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目的:研究淫羊藿苷(icariin,Ica)对体内异位组织工程化软骨细胞基质分泌和特异性基因表达的影响,以评估Ica作为软骨组织工程促进剂的可能性。方法:分离、培养新生家兔关节软骨细胞;制备封装在包埋盒中含Ica终浓度为1×10-5M的软骨细胞-胶原水凝胶构建物,体外培养1周后植入家兔背部皮下,分期取材。激光共聚焦显微镜观察Ica对构建物中软骨细胞生长活性的影响;生化方法检测糖胺聚糖(gLycosaminogLycans,GAG)的分泌量;ELISA测定II型胶原(CollagenⅡ,ColⅡ)的合成情况;实时荧光定量聚合物酶联反应检测软骨特有基因表达情况。结果 :在凝胶中载入1×10-5M浓度范围内的Ica可使细胞-凝胶构建物外观更为致密,类似于软骨组织;维持软骨细胞表型及生长活性;促进GAG和ColⅡ的分泌;提高Aggrecan、CollagenⅡ和Sox9等软骨细胞特有基因的表达。结论:Ica可维持体内异位环境下组织工程化软骨细胞表型,促进细胞外基质分泌和软骨特有基因的表达,有望成为软骨组织工程中一种安全有效的促进剂。
OBJECTIVE: To study the effect of icariin (Ica) on the secretion of ECs and the specific gene expression in ectopic tissue-engineered chondrocytes in vivo so as to evaluate the possibility of Ica acting as a cartilage tissue engineering promoter. Methods: Chondrocytes of newborn rabbits were isolated and cultured. Chondrocyte-collagen hydrogels containing Ica final concentration of 1 × 10-5M were prepared and embedded in the back of rabbits for 1 week. Staging drawn. Laser confocal microscopy was used to observe the effect of Ica on the activity of chondrocytes in the constructs. The biochemical methods were used to detect the secretion of gLycosaminogLycans (GAG), the synthesis of collagenⅡ (ColⅡ) by ELISA, Polymerase chain reaction to detect specific gene expression of cartilage. RESULTS: Loading Ica in the concentration range of 1 × 10-5 M in the gel resulted in a denser appearance of the cell-gel constructs, similar to cartilage tissue; maintenance of chondrocyte phenotype and growth activity; promotion of GAG and ColⅡ Secretion; improve expression of specific genes of chondrocytes such as Aggrecan, CollagenⅡ and Sox9. CONCLUSION: Ica can maintain the phenotype of tissue-engineered chondrocytes under ectopic environment and promote the secretion of extracellular matrix and the expression of specific genes in cartilage. It is expected to be a safe and effective accelerator for cartilage tissue engineering.