体外负压培养对hBMSCs成骨活性影响的初步研究

Effects of negative pressure on osteogenesis in human BMSCs cultured in vitro

[目的]探讨体外负压培养对hBMSCs成骨活性的影响。[方法]取第3代hBMSCs分为实验组和对照组,实验组进行间歇性负压培养,设置压力为17 kPa,30 m in/次,4次/d,干预2周;对照组于普通CO2培养箱中常规培养。倒置显微镜下观察细胞形态,MTT法和流式细胞仪检测细胞增殖和凋亡,检测ALP活性,茜素红染色观察钙节结形成,免疫组织化学检测Ⅰ型胶原和VEGF的表达。[结果]诱导2周,实验组细胞增殖能力下降,S期细胞百分比为(5.14±1.56)%,较对照组(13.45±3.51)%约下降62.4%,细胞凋亡增加。实验组ALP活性为(15.68±1.97)mU/mg,对照组为(6.34±1.21)mU/mg,两组比较差异有统计学意义(P<0.05);与对照组比较,实验组钙结节形成增多,VEGF表达较对照组显著提高;实验组Ⅰ型胶原表达呈阳性,对照组呈阴性反应。[结论]负压能抑制hBMSCs增殖,促进细胞凋亡,但可以提高细胞成骨活性。

[Objective]To investigate the effects of negative pressure on osteogenesis in human bone marrow stem cells（hBMSCs） cultured in vitro.[Methods]The third passage hBMSCs were divided into negative pressure treatment group and control group.The treatment group was induced by negative pressure intermittently（pressure：17 kPa,30 min/at a time,4/times a day）.The control group was cultured in conventional condition.The differentiation of hBMSCs was examined by invert phase contrast microscopy,the determination of alkaline phosphatase（ALP） activities,alizarin-red staining and the immunohistochemistry of collagen typeⅠand vascular endothelial growth factor（VEGF）.MTT assay and flow cytometry were applied to measure the proliferation and apoptosis.[Results]Negative pressure inhibited the cellular proliferation and facilitated the apoptosis.The percentage of phase S cells was（5.14±1.56）% in treatment group compared with（13.45±3.51）% in the control group,with a decrease of 62.4%,and the activity of ALP was（15.68±1.97） mU/mg in the treatment group increased significantly compared with（6.34±1.21） mU/mg in the control group（P0.05） after two weeks.In the treatment group,calcium nodes were seen by alizarin-red staining,the expression of collagen typeⅠwas positive and VEGF was in creased significantly,compared with that in control group.[Conclusion]Negative pressure not only can inhibit the proliferation,facilitate apoptosis but also promote osteogenesis of hBMSCs.