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...

Full description

Saved in:
Bibliographic Details
Published in复旦学报：医学版 Vol. 39; no. 6; pp. 558 - 564
Main Author 刘红 俞小芳 滕杰 邹建洲 方艺 刘少鹏 丁小强
Format Journal Article
LanguageChinese
Published 2012
Subjects
低氧
凋亡
增殖
骨髓间充质干细胞（BMSCs）
Online AccessGet full text

Cover

More Information
Summary: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、GLU-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-1a表达增加及上调其下游基因GLUT-1和VEGF表达有关。
Bibliography:bone marrow derived mesenchymal stem cells （BMSCs） ; hypoxia; proliferation; apoptosis ; necrosis
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-1 α（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
ISSN:1672-8467