低氧诱导因子1α及2α在人BMSCs成软骨分化中的表达规律  

HYPOXIA INDUCIBLE FACTOR 1α/2α GENES EXPRESSION IN CHONDROGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS

作　　者：龚铭[1] 黄胜[1] 罗嘉全[1] 黄保丁[2] 周治宇[1] 代学俊 高蔓蔓 李亮平[1] 邹学农[1] 

机构地区：[1]中山大学附属第一医院脊柱外科/骨科研究所,广州510080 [2]中山大学附属第六医院骨外科 [3]昆明市延安医院脊柱外科

出　　处：《中国修复重建外科杂志》2015年第7期857-862,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家重点基础研究发展计划(973)资助项目(2012CB619100);国家自然科学基金重点项目(31430030);东莞市高等院校科研机构和医疗卫生单位科技计划项目(20101051503501)~~

摘　　要：目的通过诱导人BMSCs(human BMSCs,hBMSCs)成软骨分化,观察低氧诱导因子1α(hypoxia inducible factor 1α,HIF-1α)、HIF-2α在分化过程中的表达趋势,为阐明HIF参与调控成软骨分化机制提供依据。方法对已消化的悬浮hBMSCs进行离心沉淀,形成细胞微球。将细胞微球分为2组,对照组加入含2%FBS的H-DMEM培养基,成软骨诱导组加入软骨诱导液,于低氧(2%O2)条件下培养。培养3周后行甲苯胺蓝及Ⅱ型胶原免疫组织化学染色观察,培养1周行Western blot检测HIF-1α、HIF-2α蛋白表达,培养1、2、3周行实时定量PCR检测成软骨分化关键转录因子及相关标志基因表达。结果甲苯胺蓝染色示,对照组染色细胞核整体分布稀疏,而成软骨诱导组分布致密;成软骨诱导组细胞外基质染色明显较对照组深。Ⅱ型胶原免疫组织化学染色示阳性信号主要位于细胞质内;与成软骨诱导组相比,对照组细胞核分布较稀疏且着色浅。Western blot检测示,培养1周成软骨诱导组HIF-1α和HIF-2α蛋白相对表达量均显著低于对照组(t=8.345,P=0.001;t=7.683,P=0.002)。实时定量PCR检测示,与对照组比较,成软骨诱导组HIF-1αmRNA相对表达量在培养1周时降低、2周时显著升高,差异均有统计学意义(P<0.05);3周时两组比较差异无统计学意义(P>0.05)。成软骨诱导组培养各时间点HIF-2αmRNA相对表达量均显著低于对照组,Sox-9 mRNA相对表达量均显著高于对照组,差异均有统计学意义(P<0.01)。培养过程中,Ⅱ型胶原及Ⅹ型胶原表达呈逐渐增加趋势,第2、3周时两者的mRNA相对表达量显著高于对照组(P<0.05)。而成软骨诱导组多聚蛋白聚糖mRNA相对表达量在各时间点均高于对照组(P<0.05)。结论 HIF-1α参与诱导hBMSCs成软骨分化过程,但HIF-2α表达在该分化过程中受抑制。Objective To observe the genes expression of hypoxia inducible factor 1α（HIF-1α） and HIF-2α by inducing chondrogenic differentiation of human bone marrow mesenchymal stem cells（hBMSCs） so as to provide a fundamental basis for HIF involving in the mechanism of chondrogenesis. Methods High density pellet of hBMSCs was obtained by centrifugation and cultured with H-DMEM medium containing 2% fetal bovine serum（control group） and with chondrogenic medium（chondrogenic induction group） under hypoxia（2%O2） for 3 weeks.Immunohistochemistry staining was utilized to identify extracellular proteogly can and collagen type Ⅱ at 3 weeks after culture. Western blot was applied for measuring HIF-1α and HIF-2α protein levels at 1 week after culture. Real-time quantitative PCR was performed to detect the genes expressions of HIF-1α, HIF-2α, Sox-9, collagen type Ⅱ, collagen type X, and Aggrecan at 1, 2, and 3 weeks after culture. Results Toluidine blue staining showed sparse nucleus in the control group, and dense nucleus in the chondrogenic induction group; extracellular matrix staining was deeper in the chondrogenic induction group than the control group. Immunohistochemical staining for collagen type Ⅱ was positive in cytoplasm; when compared with the chondrogenic induction group, the control group showed sparse and light-coloured nucleus. At 1 week after culture, the protein expression levels of HIF-1α and HIF-2α in the chondrogenic induction group were significantly lower than those in the control group（t=8.345, P=0.001; t=7.683, P=0.002）. When compared with control group, the HIF-1α mRNA expression was significantly down-regulated at 1 week and significantly up-regulated at 2 weeks in chondrogenic induction group（P〈0.05）, but no significant difference was found at 3 weeks between the 2 groups（P〉0.05）. And the mRNA expression of HIF-2α was significantly down-regulated and mRNA expression of Sox-9 was significantly up-regulated after chondrogenic differentiation when compared

关 键 词：低氧诱导因子 人BMSCs 成软骨分化 

分 类 号：R68[医药卫生—骨科学]