低氧条件下转化生长因子－β3促进大鼠骨髓间充质干细胞向软骨分化的分析

TGF-β3 improves bone mesenchymal stem cells toward chondrogenic differentiation under hypoxia environment

目的探索低氧环境下转化生长因子－β3（TGF－β3）诱导大鼠骨髓间充质干细胞（BM－MSCs）向软骨细胞分化的作用及相关机制。方法取SD大鼠的胫骨、股骨，运用全骨髓贴壁法培养BM－MSCs，通过流式细胞术检测其细胞表面抗原、定向诱导分化等方式鉴定BM－MSCs；在常氧或低氧条件下，添加TGF-β3诱导BM-MSCs，阿利新蓝染色和Ⅱ型胶原免疫荧光染色检测BM－MSCs的软骨表达情况；实时荧光定量聚合酶链反应检测软骨Ⅱ型胶原蛋白（collagen Ⅱ）、聚蛋白多聚糖（aggrecan）、Ⅹ型胶原蛋白（collagen Ⅹ）的表达情况；蛋白免疫印迹法分别检测collagen Ⅱ、低氧诱导因子－1α（HIF－1α）及β－连环蛋白的表达情况。结果BM-MSCs呈成纤维细胞状，细胞表面高表达CD90（99.26%），CD29（96.41%），CD44（95.98%），弱表达CD45（8.8%）并可诱导向成骨、成脂、成软骨分化；阿利新蓝及细胞免疫荧光染色的结果发现低氧条件下添加TGF－β3培养BM－MSCs细胞团高表达aggrecan和collagen Ⅱ。实时荧光定量聚合酶链反应的结果发现，对比对照组，低氧条件下添加TGF－β3可使BM-MSCs的collagen Ⅱ、aggrecan和collagen Ⅹ的mRNA水平分别上调2.46、2.20和1.80倍（P〈0.05）。蛋白免疫印迹法提示低氧TGF－β3组较对照组HIF-1α、collagen Ⅱ表达量增加，β－连环蛋白表达量下降。结论低氧环境下TGF－β3可以明显促进BM－MSCs成软骨分化能力，并可能与抑制Wnt/β－catenin通路相关，从而为BM－MSCs治疗关节软骨损伤提供有效的新途径。

ObjectiveTo investigate the impact of TGF-β3 on the chondrogenesis of bone marrow mesenchymal stem cells （BM-MSCs） under hypoxia environment. MethodsBM-MSCs were obtained from SD rat tibias and femora and cultured with whole bone marrow adherent method. Cell surface antigens were analyzed by flow cytometry and the multiple-directional differentiation capabilities were detected with special differentiation agents to affirm the reality of BM-MSCs. Under normoxia or hypoxia condition, BM-MSCs were induced with TGF-β3 or not. Then, alcian blue and immunofluorescence staining were performed to evaluate the expression level of aggrecan, collagen Ⅱ. qRT-PCR analysis were performed to analyze the expression of aggrecan, collagen Ⅱ and collagen Ⅹ. qRT-PCR and Western blot analysis was performed to detect the mRNA and protein level of HIF-1α, collagenⅡ and β-catenin. ResultsBM-MSCs were fibroblast-like shape and had ablities of osteogeic, adipogenic and chondrogenic differentiation, with the expression of CD29, CD44 and CD90 but not CD45. Alcian blue and immunofluorescence staining showed that BM-MSCs strongly expressed the aggrecan and collagen Ⅱ with the presence of TGF-β3 under hypoxia condition. qRT-PCR analysis showed the mRNA expression levels of collagen Ⅱ, aggrecan and collagen Ⅹ were up-regulated at 2.46, 2.20 and 1.80 folds, comparing with control group （all P〈0.05）. Western blot analysis showed that the protein levels of HIF-1α, collagenⅡ in BM-MSCs were up-regulated with the presence of TGF-β3 under hypoxia condition, but β-catenin level was down-regulated. ConclusionTGF-β3 promotes the chondrogenic differentiation ability of BM-MSCs under hypoxia condition, which may be relative with the inhibition of Wnt/β-catenin signaling pathway.