角蛋白支架联合TGF-β3基因过表达载体质粒转染的骨髓间充质干细胞对软骨缺损的修复效果

EFFECT OF KERATIN SCAFFOLD COMBINED WITH BONE MARROW MESENCHYMAL STEM CELLS TRANSFECTED WITH TRANSFORMING GROWTH FACTOR-β3 GENE OVEREXPRESSION VECTOR PLASMID IN THE REPAIR OF CARTILAGE DEFECT

目的通过构建新西兰种大白兔的软骨缺损模型,探讨角蛋白支架联合转化生长因子-β3(TGF-β3)基因过表达载体质粒转染的骨髓间充质干细胞(BMSC)复合物对软骨缺损的修复作用。方法构建大白兔的软骨缺损模型;将新西兰大白兔随机分为模型组(A组)、支架组(B组)、联合BMSC组(C组)和联合过表达质粒转染BMSC组(D组),每组5只,B组软骨缺损处填充角蛋白支架,C组软骨缺损处填充角蛋白支架联合BMSC复合物,D组软骨缺损处填充角蛋白支架联合TGF-β3基因过表达载体转染的BMSC复合物。利用苏木精-伊红(HE)染色、番红-固绿染色和免疫组织化学染色检测各组大白兔缺损软骨修复情况,并且利用改良O′Driscoll评分系统评估HE染色结果,利用改良Mankin评分系统评估番红-固绿染色结果。结果HE染色和番红-固绿染色结果显示,D组软骨缺损基本愈合,愈合处多为软骨组织。A组、B组、C组和D组O′Driscoll评分比较差异有显著性(F=9.342,P<0.05),其中D组O′Driscoll评分明显低于其他组(q=2.018~2.498,P<0.05);A组、B组、C组和D组Mankin评分比较差异均具有显著性(F=8.549,P<0.05);其中D组Mankin评分明显高于其他组(q=2.237~3.457,P<0.05)。免疫组织化学染色结果显示,C组和D组软骨缺损处填充物均可见Ⅱ型胶原蛋白表达,D组表达量更多。结论角蛋白支架联合TGF-β3基因过表达载体质粒转染的BMSC复合物可以促进缺损软骨的修复。

Objective To investigate the effect of the complex of keratin scaffold combined with bone marrow mesenchymal stem cells(BMSC)transfected with transforming growth factor-β3(TGF-β3)gene overexpression vector plasmid in the repair of cartilage defect by establishing a New Zealand white rabbit model of cartilage defect.Methods A New Zealand white rabbit model of cartilage defect was established,and then the rabbits were divided into model group(group A),scaffold group(group B),scaffold+BMSC group(group C),and scaffold+BMSC transfected with TGF-β3 gene overexpression vector plasmid group(group D),with 5 rabbits in each group.For group B,the cartilage defect was filled with keratin scaffold;for group C,the cartilage defect was filled with the complex of keratin scaffold combined with BMSC;for group D,the cartilage defect was filled with the complex of keratin scaffold combined with BMSC transfected with TGF-β3 gene overexpression vector plasmid.HE staining,safranin-fast green staining,and immunohistochemical staining were used to evaluate the repair of cartilage defect;the modified O′Driscoll score was used to evaluate the results of HE staining,and the modified Mankin score was used to evaluate the results of safranin-fast green staining.Results HE staining and safranin-fast green staining showed that the cartilage defect in group D was basically healed,mostly with cartilage tissue.There was a significant difference in modified O′Driscoll score between groups A,B,C,and D(F=9.342,P<0.05),and group D had a significantly lower O′Driscoll score than the other groups(q=2.018-2.498,P<0.05).There was a significant difference in Mankin score between groups A,B,C,and D(F=8.549,P<0.05),and group D had a significantly higher Mankin score than the other groups(q=2.237-3.457,P<0.05).Immunohistochemical staining showed that the expression of typeⅡcollagen was observed in the fillers of cartilage defect in groups C and D,with a higher expression level in group D.Conclusion The complex of keratin scaffold combined with BMSC transfected with TGF-β3 gene overexpression vector plasmid can promote the repair of cartilage defect.