骨髓间充质干细胞二维培养条件下向软骨细胞诱导分化的实验研究  

Research of inducing chondrogenic differentiation of bone marrow mesenchymal stem cells in two-dimensional culture

作　　者：邓进[1] 彭吾训[1] 王蕾[2] 李鹏[1] 

机构地区：[1]贵阳医学院附属医院急诊外科,贵州贵阳550004 [2]贵州省疾病预防控制中心,贵州贵阳550010

出　　处：《中国现代医学杂志》2008年第16期2340-2343,共4页China Journal of Modern Medicine

摘　　要：目的探讨二维培养条件下转化生长因子-β1(TGF-β1)体外诱导SD大鼠骨髓间充质干细胞(BMSC)分化为软骨细胞的方法。方法取SD大鼠的股骨及胫骨,采用贴壁培养法分离纯化大鼠BMSC,取第4代BMSC行流式细胞术检测鉴定其表面抗原,分别以含1、5、10、15和20ng/mL的TGF-β1的诱导培养基在二维培养条件下对第4代BMSC诱导培养,2周后采用免疫组化法对Ⅱ型胶原进行定性检测,采用二甲基亚甲蓝(DMB)比色法定量检测诱导后细胞外基质糖胺多糖(GAG)的表达情况。结果贴壁培养法可分离并纯化SD大鼠的BMSC,所得第4代BMSC细胞表面抗原CD44阳性,CD34、CD45阴性。经诱导培养2周后细胞形态变为不规则,Ⅱ型胶原免疫组化染色显示TGF-β15、10、15和20ng/mL组阳性,二甲基亚甲蓝(DMB)比色法检测显示实验组细胞外基质糖胺多糖(GAG)含量均明显增加(P<0.00),其中TGF-β110ng/mL组细胞分泌的GAG显著多于其他剂量组(P<0.00)。细胞分泌GAG的能力与细胞密度呈正相关(r=0.822,P<0.01)。结论贴壁培养法可以获得较纯的BMSC,该实验所用的诱导体系可成功将BMSC诱导为软骨细胞;TGF-β110ng/mL组诱导后细胞合成糖胺多糖(GAG)的能力强于其他剂量组,在该实验的细胞密度范围内其诱导后细胞合成糖胺多糖(GAG)的能力与细胞密度呈正相关。[Objective] To investigate the system that Bone Marrow Mesenchymal Stem Cells（BMSC） differentiate into chondrocytes in two-dimensional culture in vitro. [Methods] BMSC were isolated from femoral and tibia bone marrow then cultrued and purified by adherent cultivation. Cell surface antigen of BMSC at the 4th passage was analyzed by flow Cytometry. The 4th passage cells were induced by the medium contained TGF-β1 at 1, 5, 10, 15 and 20 ng/mL in two-dimensional culture,and both COL Ⅱ and glycosaminoglycan （GAG） were detected by immunohistochemical method and 1,9-dimethylmethylene blue-colofimetric method after 2 weeks. [Results] BMSC were purified by adherent cultivation. BMSC were positive for CD44, and did not express CD34 and CD45 The shapes of cells became irregular after the inducation. The groups of TGF-β1 5, 10, 15 and 20 ng/mL expressed COL Ⅱ detected by immunohistoehemical method.The experiement groups express more GAG than control group detected by 1,9-dimethylmethylene blue--colorimetric method （P 〈0.00）.The Group C （TGF-β1110 ng/mL） expressed significantly more GAG than other expefiement groups （P 〈0.00）. The capability of induced cells extemalizating GAG was closely correlated with cell densities （r =0.821, P 〈0.01）. [Con-dusion] BMSC SD-rats can be purified by adherent cultivation. BMSC can differentiate into ehondrocytes when cultured in our inducing system. Cells expressed significantly more GAG than others when they were induced by TGF-β110 ng/mL, and the capability of induced cells extemalizating GAG was positively correlated with cell densities refered in this experiment when they were induced by TGF-β110 ng/mL in two-dimensional culture.

关 键 词：转化生长因子-Β1 间充质干细胞 软骨细胞 

分 类 号：R-332[医药卫生]