低氧预处理对大鼠骨髓间充质干细胞(BMSCs)增殖、凋亡和坏死的影响

 目的 探讨低氧预处理对大鼠骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMSCs)增殖、凋亡和坏死的影响及其可能机制。方法 采用全骨髓细胞贴壁法分离培养BMSCs,通过细胞成脂、成骨分化诱导及流式细胞仪检测其表面标志物(CD29、CD90、CD45)鉴定BMSCs;运用siRNA干扰技术沉默低氧诱导因子 1α(hypoxia inducible factor,HIF 1α)基因。将细胞分成6组:正常培养组、正常培养+转染阴性对照组、正常培养+HIF 1α RNA干扰组、低氧预处理组、低氧培养+转染阴性对照组及低氧培养+HIF 1α RNA干扰组,分别采用四甲基偶氮唑盐(MTT)法、流式细胞仪和乳酸脱氢酶(lactic acid dehydrogenase,LDH)活力测定等方法,检测各组BMSCs的增殖、凋亡和坏死情况;Western blot和real time PCR 检测各组BMSCs中HIF 1α、葡萄糖转运子 1(glucose transporter,GLUT 1)及血管内皮生长因子(vascular endothelial growth factor,VEGF)的mRNA和蛋白的表达。 结果 培养的BMSCs具有成脂、成骨等多向分化潜能;流式检测呈现CD29和CD90高表达,CD45低表达;低氧预处理可促进BMSCs增殖,减轻坏死,对细胞凋亡无明显影响;低氧预处理组HIF 1α、GLUT 1及VEGF的蛋白及mRNA表达明显高于常氧培养组(P<0.05);给予低氧预处理组siRNA HIF 1α后其HIF 1α、GLUT 1及VEGF的蛋白及mRNA表达均明显低于未干扰前(P<0.05),且细胞增殖受抑制,细胞凋亡和坏死加重(与未干扰前相比,P<0.05)。 结论 低氧预处理可促进BMSCs的体外增殖,减轻坏死,其机制可能与低氧预处理后HIF 1α表达增加及上调其下游基因GLUT 1和VEGF表达有关。

Effects of hypoxia preconditioning on the proliferation, apoptosis and necrosis of bone marrow derived mesenchymal stem cells (BMSCs)

Objective To explore the effects and probable mechanism of hypoxia preconditioning on the proliferation, apoptosis and necrosis of bone marrow derived mesenchymal stem cells (BMSCs).Methods BMSCs were cultured by whole bone marrow adherence method.The characteristics of BMSCs were identified by inducing adipogenic and osteogenic differentiation and surface markers (CD29、CD90、CD45) with flow cytometry (FCM).Cultured BMSCs were divided into the following groups:normal culture group,normal transfection negative group,normal hypoxia inducible factor 1α (HIF 1α) siRNA group,hypoxia culture group,hypoxia transfection negative group and hypoxia HIF 1α siRNA group.MTT assay was used to detect the proliferation rate of cells.FCM was appliedto detect the apoptosis and lactic acid dehydrogenase (LDH) activity in supernatant was determined to evaluate cell necrosis.Theprotein and mRNA expressions of HIF 1α,glucose transporter (GLUT 1) and VEGF of BMSCs were detected by Western blot and real time PCR.Results Cultured BMSCs had the capacities for adipogenic and osteogenic differentiation,and highly expressed CD29 and CD90,and negative for CD45.Hypoxia culture group had higher proliferation rate and lower LDH activity than normal culture groups (P<0.05),but there was no significant difference of apoptosis among the above groups (P>0.05).The protein and mRNA levels of HIF 1α,GLUT 1 and vascular endothelial growth factor (VEGF) of BMSCs in hypoxia culture group were significantly higher than that in normal group.After silencing HIF 1α,the protein and mRNA levels of HIF 1α,GLUT 1 and VEGF of BMSCs in hypoxia HIF 1α siRNA group were lower than that in hypoxia culture groups.Besides,in hypoxia HIF 1α siRNA group,the proliferation rate was lower and the LDH activity was higher than that in hypoxia culture group (P<0.05).Conclusions Hypoxia preconditioning promoted the proliferation ability and inhibited the necrosis of BMSCs in vitro,which were attributable in part to the up regulation of GLUT 1 and VEGF by HIF 1α activation after hypoxia preconditioning.