CAJ | 학술논문

目的探讨TGF-β诱导前软骨干细胞(precartilaginous stem cells,PSCs)成软骨分化的可行性及软骨细胞外基质的表达机制. 方法采用藻酸盐凝胶三维培养分别以TGF-β1、TGF-β2和TGF-β3诱导PSCs向成软骨方向分化,应用免疫组化、RT-PCR、免疫沉淀、Western blot和分光光度等技术测定分化过程中特异性软骨基质成分的表达. 结果 TGF-β1、TGF-β2和TGF-β3在培养7,14,21 d均有Ⅱ型胶原表达,TGF-β3组Ⅱ型胶原表达较强;TGF-β3诱导21 d的PSCs免疫组化检测可见细胞及周围均有可聚蛋白聚糖、纤调蛋白、软骨寡聚基质蛋白表达;TGF-β3组RT-PCR检测显示在8 d内开始出现可聚蛋白聚糖、纤调蛋白、Ⅰ型胶原、X型胶原、软骨寡聚基质蛋白等的表达,8 d后开始出现Ⅱ型胶原表达.免疫沉淀及Western blot检测显示TGF-β3组7,14,21 d分别有软骨寡聚基质蛋白及X型胶原表达.分光光度检测显示TGF-β1组在14或21 d时氨基多糖含量及氨基多糖/DNA比率均低于TGF-β2组或TGF-β3组(P<0.01).结论 TGF-β能诱导PSCs向成软骨方向分化,此分化过程伴随特异性软骨细胞外基质成分的沉淀.可以通过分析关键性软骨基质成分的表达了解PSCs所处的分化阶段.
목적 탐토TGF-β유도전연골간세포(precartilaginous stem cells,PSCs)성연골분화적가행성급연골세포외기질적표체궤제. 방법채용조산염응효삼유배양분별이TGF-β1、TGF-β2화TGF-β3유도PSCs향성연골방향분화,응용면역조화、RT-PCR、면역침정、Western blot화분광광도등기술측정분화과정중특이성연골기질성분적표체. 결과 TGF-β1、TGF-β2화TGF-β3재배양7,14,21 d균유Ⅱ형효원표체,TGF-β3조Ⅱ형효원표체교강;TGF-β3유도21 d적PSCs면역조화검측가견세포급주위균유가취단백취당、섬조단백、연골과취기질단백표체;TGF-β3조RT-PCR검측현시재8 d내개시출현가취단백취당、섬조단백、Ⅰ형효원、X형효원、연골과취기질단백등적표체,8 d후개시출현Ⅱ형효원표체.면역침정급Western blot검측현시TGF-β3조7,14,21 d분별유연골과취기질단백급X형효원표체.분광광도검측현시TGF-β1조재14혹21 d시안기다당함량급안기다당/DNA비솔균저우TGF-β2조혹TGF-β3조(P<0.01).결론 TGF-β능유도PSCs향성연골방향분화,차분화과정반수특이성연골세포외기질성분적침정.가이통과분석관건성연골기질성분적표체료해PSCs소처적분화계단.
Objective To investigate the possibility of transforming growth factor β (TGF-p) inducing chondrogenesis of precartilaginous stem cells (PSCs) and discuss expression mechanism of extracel-luar matrix. Methods PSCs were induced into a chondrogenic pathway in alginate bead culture in the absence of serum and in the presence of TGF-β1, β2, or-β3. The temporal pattern of expression of cartilage-specific extracellular matrix during chondrogenesis were analyzed by immunocytochemistry, immunoflu-orescence, RT-PCR, immunoprecipitation, Western blot and spectrophotometer. Results Type Ⅱ collagen staining was positive at days 7, 14 and 21 in alginate bead culture, showing most intense staining in the TGF-p3-treated culture. Expression of type Ⅱ collagen was increased in TGF-β3 group. Immunocytochemi-cal analysis of a number of other extracellular matrix components showed widespread expressions of aggre-can, fibromodulin and COMP in alginate bead culture that presented TGF-p3 for 21 days. The expressions of Aggrcan, fibromodulin, type Ⅰ and ⅹ collagen, and COMP were detected by RT-PCR in TGF-β3 group within 8 days, while type Ⅱ collagen began expression at days 8-21. COMP or type X collagen was present in TGF-β3 group at days 7, 14 and 21 by immunoprecipitation or Western blot analysis respectively. The extracted glycosaminoglycan content or the glycosaminoglycan/DNA rate in TGF-βl group was significantly lower than those in TGF-β2 group or TGF-β3 group at days 14 and 21 (P <0.01). Conclusions TGF-β can evocate chondrogenesis of PSCs, when rapid deposition of cartilage-specific extracellular matrix is involved. The sequential events in this pathway leading from the undifferentiated stem cells to mature chon-drocytes can be investigated by analysis of key matrix elements.