摘要
目的:体外诱导大鼠脂肪间充质干细胞向软骨细胞分化,并对分化细胞进行相应鉴定,观察脂肪间充质干细胞作为软骨组织工程种子细胞的可行性。方法:实验于2005-05/2006-05在湘雅医院中心实验室完成。①取大鼠腹股沟脂肪,消化分离脂肪间充质干细胞进行原代培养。②流式细胞仪检测第3代脂肪间充质干细胞表面CD29,CD34,CD44表达。③传3代细胞进行微团培养1d,换用含体积分数为0.01的新生牛血清、10μg/L的转化生长因子β1、6.25mg/L的胰岛素、6.25mg/L的转铁蛋白、1×10-7mol/L的地塞米松、50mg/L的维生素C的高糖DMEM诱导液,为特定培养条件。④在诱导后4,7,14d应用阿尔新兰染色、蕃红O/固绿染色和Ⅱ型胶原免疫细胞化学评价软骨形成情况。⑤反转录-聚合酶链反应在诱导后0和14d检测脂肪间充质干细胞前Ⅱ型胶原蛋白和聚集蛋白聚糖mRNA的表达。结果:①脂肪间充质干细胞的形态特征:原代脂肪间充质干细胞呈短梭形、梭形及多角形,3代后呈均一长梭形。②脂肪间充质干细胞的表面标志鉴定:脂肪间充质干细胞表达CD29,CD44,基本不表达CD34。③脂肪间充质干细胞诱导后的形态特征:脂肪间充质干细胞经诱导后,由长梭形转变为三角形、多角形或短梭形,逐渐聚集成结节。④脂肪间充质干细胞诱导后的细胞化学染色结果:诱导后脂肪间充质干细胞胞外基质阿尔新兰染色、蕃红O/固绿染色、和Ⅱ型胶原免疫细胞化学着色阳性。⑤反转录-聚合酶链反应检测结果:反转录-聚合酶链反应检测未诱导脂肪间充质干细胞不表达前Ⅱ型胶原蛋白和聚集蛋白聚糖mRNA基因,而诱导后的脂肪间充质干细胞表达前Ⅱ型胶原蛋白和聚集蛋白聚糖mRNA基因。结论:脂肪间充质干细胞在由转化生长因子1、胰岛素、转铁蛋白等组成的特定培养基条件诱导下可以定向软骨细胞分化。
AIM： To induce rat adipose-derived stromal cells （ADSCs） to differentiate towards chondrocytes in vitro, and identify the differentiated cells, so as to observe the feasibility of the ADSCs as the seed cells of cartilage tissue engineering. METHODS： The experiment was carried out in the central laboratory of Xiangya Hospital between May 2005 and May 2006. （1）ADSCs were isolated from the rats＇ inguinal fat pads and cultured. （2）The CD29, CD34 and CD44 expressions of the third passage cells were detected by flow cytometry. （3）The fourth passage cells were cultured in micro-mass for 1 day and induced in medium containing fetal bovine serum of 0.01 volume fraction, 10 μg/L transforming growth factor β1 （TGF-β1）, 6.25 mg/L insulin, 6.25 mg/L transferrin,1×10^-7 mol/L dexamethasone and 50 mg/L ascorbic acid-2-phosphate. （4） Chondrogenesis was assessed with Alcian blue staining, Safranin O/Fast Green staining and collagen Ⅱ immunohistochemistry at 4, 7, and 14 days after initial chondrogenic induction. （5）The expression of type Ⅱ procollagen （Col2al） and aggrecan （Agcl） in the induced ADSCs were detected by RT-PCR at 0 and 14 days after induce. RESULTS： （1） Morphologic character of ADSCs： The primary ADSCs presented in long or short spindle-shape or multangular shape, but the third passaged ADSCs and their offspring indicated uniform long spindle-shape. （2）Surface marker identification of ADSCs： The ADSCs expressed CD29, CD44, but not CD34. （3）Morphologic character of induced ADSCs： The induced ADSCs indicated triangular or multangular shape or short spindle-shape and might form nudes. （4） Staining results of ADSCs： The extracellular matrix Alcian blue staining, Safranin O/Fast Green staining and collagen Ⅱ immunohistochemistry were positive. （5）RT-PCR results： RT-PCR showed that Col2al and Agcl were expressed in the induced ADSCs, but not in the primary ADSCs. CONCLUSION： ADSCs can differentiate into chondrocytes under the specific inducing environment composed of TGF-β1, insulin, transferrin.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第3期435-438,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
