软基质力学特性对滑膜间充质干细胞向软骨细胞分化的干预作用

Effects of soft substrates on the chondrogenic differentiation of human synovial-derived mesenchymal stem cells

背景:课题组前期研究表明,较软的培养基质对大鼠骨髓间充质干细胞的形态及细胞骨架有明显的影响。目的:探讨不同弹性模量的聚丙烯酰胺凝胶软基质对人滑膜间充质干细胞向软骨细胞分化的影响。方法:无菌条件下获取骨关节炎患者滑膜组织,有限稀释法获得原代人滑膜间充质干细胞,流式细胞术进行细胞表面标记物鉴定,多向诱导分化实验进行功能鉴定。用丙烯酰胺和甲叉双丙烯酰胺制备0.4,6,30 kPa3个弹性模量的聚丙烯酰胺凝胶软基质作为培养人滑膜间充质干细胞的基底,在转化生长因子β1存在的情况下分别培养7 d和14 d,RT-PCR方法检测软骨形成相关基因COL2A1、CRTAC1 mRNA的表达,以6孔细胞培养板作为对照组。结果与结论:在不同弹性模量上生长的人滑膜间充质干细胞表现出不同的细胞形态;软基质的弹性模量及培养时间对人滑膜间充质干细胞成软骨基因COL2A1、CRTAC1的表达有交互作用:7 d时,CRTAC1 mRNA在6 k Pa弹性模量聚丙烯酰胺凝胶上表达量最高(F=44.350,P=0.000);7 d时,COL2A1 mRNA在0.4 k Pa弹性模量聚丙烯酰胺软基质上的表达量最高(F=6.384,P=0.005)。较低弹性模量的聚丙烯酰胺凝胶软基质比常规细胞培养板更具有促进滑膜间充质干细胞向软骨细胞分化的作用。

BACKGROUND： Our previous studies have shown that a soft substrate has a significant effect on morphology and cytoskeleton of rat bone marrow mesenchymal stem cell. OBJECTIVE： To explore the effect of polyacrylamide gels as soft substrates with different elastic moduli on the chondrogenic differentiation of human synovial-derived mesenchymal stem cells. METHODS： The synovium was harvested from patients with osteoarthritis under sterile conditions, and primary human synovial-derived mesenchymal stem cells were separated using limiting dilution assay. The flow cytometry and multi-directional differentiation experiments were used to identify the cell surface markers and function of the human synovial-derived mesenchymal stem cells, respectively. The polyacrylamide gels with the elastic modulus of 0.4, 6, 30 kPa, which were made using various amounts of acrylamide and bis-acrylamide, were used to culture human synovial-derived mesenchymal stem cells under induction with transforming growth factor-β1 for 7 and 14 days. RT-PCR was used to test the expression of chondrogenic genes, type II collagen gene and cartilage acidic protein 1. The 6-well cell culture plates served as controls. RESULTS AND CONCLUSION： The human synovial-derived mesenchymal stem cells showed different cell morphology in the different elastic modulus of polyacrylamide gels. The expression of type II collagen gene and cartilage acidic protein 1 were affected by the different elastic modulus of polyacrylamide gels and culture time, and there was an interaction between these two factors. At 7 days of induction, the expression of cartilage acidic protein 1 gene on 6 kPa polyacrylamide gels was the highest（F=44.350, P=0.000）; meanwhile, the expression of type II collagen gene on 0.4 kPa polyacrylamide gels was the highest（F=6.384, P=0.005）. These findings indicate that polyacrylamide gels with lower elastic modulus are superior to routine culture plates to promote the chondrogenic differentiation of human synovial-derived mesenchymal stem cells.