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  

Experimental research on differentiation of immortalized human precartilaginous stem cells to nucleus pulposus-liked cells induced by TGF-β1

作　　者：顾晓峰[1] 王琼[1] 夏仁云[2] 方煌[2] 程力[1] 王俊芳[1] 

机构地区：[1]南京医科大学附属无锡市人民医院骨科,无锡214023 [2]华中科技大学同济医学院附属同济医院骨科

出　　处：《中华骨科杂志》2012年第3期271-276,共6页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金青年科学基金项目（81101374）;南京医科大学科技发展基金重点项目（2010NJMUZ64,2011NJMU205）

摘　　要：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，加人含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AlcianBlue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（Collagen11）和蛋白聚糖（Aggrecan）的表达情况，WesternBlot检测诱导前后细胞内B．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，AlcianBlue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；West-eYnBlot证实细胞内p-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B—catenin的表达诱导IPSCs向髓核样细胞分化。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/glyc- erophosphate （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 character- istic 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.

关 键 词：转化生长因子Β1 软骨细胞 干细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]