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目的研究BMP-14与软骨细胞共同诱导脂肪来源干细胞(adipose-derived stem cells,ADSCs)向软骨细胞分化是否具有协同作用,优化软骨组织工程种子细胞来源。方法取2只雄性新西兰大白兔(体重1.5、2.0 kg)关节软骨和皮下脂肪组织,分离、培养软骨细胞和ADSCs,取第3代细胞进行实验。对ADSCs行成骨(茜素红染色)、成软骨(阿利新蓝染色)和成脂(油红O染色)诱导鉴定。测定Ad-巨细胞病毒-BMP-14-内部核糖体进入位点-人源化海肾绿色荧光蛋白1转染的最佳感染复数(multiplicity of infection,MOI)并以其转染ADSCs。实验分为5组:A组,采用Transwell小室将BMP-14转染的ADSCs与软骨细胞共培养(1∶1);B组,采用Transwell小室将未转染的ADSCs与软骨细胞共培养(1∶1);C组,单纯BMP-14转染的ADSCs培养;D组,单纯软骨细胞培养;E组,单纯ADSCs培养。培养3周后,取细胞采用阿利新蓝法检测糖胺聚糖(glycosaminoglycan,GAG)含量,Western blot检测BMP-14和Ⅱ型胶原蛋白表达,RT-PCR检测Sox-9基因表达。结果培养细胞经鉴定提示为ADSCs。倒置荧光显微镜观察示,MOI值为150时转染效果最好。A、C组GAG含量、BMP-14和Ⅱ型胶原蛋白表达以及Sox-9基因表达均明显高于其余3组,A组高于C组,差异均有统计学意义(P<0.05);B、D组均明显高于E组(P<0.05),B、D组间差异均无统计学意义(P>0.05)。结论 BMP-14与软骨细胞能共同诱导并促进兔ADSCs向软骨细胞分化,两者之间具有协同作用。
OBJECTIVE: To investigate whether BMP-14 and chondrocytes co-induce adipose-derived stem cells (ADSCs) differentiating into chondrocytes with synergistic effect and optimize the source of cartilage tissue engineering seed cells. Methods Articular cartilage and subcutaneous adipose tissue were obtained from two male New Zealand white rabbits. The chondrocytes and ADSCs were isolated and cultured. The third generation cells were used for the experiment. ADSCs were induced to osteogenic (Alizarin red staining), cartilage (Alitime blue staining) and adipogenic (Oil red O staining). The optimal multiplicity of infection (MOI) for transfection of Ad-CMV-BMP-14-internal ribosome entry site-humanized renina green-fluorescent protein 1 was determined and transfected into ADSCs. The experiment was divided into five groups: Group A, BMP-14-transfected ADSCs were co-cultured with chondrocytes (1: 1) in Transwell chamber; Group B, untransfected ADSCs and chondrocytes were co- : 1); Group C, BMP-14-transfected ADSCs; Group D, chondrocytes alone; Group E, ADSCs alone. After cultured for 3 weeks, the cells were harvested for the determination of glycosaminoglycan (GAG) content by Western Blot. The expression of BMP-14 and collagen Ⅱ was detected by Western blot. The expression of Sox-9 gene was detected by RT-PCR. Results The cultured cells were identified as ADSCs. Inverted fluorescence microscopy showed that MOI value of 150 transfection was the best. The GAG content, the expression of BMP-14 and type II collagen, and the expression of Sox-9 in group A and group C were significantly higher than those in the other three groups (P <0.05). The difference was statistically significant (P <0.05) , D group were significantly higher than the E group (P <0.05), B, D group differences were not statistically significant (P> 0.05). Conclusion BMP-14 and chondrocytes can induce ADSCs to differentiate into chondrocytes in a cooperative manner.