骨软骨前体细胞分离鉴定及转化生长因子β3对其成软骨分化的影响

摘要

背景:关节软骨中存在着前软骨干细胞,可能成为软骨组织工程潜在的种子细胞,转化生长因子β3对前软骨干细胞的增殖及成软骨分化具有正向调节作用.目的:分析转化生长因子β3对骨软骨前体细胞成软骨的分化作用.方法:分离筛选出晚期骨关节炎患者软骨组织 CD146+软骨细胞并鉴定.培养 CD146+软骨细胞团块分为4组:为普通培养基组,转化生长因子β3-诱导组,转化生长因子β3+诱导组(含2.5 μg/L重组人转化生长因子β3)和转化生长因子β3++诱导组(含10 μg/L重组人转化生长因子β3).培养4周后行组织块Ⅱ型胶原、Aggrecan免疫组织化学及相关基因实时荧光定量PCR检测.结果与结论:①成软骨诱导培养时,转化生长因子β3++诱导组所形成软骨块＞转化生长因子β3+诱导组＞转化生长因子β3-诱导组;②免疫组织化学结果显示,转化生长因子β3++诱导组Ⅱ型胶原和Aggrecan的表达明显高于转化生长因子β3+诱导组(P＜ 0.05),转化生长因子β3+诱导组表达明显高于转化生长因子β3-诱导组(P＜ 0.05);③实时荧光定量PCR检测显示,转化生长因子β3++诱导组Ⅱ型胶原和Aggrecan mRNA的表达明显强于转化生长因子β3+诱导组(P＜ 0.05),转化生长因子β3+诱导组2指标表达明显强于转化生长因子β3-诱导组(P＜ 0.05);转化生长因子β3++诱导组和转化生长因子β3+诱导组性别决定区Y框蛋白-9的表达均明显强于转化生长因子β3-诱导组(P＜ 0.05);④结果表明,晚期骨关节炎患者残余关节软骨中存在着具有干细胞特性的骨软骨前体细胞;转化生长因子β3具有较强促骨软骨前体细胞成软骨分化的能力,其有可能成为软骨组织工程中理想的细胞因子.%BACKGROUND: Precartilaginous stem cells exist in articular cartilage, which may become potential seed cells for cartilage tissue engineering. Transforming growth factor-β3 (TGF-β3) has positive regulation effect on the proliferation and chondrogenic differentiation of precartilagious stem cells. OBJECTIVE: To investigate the effect of TGF-β3 on the chondrogenesis of osteochondroprogenitor cells. METHODS: CD146+chondrocytes were isolated from the patients with advanced osteoarthritis, and were then identified. CD146+chondrocytes were cultured in the normal medium (blank control group), chondrogenic induced medium (control group), chondrogenic induced medium containing 2.5 and 10 μg/L recombinant human TGF-β3, respectively. The immunohistochemistry of collagen Ⅱ and aggrecan was performed, and the related gene expression was tested by real-time quantitative PCR after 4 weeks of culture. RESULTS AND CONCLUSION: When chondrogenic differentiation was performed, the number of cell pellets in the 10 μg/L TGF-β3 group was greater than that in the 2.5 μg/L TGF-β3 group, and the number of cell pellets in the 2.5 μg/L TGF-β3 group was greater than that in the control group. The expression levels of collagen Ⅱ and aggrecan in the 10 μg/L TGF-β3 group was significantly higher than that in the 2.5 μg/L TGF-β3 group (P＜ 0.05). The expression levels of collagen Ⅱ and aggrecan in the 2.5 μg/L TGF-β3 group were significantly higher than those in the control group (P＜ 0.05). Real-time quantitative PCR results showed that the expression levels of collagen Ⅱ and aggrecan mRNA in the 10 μg/L TGF-β3 group were significantly higher than those in the 2.5 μg/L TGF-β3 group (P＜ 0.05), but the expression level of SOX-9 showed insignificant difference between two groups (P ＞ 0.05), and the expression level of SOX-9 in the 10 and 2.5 μg/L TGF-β3 groups was significantly higher than that in the control group (P＜ 0.05); the expression levels of collagen Ⅱ and aggrecan mRNA in the 2.5 μg/L TGF-β3 group were higher than those in the control group (P＜ 0.05). Our findings suggest that osteochondroprogenitor cells with stem cell characteristics exist in the residual articular cartilage of the patients with advanced osteoarthritis. TGF-β3 has the ability of promoting chondrogenic differentiation of osteochondroprogenitor cells, which may be an ideal cytokine for cartilage tissue engineering.