CAJ | 학술논문

目的观察转化生长因子(TGF)-β1对骨髓干细胞向软骨细胞分化过程中Wnt信号通路相关基因表达的影响.方法 TGF-β1软骨诱导液培养骨髓干细胞(BMSCs),倒置相差显微镜进行形态学观察,甲苯胺蓝染色、鉴定.酶联免疫吸附试验(ELISA)法检测不同浓度TGF-β1对糖胺聚糖(GAG)表达量的影响,实时荧光定量聚合酶链反应(FQ-PCR)检测目的基因Wnt1、Wnt3a、β-连环蛋白(β-catenin)、Sox9和胶原蛋白Ⅱ(Collagen Ⅱ) mRNA表达.结果 TGF-β1诱导培养第21天,甲苯胺蓝染色显示,95％以上细胞为软骨细胞；2μg/L TGF-β1诱导液组细胞培养液中GAG表达量为(69.16±1.18) mg/L,明显高于其余各组(P＜0.05)；FQ-PCR结果表明,与对照组比较,2μg/LTGF-β1诱导液组的Wnt1、Wnt3a mRNA的表达量分别为0.0266±0.0047、0.0154±0.0033,其表达受到了抑制(P＜0.05),β-catenin mRNA的表达量为0.0187 ±0.0021,明显低于对照组(P＜0.01)；相反,Sox9、Collagen Ⅱ mRNA表达量分别为0.0682±0.0024、0.1134±0.0048,表达有所升高(P＜0.05).结论 TGF-β1促进BMSCs向软骨细胞分化,Wnt/β-catenin信号通路相关基因表达受到抑制,而软骨细胞特异基因表达增加.
목적 관찰전화생장인자(TGF)-β1대골수간세포향연골세포분화과정중Wnt신호통로상관기인표체적영향.방법 TGF-β1연골유도액배양골수간세포(BMSCs),도치상차현미경진행형태학관찰,갑분알람염색、감정.매련면역흡부시험(ELISA)법검측불동농도TGF-β1대당알취당(GAG)표체량적영향,실시형광정량취합매련반응(FQ-PCR)검측목적기인Wnt1、Wnt3a、β-련배단백(β-catenin)、Sox9화효원단백Ⅱ(Collagen Ⅱ) mRNA표체.결과 TGF-β1유도배양제21천,갑분알람염색현시,95％이상세포위연골세포；2μg/L TGF-β1유도액조세포배양액중GAG표체량위(69.16±1.18) mg/L,명현고우기여각조(P＜0.05)；FQ-PCR결과표명,여대조조비교,2μg/LTGF-β1유도액조적Wnt1、Wnt3a mRNA적표체량분별위0.0266±0.0047、0.0154±0.0033,기표체수도료억제(P＜0.05),β-catenin mRNA적표체량위0.0187 ±0.0021,명현저우대조조(P＜0.01)；상반,Sox9、Collagen Ⅱ mRNA표체량분별위0.0682±0.0024、0.1134±0.0048,표체유소승고(P＜0.05).결론 TGF-β1촉진BMSCs향연골세포분화,Wnt/β-catenin신호통로상관기인표체수도억제,이연골세포특이기인표체증가.
Objective To observe the transforming growth factor-β1 (TGF-β1) on the expression of Wnt signaling pathway related genes during the chondrogenesis of bone marrow stromal cells (BMSCs) in vitro.Methods BMSCs cultured in TGF-β1 medium were observed by inverted phase contrast microscope and identified by toluidine blue staining.The expression levels of glycosaminoglycan (GAG) and target genes including Wnt1,Wnt3a,β-catenin,Sox9 and collagen Ⅱ mRNA under different concentrations of TGF-β1 were detected by using enzyme linked immunosorbent assay (ELISA) and real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) respectively.Results At day 21 after TGF-β1 induction,toluidine blue staining showed that more than 95％ cells differentiated into chondrocytes; the amount of GAG in the cell cultured medium in 2 μg/L TGF-β1-induced group was (69.16 ± 1.18) mg/L,significantly higher than other groups (P ＜ 0.05).FQ-PCR results showed that Wnt1,Wnt3a and β-catenin mRNA expression in 2 μg/L TGF-β1-induced group was inhibited [0.0266 ±0.0047,0.0154 ±0.0033,0.0187 ±0.0021,(P ＜0.05)],but in contrast Sox9 and collagen Ⅱ mRNA expression levels were increased [0.0682 ± 0.0024,0.1134 ± 0.0048 (P ＜ 0.05)] as compared with the control group.Conclusion TGF-β1 could promote BMSCs chondrogenic differentiation,and the expression of Wnt/β-catenin signaling pathway related genes Wnt1,Wnt3a and β-catenin mRNA was inhibited and the expression of chondrocyte specific genes Sox9 and collagen Ⅱ mRNA was increased.