淫羊藿苷对胶原支架中软骨细胞生长及胞外基质分泌的影响

The effect of icariin on growth and ECM secretion of chondrocytes cultured in collagen scaffold

目的:研究淫羊藿苷(icariin,ICA)对体外二维及三维培养条件下家兔关节软骨细胞增殖、形态及胞外基质分泌的影响,以期为淫羊藿苷应用于软骨组织修复提供实验基础。方法:分离、培养新生家兔关节软骨细胞;制备含5×10-2mg/ml终浓度ICA的胶原膜,与家兔关节软骨细胞进行二维共培养,观察细胞生长情况,MTT法检测ICA对细胞增殖的影响;制备含6.8×10-4mg/ml终浓度ICA的胶原水凝胶三维支架,激光共聚焦显微镜观察ICA对支架中家兔软骨细胞生长形态的影响,HE及甲苯胺蓝染色定性观察ICA对细胞胞外基质分泌的影响,定量测定糖胺聚糖(glycosaminoglycans,GAG)及细胞总DNA比值,以考察ICA对主要胞外基质GAG分泌的影响。结果:ICA对家兔软骨细胞具有良好的生物相容性,且在6d可促进其增殖;含ICA的胶原水凝胶支架中的家兔软骨细胞增殖较快且表型维持更好,更多细胞趋向于成簇生长,且具有更高的GAG\DNA比值,提示ICA可促进软骨细胞胞外基质的分泌,从而有助于软骨细胞的存活及向组织的转化。结论:ICA对软骨细胞具有良好的生物相容性,可维持细胞表型,促进细胞胞外基质分泌,有助于细胞向组织的演进,可望成为软骨组织工程安全有效的促进剂。

Objective： In order to provide experimental foundation for application of icariin（Ica） to cartilage tissue engineering,the effect of icariin on the proliferation,morphology and ECM secretion of rabbit cartilage chondrocytes cultured in 2D and 3D conditions was evaluated.Methods ：Rabbit chondrocytes were isolated from articular cartilage and cultured in medium containing α-MEM.The chondrocytes were seeded onto sterilized Ica/Col films and the morphology of cells cultured for 2 days was visualized using inverted fluorescence microscope.MTT method was used to examine the effect of Ica on cell proliferation.Chondrocytes-Ica/Col constructs were prepared and cultured under static conditions in a humidified atmosphere with 5% CO2 at 37℃.The chondrocytes cultured in hydrogel constructs were observed by confocal laser scanning microscopy to evaluate the cytocompatibility of hydrogels.HE,toluidine blue staining and biochemical assays were employed to evaluate the effect of Ica on ECM secretion of chondrocytes.Results： The chondrocytes cultured on Ica/Col films showed good cell morphology and higher proliferation compared to Col films after cultured for 6days,suggesting no obvious cytotoxicity of icariin toward chondrocytes.The amount of chondrocytes appeared the tendency of clustered growth in the Ica/Col hydrogel constructs was more than that of in Col hydrogel constructs and the clustering cells possessed spherical morphology similar as the cells in normal cartilage.And the Ica/Col hydrogels improved rabbit chondrocytes secreting the ECM compared with the collagen hydrogels,suggesting that Ica could promote chondrocytes secreting ECM.Conclusion： icariin had good biocompatibility to chondrocytes and could maintain the chondrocytic phenotype and promot the cell secreting ECM.Ica had potential to be used as an accelerant for cartilage tissue engineering.