转染人血管内皮生长因子基因的组织工程骨修复眼眶壁骨缺损的实验研究

VEGF Gene Transfected Human BMSCs Acting as Seeding Cell for Tissue Engineering Bond in Orbital Wall Bone Defects

目的探讨以转染人血管内皮生长因子(Vascular endothelial growth factor,VEGF)基因的骨髓基质干细胞(Bone marrow stromal cells,BMSCs)构建的组织工程骨在犬眼眶壁骨缺损中的修复效果。方法体外分离扩增犬自体BMSCs至第2代,用腺病毒转染VEGF基因。采用Real-time PCR和Western Blot检测目的基因和蛋白表达情况。细胞接种在珊瑚支架材料上构建组织工程骨,Elisa检测转基因细胞在支架材料上的蛋白持续表达情况。成年比格犬24只,双侧眼眶内侧壁制造直径12mm圆形骨缺损模型,随机分为4组:A组植入转染VEGF的组织工程骨,B组植入未转染基因的组织工程骨,C组植入单纯珊瑚材料,D组为旷置组。分别在手术后4周、12周、24周取材,行大体观察、Micro-CT分析、免疫组化方法检测血管形成情况,以及组织学观察和组织形态学检测比较骨缺损修复效果。结果VEGF基因修饰的BMSCs能够高表达目的基因和蛋白,构建组织工程骨后能够在体外持续分泌VEGF蛋白22d。C组和D组均未修复眶壁骨缺损。A组在骨缺损修复过程中,4周时的新生血管形成量和新生骨体积明显高于B组(P<0.05),但12周、24周时两组的新生血管量和新生骨量均无显著性差异(P>0.05)。结论转染VEGF的BMSCs构建的组织工程骨体内回植后早期能够促进新生血管形成,加速新骨生成。

Objective To investigate the effect VEGF-gene transfected BMSCs to construct tissue engineered bone in the treatment of orbital wall bone defects. Methods In vitro isolated and expanded BMSCs of passage 2 were transfeeted with adenoviruses containing human VEGF gene, and the target gene expression was evaluated by Real-time PCR and Western Blot. BMSCs were harvested and seeded into the coral scaffolds to construct tissue engineered bone. VEGF protein secretion from the cells was detected using Elisa method. Bilateral orbital wall circular defects （12 mm in diameter） were created in 24 adult beagles dogs. The dogs were divided into 5 groups, the group trasfected by VEGF （Group A）, the non-transfeeted group （Group B）, the group implanted coral alone （Group C）, and blank group （Group D）. Animals were sacrificed at 4, 12 and 24 weeks post-implantation, and the repairing effect was evaluated by gross observation, Miero-CT, immunohistochemistry, and histomorphometric analysis. Results VEGF gene expression was regulated after gene transfeetion, and protein secretion was detected 22 days after cell seeding. No repair was found in Groups C and D. The number of new blood vessel and volume of new bone were significantly higher in Group A than in Group B at 4 weeks after implantation （P〈0.05）, but no statistically significant at 12 and 24 weeks （P〉0.05）. Conclusion Tissue-engineered bone composed of VEGF-expressing BMSCs could enhance the formation of blood vessel and bone at early periods in vivo.