骨形成蛋白2基因修饰的骨髓基质细胞与纤维蛋白复合物修复兔关节软骨缺损  

APPLICATION OF ADENOVIRUS BONE MORPHOGENETIC PROTEIN 2 GENE TRANSFER INDUCING MESENCHYMAL STEM CELLS WITH FIBRIN GEL IN TREATING RABBIT CARTILAGE DEFECT

作　　者：王立春[1] 刘明辉[2] 张宏颖 石义钢[1] 韩剑锋[1] 韩竹[1] 刘建宇[1] 林欣[1] 

机构地区：[1]哈尔滨医科大学附属第二医院脊柱外科,哈尔滨150086 [2]四川大学华西医院骨科 [3]哈尔滨市穆斯林医院内科

出　　处：《中国修复重建外科杂志》2005年第12期960-965,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：哈尔滨市青年科学研究基金资助项目(2004AFQXJ006);黑龙江省自然科学基金资助项目(D01-11)~~

摘　　要：目的研究骨形成蛋白2(bone mophogenetic protein 2, BMP-2)重组腺病毒(adenovirus)转染骨髓基质细胞(mesenchymal stem cells, MSCs)后,与纤维蛋白凝胶构成的复合物(Ad-BMP-2+MSCs-纤维蛋白凝胶)对兔关节软骨缺损修复的影响. 方法①Ad-BMP-2转染原代培养的MSCs,通过RT-PCR、细胞免疫组织化学染色、甲苯胺蓝染色等观察转染后3～9 d的MSCs其BMP-2、Ⅱ型胶原及蛋白多糖转录、表达水平的变化.②转染后的MSCs种植于纤维蛋白凝胶,在体外培养1～9 d,通过上述指标及透射电镜观察三维培养条件下其软骨基质的产生.③42只日本大耳白兔先制成直径4.5 mm全层软骨缺损模型,随机分为3组(n=14):A组为Ad-BMP-2+MSCs-纤维蛋白凝胶修复组,B组为MSCs-纤维蛋白凝胶修复组,C组为空白对照组.术后4、8和12周取材行大体观察、HE染色、甲苯胺蓝染色、Ⅱ型胶原免疫组织化学及12周关节软骨弹性常数检测. 结果①Ad-BMP-2转基因MSCs的BMP-2、Ⅱ型胶原 mRNA RT-PCR检测分别在3、5 d呈阳性,与对照组比较差异有统计学意义(P<0.05).②转基因MSCs三维培养物免疫组织化学和甲苯胺蓝染色呈阳性,电镜显示细胞生长良好,有基质合成.③A组各时间点大体、组织形态学和组织化学染色均显著优于B组和C组,12周时力学和组织学已接近正常关节软骨. 结论 Ad-BMP-2能通过促进MSCs分泌BMP-2,诱导Ⅱ型胶原和蛋白多糖的表达,在纤维蛋白三维支架中形成软骨基质,移植入兔关节软骨缺损区可修复直径4.5 mm缺损,其修复组织成分接近正常软骨.Objective To study the effect of adenovirus bone morphogenetic protein 2 gene （Ad-BMP-2） transfer inducing mesenchymal stem cells （MSCs） compounded with fibrin gel on repair of rabbit cartilage defect. Methods ①BMP-2 and collagen type Ⅱ in MSCs transferred by Ad-BMP 2 were examined by RT-PCR, aniline dyeing and immunohistochemical analysis in vitro. ②MSCs were cultured in fibrin gel for 9 days, and were examined with electron microscope. ③Forty-two rabbits suffering from cartilage defect were divided into 3 groups：the defects were treated with Ad BMP 2 transfer inducing MSCs compounded with fibrin in group A, with MSCs compounded with fibrin gel in group B and with no implants in group C as control. HE and aniline dyeing, immunohistochemical analysis and biomechanics study were carried out in the 4th, 8th and 12th weeks. Results ①The positive results were observed for BMP-2 and collagen type Ⅱ with RT-PCR on the 3rd day and 5th day respectively, being statistically significant difference when compared with control group （P〈0.05）. ② Ad-BMP-2 transfer inducing MSCs cultured in fibrin gel were positively stained by aniline dyeing and immunohistochemstry. ③The therapy effect of group A was better than that of the other two groups in histology, biochemistry and biomechanics, and the biomechanic and histological features of repaired cartilage were similar to those of the natural cartilage. Conclusion Ad-BMP-2 can induce the expression of collagen type Ⅱ and mucopolysaccharide in MSCs by secreting BMP-2, and can reconstruct articular cartilage defects better when compounded with fibrin gel.

关 键 词：关节软骨缺损 骨形成蛋白2 转基因 骨髓基质细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]