TGFBI基因调控人脐带间充质干细胞向成骨细胞的分化

Role of TGFBI gene in regulating osteoblastic differentiation of human umbilical cord-derived mesenchymal stem cells

目的探讨转化生长因子β诱导蛋白(TGFBI)基因调控人脐带来源间充质干细胞(huc-MSC)的成骨分化作用。方法培养huc-MSC,流式细胞术检测细胞表面标志物CD14、CD34、CD45、CD73、CD90、CD105,油红O染色和碱性磷酸酶(ALP)染色检测体外成脂肪细胞和成骨细胞的诱导分化能力,实时荧光定量PCR(q-PCR)检测成脂和成骨关键转录因子的表达。采用si-RNA瞬时转染技术敲低huc-MSC的TGFBI基因,并研究其对huc-MSC生物学特性的影响;进一步利用慢病毒载体构建稳定敲低TGFBI基因的huc-MSC(TGFBI-/-huc-MSC),并对TGFBI-/--hucMSC进行成骨细胞诱导分化,ALP染色鉴定细胞ALP活性,q-PCR检测成骨分化关键转录因子ALP及骨桥蛋白(OPN)的表达差异。结果培养的huc-MSC表达MSC表面标志物CD14-CD34-CD45-CD73+CD90+CD105+,TGFBI基因敲低后不影响huc-MSC表面标志物的表达;成脂肪诱导分化后,出现大量红色脂滴,成脂分化关键转录因子Adi和过氧化物酶体增殖物激活受体(PPARγ)表达显著增加(P<0.001);成骨诱导分化后,ALP活性增加,成骨分化关键转录因子OPN和ALP表达亦显著增加(P<0.001),证明敲低TGFBI基因的huc-MSC仍具有向成脂、成骨分化的特性,但其成骨诱导分化能力较未敲除组增强,其中ALP活性、ALP基因(P<0.001)及OPN基因(P<0.001)的表达均显著增高。结论TGFBI基因能抑制huc-MSC的成骨分化。

Objective To explore the role of transforming growth factor beta-induced protein(TGFBI)genes in regulating osteogenic differentiation of human umbilical cord-derived mesenchymal stem cells(huc-MSCs).Methods huc-MSCs were cultured and the cell surface markers of CD14,CD34,CD45,CD73,CD90 and CD105 were analyzed by flow cytometry.The differentiation ability of huc-MSCs into adipocytes or osteocytes was detected by oil red 0 staining and alkaline phosphatase(ALp)staining.The expressions of adipogenic and osteogenic key transcription factors were detected by quantitative real-time fluorescence PCR(q-PCR).To assess the effect of TGFBI gene on the biological characteristics of huc-MSCs,transient siRNA transfection technology was used to knock down TGFBI in huc-MSCs.Furthermore,the lentiviral vectors were constructed with stable knockdown of TGFBI gene in huc-MSCs(TGFBI-/-huc-MSCs)and TGFBI-/-huc-MSCs were induced to differentiate into osteoblasts.ALP was performed to identify the activity of TGFBI-/-huc-MSCs ALP.The expressions of osteogenic key transcription factors ALP and OPN were detected by q-PCR.Results The cultured huc-MSCs and TGFBI-/-huc-MSCs expressed the MSCs surface markers CD14-CD34-CD45-CD73+CD90+CD105+,suggesting that TGFBI gene knockdown did not affect the expression of huc-MSCs surface markers.After adipogenic differentiation of hucMSCs and TGFBI-/-huc-MSCs,numerous intracellular lipid droplets could be observed by oil red 0 staining.The q-PCR results showed that the expressions of Adi and PPARγin the induction group were significantly up-regulated.Osteogenic differentiation results demonstrated that the activity of ALP was enhanced in the induced group(P<0.001)and the expressions of OPN and ALP in the induced group also showed a significant increase with q-PCR detection(P<0.001).These results indicated that TGFBI-/-huc-MSCs were still capable of adipogenic and osteogenic differentiation.While the osteogenic differentiation ability was significantly increased compared with the huc-MSCs group,ALP activity,ALP gene(P<0.001)and OPN gene(P<0.001)expressions were elevated.Conclusion TGFBI gene could inhibit huc-MSCs osteoblastic differentiation.