表达HIF1α^(mu) EPCs增强BMP2转染BMSCs成骨及成血管能力

EPCs expressing hypoxia inducible factor 1α^(mu) enhances the osteogenesis and angiogenesis of BMSCs transfected with bone morphogenetic protein 2

目的研究表达突变型低氧诱导因子1α的内皮祖细胞(EPCs)对骨形态形成蛋白2(BMP2)转染的骨髓基质细胞(BMSCs)成骨及成血管的影响。方法密度梯度离心法分离培养兔BMSCs和EPCs并鉴定,MTT法检测不同比例BMSCs/EPCs混合培养时增殖作用,以确定混合细胞比例。实验分为6组:A组(BMSCs);B组(Adv-BMP-2BMSCs);C组(BMSCs+EPCs);D组(Adv-BMP-2 BMSCs+EPCs);E组(BMSCs+Adv-HIF1αmu-EPCs);F组(AdvBMP-2 BMSCs+Adv-HIF1αmu-EPCs)。以Adv-HIF1αmu和Adv-BMP2的最佳转染指数(MOI)分别转染EPCs和BMSCs。Western blot检测HIF-1α和BMP-2蛋白表达。行碱性磷酸酶活性检测;用qRT-PCR检测相关基因的表达,以评价共培养细胞成骨和成血管情况。结果 BMSCs与EPCs以1∶1混合培养增殖能力优于1∶2及2∶1,故该实验混合细胞比例确定为1∶1。Adv-BMP-2 BMSCs和BMSCs组可见BMP-2蛋白表达且表达量高于EPCs和AdvHIF1αmu-EPCs组(P<0.05)。Adv-HIF1αmu-EPCs组可见HIF1α蛋白表达且表达量明显高于Adv-BMP-2 BMSCs、BMSCs及EPCs组(P<0.05)。F组ALP活性以及成骨和成血管相关基因表达高于其他组(P<0.05)。结论表达HIF1αmu的EPCs能够增强Adv-BMP-2 BMSCs成骨和成血管潜能。

Objective To study the effect of endothelial progenitor cells（ EPCs） expressing hypoxia inducible factor 1αmuon osteogenesis and angiogenesis of bone morphogenetic protein 2 transfected bone marrow stromal cells（ BMSCs）. Methods BMSCs and EPCs were collected and identified from rabbit bone marrow by density gradient centrifugation. To define the best ratio of EPCs and BMSCs,MTT assay was used to detect the proliferation of different proportion of BMSCs / EPCs. Samples were divided into 6 groups according to different cells and genes transfected： group A： BMSCs; group B： Adv-BMP-2 BMSCs; group C： BMSCs plus EPCs; group D： Adv-BMP-2 BMSCs plus EPCs; group E： BMSCs plus Adv-HIF1αmu-EPCs; group F： Adv-BMP-2 BMSCs plus Adv-HIF1αmu-EPCs. AdvHIF1αmuand Adv-BMP2 were respectively transfected into EPCs and BMSCs with the optimum multiplicity of infection（ MOI）. The expressions of BMP2 and HIF-1α proteins were detected by Western blot. The osteogenesis andangiogenesis potential of co-cultured cells were detected by alkaline phosphatase（ ALP） activity and qRT-PCR.Results The co-culture proportion of BMSCs and EPCs was 1∶ 1. Western blot showed that expression of BMP2 protein in groups of Adv-BMP-2 BMSCs、BMSCs was higher than that in the groups of Adv-HIF1αmuEPCs and EPCs（ P 0. 05）,expression of HIF-1α protein in group of Adv-HIF1αmuEPCs was higher than that in the groups of AdvBMP2-BMSCs、BMSCs and EPCs（ P 0. 05）. Alkaline phosphatase activity and osteogenesis and angiogenesis related gene expression in group F were higher than that in other groups（ P 0. 05）. Conclusions Adv-HIF1αmuEPCs co-cultured with Adv-BMP2-BMSCs can promote osteogenesis and angiogenesis of Adv-BMP-2 BMSCs.