【摘要】 目的探究野生型p53诱导磷酸酶1（Wip1）基因调控小鼠骨实质来源间充质干细胞（MSC）成脂肪细胞分化的作用与调控机制。方法分离培养Wip1基因敲除小鼠的MSC,观察细胞形态,流式细胞术检测细胞表型和体外诱导分化鉴定MSC。利用油红O染色和实时荧光定量PCR（RT-PCR）比较野生型MSC（WT-MSC）和Wip1敲除MSC(Wip1^-/-MSC)成脂分化后的脂滴数和成脂关键转录因子的表达。Western印迹（WB）检测脂肪细胞分化关键蛋白蛋白质磷酸酶2A（PP2A）的表达。分别收集MSC成脂诱导分化后第0、2、4和5天样本,RT-PCR检测成脂关键转录因子过氧化物酶体增殖物激活受体（PPARγ）与PP2A的表达相关性。构建EGFP-Wip1质粒转染至293T细胞,WB检测过表达Wip1后细胞表达PP2A的差异。采用si RNA-PP2A瞬时转染MSC,检测PP2A敲低后PPARγ在MSC中的表达。结果分离培养的细胞形态呈成纤维样,细胞表型为Sca-1⁺CD90⁺CD105⁺CD29⁺MHCⅡ^-CD11b^-CD34^-CD45^-,体外可诱导分化为成脂肪细胞和成骨细胞,符合MSC判定标准。Wip1^-/-MSC成脂分化后脂滴数少于WT-MSC组,同时RT-PCR检测成脂分化关键转录因子PPARγ的表达也显著低于WT-MSC（P<0.001）;WB检测Wip1^-/-MSC中PP2A蛋白表达亦显著下降,在293T细胞中过表达EGFP-Wip1可使PP2A蛋白表达显著上升;而PP2A-si RNA瞬时转染MSC后,成脂关键转录因子PPARγ表达则显著减少。结论 Wip1通过调控PP2A的表达影响MSC的成脂分化,为深入探讨MSC成脂分化机制提供了理论与实验依据。更多还原

【Abstract】 Objective To investigate the effect and regulatoiy mechanism of Wipl gene on adipogenic differentiation of murine bone parenchymal mesenchymal stem cells（MSCs）.Methods Wipl knockout mice bone parenchymal-derived MSCs were isolated and cultured.The wild type MSss（WT-MSCs） and Wipl knockout MSss(Wip1^-/-MScs) were cultured respectively.Cell morphology was observed under a microscope and cell phenotype was assessed by flow cytometry.Then,both types of MSC.s were induced into adipocytes or osteocytes.The number of lipids drops and the expressions of adipogenic key transcription factors were measured by oil red 0 staining and quantitative real-time fluorescence PCR（RTPcR）.Western blot（WB）was used to assess the expression of adipogenic key transcription protein PP2A.Furthermore,MSC.s induced adipogenic differentiation samples on days 0,2,4,and 5 were collected,before the correlations between the expressions of adipogenic key transcription factors peroxisome proliferator-activated receptor gamma（PPARγ） and PP2A were analyzed by RT-PCR.EGFP-Wip1 plasmid was constructed and transfected into 293 T cells.The expression of PP2A in 293T cells with EGFP-Wipl overexpression was examined by WB.Moreover,MSCs were transduced with PP2A-siRNA,while PPARγ expression in MSC.s with PP2A knockdown was examined by WB.Results The murine compact bone-derived MSC.s exhibited the typical fibroblast-like cell morphology and the cell phonotypes were Sca-1⁺CD90⁺CD105⁺CD29⁺MHC Ⅱ^-GD11 b^-CD34^-CD45^-.These cells could induce differentiation to adipocytes and osteoblasts.All the data indicated that the cultured cells were MSCs.Compared with WT-MSCs,the number of lipids drops and the key transcription factors of adipocyte PPARγ expression in Wip1^-/- MSCs were significantly decreased by RT-PCR（P<0.001）.The expression of PP2A protein in Wip1^-/-MScs was also significantly decreased by WB.Furthermore,WB results showed that overexpression of EGFP-Wip1 in 293T cells could promote the expression of PPZA protein.Conclusion Wipl can affect the adipogenic differentiation of MSCs by regulating the expression of PP2A,which can provide data for the study on the mechanism of adipogenic differentiation of MSCs.更多还原