中文摘要：

目的：构建人骨形态发生蛋白2及血管内皮生长因子基因共表达腺病毒并观察其在兔骨髓基质干细胞中的表达。 方法：实验于2005-03／12在解放军第四军医大学唐都医院全军骨肿瘤研究所完成。①分别克隆人骨形态发生蛋白2及血管内皮生长因子基因，分别依次亚克隆至穿梭载体构建重组穿梭质粒pAdT-B2V并酶切鉴定；后经PmeI酶切线性化后与骨架质粒感受态细胞AdEasier-1 Cells经电穿孔法同源重组得到腺病毒质粒pAdBMP-7并酶切鉴定。②PacI酶切线性化后的pAdBMP-7腺病毒质粒转染293细胞包装后获得复制缺陷型重组腺病毒AdBMP-7，在荧光显微镜下计算表达绿色荧光蛋白细胞个数，测定AdB2V滴度。③将AdBMP-7体外感染兔骨髓基质干细胞后，以荧光显微镜观察绿色荧光蛋白表达及反转录-聚合酶链反应方法鉴定外源基因的表达，以B-肌动蛋白为内参照，未感染细胞为对照。④AdB2V感染兔骨髓基质干细胞后72h以免疫细胞化学染色方法检测外源基因的表达，以未感染组为对照。 结果：①酶切鉴定表明外源基因已插入到pAdTrack—CMV穿梭质粒且腺病毒重组质粒pAdBMP-7构建成功。②经293细胞包装3d后可观察到绿色荧光蛋白表达，氯化铯梯度离心法最终获得约3×10^9 efu／L滴度的重组腺病毒。③体外感染兔骨髓基质干细胞后荧光显微镜观察及反转录-聚合酶链反应证实外源基因可在兔骨髓基质干细胞中表达，未感染组为阴性。④AdB2V感染兔骨髓基质干细胞后以免疫细胞化学染色均观察到外源基因的阳性表达，未感染组呈阴性表达。 结论：成功构建人骨形态发生蛋白2及血管内皮生长因子165基因共表达重组腺病毒，证实了其在兔骨髓基质干细胞的表达，为骨再生的局部联和基因治疗打下基础。

英文摘要：

AIM：To construct the recombinant adenovirus bearing human bone morphogenetic protein 2 and vascular endothelial growth factor （VEGF） gene, and observe its expression in rabbit bone marrow-derived stromal stem cells. METHODS：The experiment was accomplished at the Orthopedics Oncology Institute of Chinese PEA , Tangdu Hospital of Fourth Military Medical University of Chinese PLA from March to December 2005. ① Human bone morphogenetic protein 2 gene and VEGF gene were cloned and sub-cloned into Shuttle vector to construct the recombinant shuttle plasmid pAdT-B2V and conduct restriction enzyme, which was linearized by PmeI and co-transformed into adenoviral backbone plasmid competent cells-AdEasier-1 cells to obtain recombinant adenoviral plasmid pAdBMP- 7 by homologous recombination. ②The recombinant adenovirus AdBMP-7 of replication defect was obtained after pAdBMP-7 was then linearized with PacI enzyme and transfected into 293 cells to get packed adenoviruses. The number of green fluorescent protein was calculated under fluorescence microscope, and the virus titre was determined. ③The AdBMP-7 was infected by rabbit bone marrow stromal cells in vitro and the expression of green fluorescent protein was observed under fluorescence microscope, and exogenous genes expression were detected by reverse transcriptionpolymerase chain reaction method. The uninfected cells were taken as control and β-actin was used as inner-control. ④Expression of exogenous genes was detected by immunocytochemical staining methods at 72 hours after AdB2V infection by rabbit bone marrow stromal cells, and the uninfected group was taken as control. RESULTS： ① The restriction endonuclease digestion showed that exogenous gene was inserted into recombinant plasmid pAdTrack-CMV and pAdBMP-7 was successfully constructed. ② Green fluorescent protein expression could be observed on the third day after packing of the linearized pAdBMP-7 in 293 cells and 3×10^9 efu/L titer of Ad-BMP-7 was obtained by CsCl gradient purificatio