目的：明确在体内Sox9基因过表达的兔骨髓间充质干细胞对于关节软骨损伤修复的作用。方法：以慢病毒介导的Sox9基因转染兔骨髓间充质干细胞（BMSCs），体外检测软骨特异性分子，将新西兰大白兔24只48个膝关节随机分为3组，动物麻醉后，双侧股骨滑车处的关节面上用直径4 mm的钻头钻孔，深度3 mm，穿透软骨下骨，造成全层关节软骨损伤，将转染后的细胞植入体内用以修复全层关节软骨损伤，实验组植入BMSCs-（Lenti-Sox9-EGFP）-藻酸钙复合物，实验对照组植入BMSCs-藻酸钙复合物，空白对照组只钻孔。术后6、12周分别进行光镜、电镜观察，以及HE、免疫组织化学染色检测软骨的修复程度。结果：经Sox9基因转染后的细胞在3 d时，Sox9基因表达最高，随后下降。转染后3 d，Ⅱ型胶原开始表达，到14 d时达到最高。表明Sox9过表达启动了兔骨髓间充质干细胞的软骨分化。组织学观察显示，实验组术后6周缺损处有透明软骨样组织填充，术后12周缺损处软骨和软骨下骨修复良好。两对照组，缺损处由纤维组织填充。免疫组织化学显示，修复组织内Ⅱ型胶原，免疫组化染色结果阳性强于两对照组。组织学评分结果显示实验组软骨损伤修复各时间点效果明显优于两对照组，差异有统计学意义。结论：Sox9基因过表达的兔骨髓间充质干细胞（BMSCs）促进软骨损伤的修复。%Objective:To study the overexpression of Sox9 gene on rabbit bone marrow mesenchymal stem cells for re⁃pairing articular cartilage injury in vivo. Methods:Rabbit bone marrow mesenchymal stem cells(BMSCs)were transduced with lentivirus vector containing Sox9 gene and then cartilage specific molecule was detected by RT-PCR in vitro. Total 48 knee joints of 24 mature New Zealand white rabbits were randomly divided into 3 groups according to different defect treat⁃ment. After animals anesthesia,a full thickness cylindrical cartilage defect of 4 mm diameter and 3 mm deep was created in the patellar groove using a stainlesssteel punch. Meanwhile,the transfected cells were implanted to repair the rabbit model with full thickness cartilage defects. Cartilage defects tissue was observed with light microscope,electron microscope,HE and im⁃munohistochemistry staining to assess the repair of defects by the complex at 6 weeks or 12 weeks after the implantation. Re⁃sults:At 3 days after the transfection,Sox9 gene expression was highest and Sox9 gene expression decreased with the increase of time. At 3 days after the transfection,the expression of collagen typeⅡbegan and reached the peak at 14 days. It showed that the bone marrow mesenchymal stem cells went into chondrogenic differentiation after transfected by Sox 9 gene. Histologi⁃cal observation showed that at 6 weeks after the operation,the defects in the experimental group was filled with hyaline like cartilage tissue,12 weeks after operation,the defects of cartilage and subchondral bone had satisfactory healing. Both at 6 and 12 weeks postoperatively,the defects were filled with fibrous tissues in control groups. Meanwhile,immunohistochemical stain⁃ing of sections with typeⅡcollagen antibodies showed the proteins in the regenerated tissue stained positive for typeⅡcolla⁃gen and stronger than the control groups. The histological scoring system indicated that the cartilage repair of experiment groups were better than the two control groups with statistical significances. Conclusion:Overexpression of Sox9 gene on rab⁃ bit bone marrow mesenchymal stem cells(BMSCs)promote the repair of cartilage defect.
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