hBMP2与hVEGF165双基因腺病毒载体转染BMSCs复合n-HA/PA66组织工程骨构建及检测  

Construction and observation of tissue engineering bone with recombinant adenovirus vectors co- expressing hBMP2 and hVEGF165 infected rabbit bone mesenchymal stem cells and n- HA / PA66 scaffold in vitro

作　　者：张聪[1] 刘洪美[2] 李庆伟[1] 陈国武[1] 梁啸[1] 孟纯阳[1] 

机构地区：[1]济宁医学院附属医院脊柱外科,山东济宁272000 [2]济宁医学院病理学教研室,山东济宁272000

出　　处：《中国矫形外科杂志》2015年第3期270-275,共6页Orthopedic Journal of China

基　　金：山东省自然科学基金资助项目(编号:ZR2010HM090);山东省教育厅资助项目(编号:J14LL04)

摘　　要：[目的]探讨腺病毒介导共表达人骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)和血管内皮生长因子165(vascular endothelia growth factor 165,VEGF165)载体转染兔骨髓间充质干细胞复合纳米羟基磷灰石/聚酰胺66(nano-hydroxyapatite/polyamide66,n-HA/PA66)体外构建组织工程骨的可行性。[方法]采用密度梯度离心法分离骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),取第3代BMSCs进行细胞表型鉴定;第3代BMSCs作为细胞实验对象,实验共分2组:Ad-BMP2-VEGF165为实验组和Ad-GFP为对照组;首先用不同感染复数感染BMSCs,并进行ELISA和Western blot检测细胞h BMP2和h VEGF165的表达;其次将感染后的细胞接种到n-HA/PA66生物材料上,7 d后扫描电镜观察细胞贴附、生长状况;同时,7 d后消化收集贴附n-HA/PA66上的细胞,Western blot检测贴附生物材料细胞中骨钙素(osteocalcin,OC)和骨桥蛋白(osteopontin,OPN)的表达,以及3 d和7 d后的碱性磷酸酶(alkaline phosphatase,ALP)活性测定。[结果]第3代细胞表面高表达CD29(99.82%)和CD44(94.14%),低表达CD14(3.11%)和CD34(0.34%);腺病毒感染后h BMP2和h VEGF165蛋白水平高表达;扫描电镜见感染细胞分布均匀,生长状态良好。实验组与对照组相比,复合n-HA/PA66后Western Blot检测显示OC和OPN表达和ALP活性均较高,两组差异均有统计学意义(P<0.05)。[结论]Ad-BMP2-VEGF165双基因腺病毒感染BMSCs后目的蛋白较高量表达,同时感染后BMSCs可以在n-HA/PA66进行良好的黏附、生长、增殖、矿化,Ad-BMP2-VEGF165双基因表达组织工程骨构建成功。[Objective] To investigate the feasibility and advantages of constructing a tissue engineered bone through adenovirus infection using n- HA / PA66 and bone marrow mesenchymal stem cells and modification with human bone morphogenetic protein 2 gene and human vascular endothelia growth factor 165 gene in vitro. [Methods] The method of density gradient centrifugation was used to separate from bone marrow mesenchymal stem cells and the cell phenotype of third generation of cells was identified. The third generation of BMSCs was selected as subjects,vectors of Ad- BMP2- VEGF165 and Ad- GFP infected BMSCs were regarded as experimental group and control group,respectively. Bone marrow mesenchymal stem cells was transfected by different multiplicity of infection and the expression of BMP2 and VEGF165 was analyzed by the means of ELISA and Western blot. Cells were inoculated into the n- HA / PA66 and the growth conditions state of cells was obsenved with scanning electron microscope after 7 days. Meanswhile,after 7 days of coulping with n- HA / PA66,BMSCs were collected and protein of osteocalcin and osteopontin were detected by Western Blot,and at 3 and 7 days later. Alkaline phosphatase activity were also determinated. [Results] The results of cell surface antigen detected by flow cytometry after 3rd passage showed that BMSCs were hypso- expression CD29（ 99. 82%） and CD44（ 94. 14%）, and then the CD14（ 3. 11%） and CD34（ 0. 34%） were hypo- expression. The protein levels of BMP2 and VEGF165 in the cells and cultured medium after infection was detected. In addition,SEM examination revealed extensive cellular attachment and growth on the n- HA / PA66. Detection of ALP activity at 3 and 7 days and protein expression of OC and OPN in experimental group at 7 days showed higher expression than in control group. [Conclusion] Ad- BMP2- VEGF165 could infect BMSC with high efficiency and interesting proteins are higher expression than in control groups. The cells after infection grew well on a n- HA / PA66 scaffold a

关 键 词：骨形态发生蛋白2 血管内皮生长因子165 骨髓间充质干细胞 生物支架 组织工程骨 

分 类 号：R318.08[医药卫生—生物医学工程]