外源性透明质酸对兔骨髓间充质干细胞定向分化为软骨细胞的影响

Influence of exogenous hyaluronic acid on chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells

背景:透明质酸是关节腔滑液最主要的成分,对细胞的形态发生起着非常重要的作用,但其用于软骨缺损修复时对骨髓间充质干细胞的影响如何呢?目的:通过分析外源性透明质酸对兔骨髓间充质干细胞体外增殖及定向分化为软骨细胞的影响,探讨关节腔内环境对骨髓间充质干细胞的作用。方法:全骨髓法+贴壁培养法分离培养兔骨髓间充质干细胞,取第4代细胞用于实验,实验组细胞加入透明质酸诱导液,以转化生长因子β3诱导组作为阳性对照,阴性对照组加入常规培养液。分别于诱导后第7,14,21d行甲苯胺蓝染色检测蛋白聚糖表达,免疫组化染色及RT-PCR检测细胞Ⅱ型胶原表达。结果与结论:经透明质酸诱导后,细胞增殖速度减慢,由长梭形变为多角形、椭圆形,细胞外基质呈甲苯胺蓝异染性和Ⅱ型胶原免疫组化阳性,RT-PCR检测示Ⅱ型胶原mRNA表达阳性,表现出软骨细胞的分化特点,但表达均比阳性对照组弱。结果提示,外源性透明质酸具有诱导兔骨髓间充质干细胞向软骨细胞分化的能力,但比转化生长因子β3的诱导能力弱,关节腔内环境对骨髓间充质干细胞向软骨细胞分化有正性促进作用,支持透明质酸作为软骨组织工程基质使用。

BACKGROUND： Hyaluronic acid is the most important component of intra-articular synovial fluid and plays a very important role on the cell morphogenesis, but its effect on bone marrow mesenchymal stem cells （MSCs） in the repair of cartilage defects remains unclear. OBJECTIVE： To investigate the role of intra-articular environment to the MSCs by studying the influence of exogenous hyaluronic acid on proliferation and chondrogenic differentiation of rabbit bone marrow MSCs. METHODS： The rabbit MSCs were isolated and cultured in the method of whole bone marrow and adherent culture. The fourth passage of cells were used for the experiment. The experimental group cells were induced by hyaluronic acid solution, with transforming growth factor-β3 induced group served as a positive control. The negative control group was joined regular medium. The features of chondrocytes were identified by toluidine blue staining, immunohistochemistry and RT-PCR to detect the expression of collagen Ⅱ after 7, 14, 21 day’s induction respectively. RESULTS AND CONCLUSION： After induced by hyaluronic acid, the speed of cell proliferation slowed down. The cell morphology changed from long spindle to polygonal, oval. The extracellular matrix showed metachromasia with toluidine blue and positive with type Ⅱ collagen immunohistochemical staining. RT-PCR detection also showed the expression of type Ⅱ collagen mRNA. All the above showed the characteristics of cartilage cell differentiation. But their expressions were weaker than the positive control group. The results indicated that the exogenous hyaluronic acid can induce the rabbit MSCs differentiate to chondrocytes. But its capacity is weaker than transforming growth factor-β3. Therefore, the intra-articular environment plays a positive role in promoting chondrogenic differentiation of MSCs and supported that hyaluronic acid could be used as a matrix for cartilage tissue engineering.