软骨形态发生蛋白1诱导瘢痕成纤维细胞表达软骨表型的实验研究

Cartilage-Derived Morphogenetic Protein 1 in the Induction of Chondrogenic Differentiation of Scar Fibroblasts in Vitro

目的应用软骨形态发生蛋白1(CDPM1)诱导瘢痕成纤维细胞向软骨细胞分化,探讨其作为烧伤后耳软骨缺损修复的种子细胞的可行性。方法取瘢痕切除术患者的增生性瘢痕组织,提取瘢痕成纤维细胞,CDPM1诱导培养(2%胎牛血清F-12培养液,CDMP1终浓度为100 ng/mL),设阴性、阳性对照组。Image Plus图像软件观察细胞形态变化;免疫荧光、RT-PCR及Western Blot分析诱导前后的软骨相关Ⅰ、Ⅱ型胶原、Sox9、Aggrecan的表达;对第4代细胞行Micromass法培养诱导,HE染色、免疫组织化学染色及Safranine-O染色对诱导结果进行检测。结果诱导后瘢痕成纤维细胞由长梭形向多角形转变,Image Plus软件分析示诱导后细胞长宽比例为(1.48±0.21):1,与诱导前的(7.21±1.54):1相比,差异有统计学意义(P<0.05);与软骨细胞的(1.31±0.13):1相比,差异无统计学意义(P>0.05)。免疫荧光、RT-PCR、Western Blot结果显示,诱导后瘢痕成纤维表达软骨相关指标Ⅱ型胶原、Sox9、Aggrecan。Micromass诱导后行HE染色显示,诱导后部分细胞出现软骨特征性陷窝结构,免疫组织化学染色显示Ⅱ型胶原阳性染色,Safranine-O染色显示微团块诱导后细胞基质红染。结论瘢痕成纤维细胞在软骨形态发生蛋白1诱导下可以向软骨细胞分化,有望成为烧伤后耳软骨缺损修复的种子细胞。

Objective To investigate the feasibility of the differentiation of scar fibroblasts into chondrogenic phenotype in vitro induced by Cartilage-derived morphogenetic protein 1(CDMP1) for the reconstruction of ear defect.Methods Scar fibroblasts were extracted from hypertrophic scar and cultured in momo-layer in vitro.Scar fibroblasts of the 4th passage were induced by CDMP1(100 ng/mL in medium of F12+2%FBS).Negative and positive control groups were also set up.After 7 days of induction,morphological change of cells was observed by Image Plus software.Expression of col Ⅰ,col Ⅱ,sox 9 and aggrecan were detected by immunofluorescence,RT-PCR and western blot.Micromass cultured scar fibroblasts were induced and cultured.The results were analyzed by HE staining,immunohistochemistry and Safranine-O staining.Results After induction,the morphology of the cells changed from spindle to polygon shape.By Image Plus software analysis,the length/width ratio changed from(7.21±1.54):1 of pre-induction to(1.48±0.21):1 of post-induction(P<0.05).No significant difference was found between post-induction fibroblasts and normal chondrocytes(P>0.05).Col Ⅱ,sox9,aggrecan were expressed in post-inductive cells by immunofluorescence,RT-PCR and western blot.HE staining,immunohistochemistry and Safranine-O staining revealed cartilage specific structure and deposition of cartilage extracellular matrix in micromass cultured scar fibroblasts.Conclusion Scar fibroblasts could be differentiated into chondrogenic phenotype by CDMP1 induction and induced scar fibroblasts can be used as the seed cells for the reconstruction of ear defect.