腺病毒介导BMP-4转染兔SMSCs软骨分化研究

目的 研究腺病毒介导BMP-4并带有绿色荧光蛋白(EGFP)基因转染兔滑膜间充质干细胞(SMSCs)向软骨细胞分化的可行性。方法 兔膝关节腔取材，用Ⅰ型胶原酶消化分离获取SMSCs，采用流式细胞技术检测表面标记物。构建包含BMP-4基因和EGFP基因的腺病毒载体(Ad-BMP-4)并包装病毒。用不同感染复数(MOI分别为50、100、200、300、400、500)的病毒感染SMSCs，观察各组转染效率及检测不同MOI条件下细胞生长曲线，确定最合适的病毒感染滴度。进而应用ELISA检测分析染后的SMSCs是否进入软骨细胞分化谱系。结果 经分离纯化后获取的SMSCs形态学表现出间充质干细胞应有的结构，表面标记蛋白CD44、CD90高度表达，但CD34、CD45基本不表达，符合SMSCs的特征。分别以50、100、200、300、400、500MOI感染后，感染效率分别为54.3%、72.2%、77.8%、85%、86.6%、89.3%，而且感染效率依赖MOI高低，结合生长曲线300MOI为最佳感染滴度。ELISA法检测发现BMP-4显著高表达，同时软骨基因相关蛋白SOX9、COLⅡ及AGC蛋白也高表达。结论 应用300MOI重组腺病毒感染兔SMSCs细胞，能维持细胞正常增殖水平，且能快速高效地表达BMP-4并激活其他软骨相关基因，可促使SMSCs向软骨细胞分化。

Feasibility Study of Adenovirus Mediated BMP-4 Gene Transfection in Rabbit SMSCs to Cartilage

Objective To study the feasibility of adenovirus-mediated BMP-4 transfected rabbit synovial mesenchymal stem cells (SMSCs) with green fluorescent protein (EGFP) gene into chondrocytes. Method The rabbit knee joint capsule was taken, and the type I collagenase was used to digest and separate the SMSCs. The surface markers were detected by flow cytometry. The recombinant adenovirus (Ad-BMP-4) containing BMP-4 gene and EGFP gene was transfected into the SMSCs by different multiplicity of infection (50,100,200,300,400,500 MOI respectively) Efficiency, find the most suitable transfection conditions, and test the cell growth curve under different transfection conditions. The EGFP-labeled cells were observed by ELISA to determine if the transfected SMSCs entered the chondrocyte differentiation lineage. Result The morphology of SMSCs obtained after isolation and purification showed the proper structure of mesenchymal stem cells. The surface markers highly expressed CD45 and CD90, and basically did not express CD34 and CD45, which was consistent with many features of SMSCs. The transfection efficiencies were 54.3%, 72.2%, 77.8%, 85%, 86.6%, 89.3% after transfection with MOI of 50,100,200,300,400,500, respectively, and the fluorescence microscopy was more and more High green fluorescence expression, MOI=300 for transfection suitable conditions. The result of ELISA showed that BMP-4 was highly expressed, and cartilage genes such as SOX9, COLⅡ and AGC protein were also highly expressed. Conclusion This study shows that recombinant adenovirus transfected rabbit SMSCs, MOI amount of 300 can quickly and efficiently express BMP-4 and activate other cartilage-related genes, prompting SMSCs to chondrocytes.