缺氧诱导因子-1α调控骨形态发生蛋白9对间充质干细胞成骨分化的影响

Effect of hypoxia inducible factor-1αregulating bone morphogenetic protein 9 on osteogenic differentiation of mesenchymal stem cells

目的探讨缺氧诱导因子-1α(HIF-1α)对骨形态发生蛋白9(BMP9)诱导间充质干细胞(MSCs)C3H10T1/2成骨分化的影响。方法体外培养小鼠MSCs C3H10T1/2细胞系,分为5组:NC组(正常培养C3H10T1/2细胞)、Ad-GFP组(转染腺病毒液)、Ad-BMP9组(转染BMP9过表达的腺病毒液)、pcDNA3.1-mRFP组(转染Ad-BMP9+pcDNA3.1空载质粒)、pcDNA3.1-HIF-1α组(转染Ad-BMP9+HIF-1α过表达质粒)。采用碱性磷酸酶(ALP)显色及活性检测试剂盒检测各组C3H10T1/2细胞中ALP活性;采用茜素红S染色检测各组C3H10T1/2细胞钙盐沉积情况,并用吸光度(A)值进行定量分析;采用免疫印迹法检测各组C3H10T1/2细胞中BMP9、HIF-1α及成骨分化标志物骨钙素(OC)、骨桥蛋白(OPN)及Runt相关转录因子2(Runx2)蛋白表达。结果与NC组、Ad-GFP组比较,Ad-BMP9组ALP活性、钙盐沉积、A值及BMP9、HIF-1α、OC、OPN、Runx2蛋白表达增加(P<0.05)。与Ad-BMP9组、pcDNA3.1-mRFP组比较,pcDNA3.1-HIF-1α组ALP活性、钙盐沉积、A值及BMP9、HIF-1α、OC、OPN、Runx2蛋白表达增加(P<0.05)。结论HIF-1α可促进BMP9诱导MSCs C3H10T1/2成骨分化,可能与上调OC、OPN及其下游Runx2蛋白表达有关。

Objective To investigate the effect of hypoxia inducible factor-1α(HIF-1α)on the osteogenic differentiation of mesenchymal stem cells(MSCs)C3H10T1/2 induced by bone morphogenetic protein 9(BMP9).Methods MSCs C3H10T1/2 cells line of the rats were cultured in vitro and divided into 5 groups:NC group(normal cultured C3H10T1/2 cells),Ad-GFP group(transfected with adenovirus control),Ad-BMP9 group(transfected with adenovirus over expression of BMP9),pcDNA3.1-mRFP group(transfected with Ad-BMP9+pcDNA3.1 empty plasmid),pcDNA3.1-HIF-1αgroup(transfected with Ad-BMP9+HIF-1αover expression plasmid).Alkaline phosphatase(ALP)activity in C3H10T1/2 cells of each group was detected by ALP staining and activity detection kit;the calcium deposition of C3H10T1/2 cells was detected by Alizarin Red S staining,and the absorbance(A)value was used for quantitative analysis;and the expressions of BMP9,HIF-1α,osteogenic differentiation markers osteocalcin(OC),osteopontin(OPN)and Runt related transcription factor 2(Runx2)in C3H10T1/2 cells were detected by Western blot method.Results Compared with NC group and Ad-GFP group,the ALP activity,calcium deposition,A value and protein expressions of BMP9,HIF-1α,OC,OPN and Runx2 in Ad-BMP9 group were increased(P<0.05).Compared with Ad-BMP9 group and pcDNA3.1-mRFP group,the ALP activity,calcium deposition,A value and protein expressions of BMP9,HIF-1α,OC,OPN and Runx2 in pcDNA3.1-HIF-1αgroup were increased(P<0.05).Conclusions HIF-1αcan promote the osteogenic differentiation of MSCs C3H10T1/2 induced by BMP9,which may be related to the up-regulation of OC,OPN and their downstream Runx2 protein expression.