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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GAGDNA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: To study the effect of icariin (icariin) on the proliferation, morphology and extracellular matrix secretion of rabbit articular chondrocytes in two-dimensional and three-dimensional culture in vitro in order to provide experimental evidence for the application of icariin in cartilage tissue repair basis. Methods: Articular chondrocytes were isolated and cultured. Collagen membrane containing 5 × 10-2mg / ml ICA was prepared and cultured with articular chondrocytes of rabbits for two-dimensional co-culture. The cell growth was observed by MTT assay. The effect of ICA on the growth of rabbit chondrocytes in the scaffolds was observed by laser confocal microscopy. HE and toluidine blue staining were used to characterize the effects of ICA on the proliferation of rabbit chondrocytes. The effect of ICA on the secretion of extracellular matrix was observed, and the ratio of glycosaminoglycans (GAG) and total DNA was quantitatively determined to investigate the effect of ICA on the secretion of GAG. RESULTS: ICA had good biocompatibility to rabbit chondrocytes and promoted its proliferation at 6 days. Rabbit chondrocytes in ICA-containing collagen hydrogel scaffold proliferated faster and maintained a better phenotype Cells tend to cluster and have a higher GAG DNA ratio, suggesting that ICA can promote the secretion of extracellular matrix of chondrocytes and thus contribute to the survival and transformation of chondrocytes. CONCLUSION: ICA has good biocompatibility to chondrocytes, maintains the phenotype of cells, promotes the secretion of extracellular matrix and contributes to the evolution of cells. It is expected to be a safe and effective accelerator for cartilage tissue engineering.