CAJ | 학술논문

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.
목적 연구전화생장인자β1 (TGF-β1)유도영생화인전연골간세포(IPSCs)향수핵양세포분화적가행성.방법 장체외배양적IPSCs접충우온민형각취당수응효(C/GP)삼유지가상,가입함TGF-β1적유도배양기,저양조건하진행유도배양,관찰삼유지가상IPSCs적생장급분화정황.7d후행Alcian Blue염색,분석세포외기질당알취당(GAG)합성정황,병수집세포,제취RNA.RT-PCR검측유도전후수핵양세포표지기인Ⅱ형효원(CollagenⅡ)화단백취당(Aggrecan)적표체정황,Western Blot검측유도전후세포내β-catenin단백표체수평적변화.결과 IPSCs재삼유지가상생장상태량호,유도7d후,Alcian Blue염색표명,세포외기질GAG적합성명현증다,실험조(유도배양기)명현다우대조조(보통배양기).RT-PCR증실Ⅱ형효원화Aggrecan적기인표체수평명현증고,실험조명현고우대조조；Western Blot증실세포내β-catenin단백표체수평명현상조,실험조명현고우대조조.결론 IPSCs경TGF-β1유도가재체외정향분화위수핵양세포,유도분화후적세포구유량호적분비공능,능구유효지분비세포외기질성분.TGF-β1가능통과상조세포내β-catenin적표체유도IPSCs향수핵양세포분화.
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.