfected with overexpression vector pcDNA-Fad24 and empty vector pcDNA,and lipogenic differentiation was induced in a high concentration of Dex(1μmol/L)lipogenic induction solution,and then the induced differentiation was examined according to morphological observations,and the effects on GR and the expression of Hsp90 and FKBPs were examined by real-time PCR and Western blot techniques.The results showed that the pcDNA empty vector group could induce lipogenic cells after 5 d of induction,while the pcDNA-Fad24 overexpression group completely inhibited lipogenic differentiation,with cells accumulating to form multiple small nodules and forming typical osteogenic nodules after 7 d of induction,and alizarin red staining confirmed the deposition of mineralized material on the nodule surface.The expression of GRαmRNA was significantly up-regulated at 48hof lipogenesis induction(P<0.01),while that of GRβmRNA was significantly up-regulated at 12hof lipogenesis induction(P<0.01);the expression of Hsp90mRNA,a high affinity helper molecule of GR,was significantly up-regulated at 48hof lipogenesis induction(P<0.01);the expression of FKBP 51mRNA was significantly up-regulated at 24hof lipogenesis induction.The expression of FKBP 51mRNA was significantly upregulated at 24hof lipogenesis induction(P<0.01),while the expression of FKBP52mRNA was significantly upregulated at 48hof lipogenesis induction(P<0.01).The effect of Fad24overexpression on the transformation of porcine DFAT cells from lipogenic to osteogenic differentiation was associated with the upregulation of GRαand its Hsp90and FKBP 52expression at 48hof lipogenic induction,and the downregulation of GRβand FKBP 51expression.