转化生长因子β1诱导永生化人前软骨干细胞向髓核样细胞分化的实验研究

摘要

Objective To explore the possibility of immortalized human precartilaginous stem cells (IPSCs) differentiating into nucleus pulposus-liked cells induced by transforming growth factor-β1 (TGF-β1)and examine its biological characters.Methods The IPSCs were seeded on the thermosensitive chitosan/glycerophosphate (C/GP) scaffolds and induced into nucleus pulposus-like cells in culture medium with the adding of TGF-β1 under hypoxia condiction.The growth and differrentiation of IPSCs on C/GP scaffolds were observed.Seven days later,Alcian blue staining was used to detect the formation of glycosaminoglycans (GAG) of extracellular matrix by the differentiating cell.RT-PCR was carried out to identify the expression of characteristic genes of nucleus pulposus-liked cells,including collagen Ⅱ and Aggrecan.Western blot were used to examine the expression of β-catenin.Results IPSCs grew well on the thermosensitive C/GP scaffolds.After 7 days,Alcian blue staining exhibited more formation of GAG in experimental group as compared with control group.RT-PCR manifested that the gene expression of collagen Ⅱ and Aggrecan were upregulated.Likewise,Western blot manifested that the expression of β-catenin was upregulated.Respectively,all of the content in the induction group obviously increased compared with that of the control group.Conclusion IPSCs can be differentiated into nucleus pulposus-like cells under the induction of TGF-β1,the differentiating cells have a favourable secretory function,which can secrete extracellular matrix effectively.Differentiation of IPSCs to nucleus pulposus-liked cells may be through upregulating the expression of β-catenin in cells.%目的 研究转化生长因子β1 (TGF-β1)诱导永生化人前软骨干细胞(IPSCs)向髓核样细胞分化的可行性.方法 将体外培养的IPSCs接种于温敏型壳聚糖水凝胶(C/GP)三维支架上,加入含TGF-β1的诱导培养基,低氧条件下进行诱导培养,观察三维支架上IPSCs的生长及分化情况.7d后行Alcian Blue染色,分析细胞外基质糖胺聚糖(GAG)合成情况,并收集细胞,提取RNA.RT-PCR检测诱导前后髓核样细胞标志基因Ⅱ型胶原(CollagenⅡ)和蛋白聚糖(Aggrecan)的表达情况,Western Blot检测诱导前后细胞内β-catenin蛋白表达水平的变化.结果 IPSCs在三维支架上生长状态良好,诱导7d后,Alcian Blue染色表明,细胞外基质GAG的合成明显增多,实验组(诱导培养基)明显多于对照组(普通培养基).RT-PCR证实Ⅱ型胶原和Aggrecan的基因表达水平明显增高,实验组明显高于对照组；Western Blot证实细胞内β-catenin蛋白表达水平明显上调,实验组明显高于对照组.结论 IPSCs经TGF-β1诱导可在体外定向分化为髓核样细胞,诱导分化后的细胞具有良好的分泌功能,能够有效地分泌细胞外基质成分.TGF-β1可能通过上调细胞内β-catenin的表达诱导IPSCs向髓核样细胞分化.