hVEGF_(165)及hBMP-7共表达重组腺相关病毒载体的体外生物学活性研究  

STUDY ON BIOLOGICAL ACTIVITY OF RECOMBINANT ADENO-ASSOCIATED VIRUS VECTOR COEXPRESSING HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR 165 AND HUMAN BONE MORPHOGENETIC PROTEIN 7 GENES IN VITRO

作　　者：时志斌[1] 樊立宏[1] 强辉[1] 唐一仑[1] 王坤正[1] 党晓谦[1] 

机构地区：[1]西安交通大学医学院第二附属医院骨一科,西安710004

出　　处：《中国修复重建外科杂志》2010年第4期415-419,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30600624);陕西省重点科研资助项目[2005k14-G1(1)]~~

摘　　要：目的通过体外实验探讨hVEGF165和hBMP-7共表达重组腺相关病毒载体(recombinant adeno-associated virus,rAAV)体外生物学活性,为体内应用其进行骨坏死的基因治疗奠定理论基础。方法分别采用rAAV-hVEGF165-内部核糖体进入位点序列(internal ribosome entry site,IRES)-hBMP-7(实验组)、绿色荧光蛋白(green fluorescent protein,GFP)标记的rAAV-IRES-GFP(对照组)转染体外培养的第3代兔BMSCs,于转染后1、2、3、7及14d应用ELISA法及转染后14d应用Western blot法鉴定两组hVEGF165和hBMP-7表达;转染后14d行细胞免疫荧光染色观察hVEGF165和hBMP-7表达一致性。应用第3代人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)管状血管形成实验检测hVEGF165蛋白活性。取第3代BMSCs行诱导成骨实验,Gomori钙钴法ALP染色及茜素红法钙盐染色检测hBMP-7蛋白活性。结果ELISA检测示随转染时间延长,两组hVEGF165和hBMP-7表达逐渐升高;实验组各时间点hVEGF165和hBMP-7表达量均明显高于对照组(P<0.05)。Western blot检测示实验组可见hVEGF165和hBMP-7表达,对照组未见阳性表达。细胞免疫荧光染色观察示实验组hVEGF165和hBMP-7染色呈阳性,表达部位和强度具有较好一致性;对照组未见阳性表达。HUVEC管状血管形成实验示实验组HUVEC拉长变形、出芽且相互交联成管状样结构;与对照组相比,管状血管数比较差异有统计学意义(P<0.05)。ALP染色见实验组细胞出现深染颗粒,对照组细胞内浅着色;与对照组相比,矿化结节数比较差异有统计学意义(P<0.05)。结论双基因共表达rAAV-hVEGF165-IRES-hBMP-7体外具有良好的生物学活性。Objective To study the biological activity of recombinant adeno-associated virus vector（rAAV）coexpressing human vascular endothelial growth factor 165（hVEGF165）and human bone morphogenetic protein 7（hBMP-7） genes in vitro so as to provide a new method for the therapeutics of osteonecrosis.Methods The 3rd passage rabbit bone marrow mesenchymal stem cells（BMSCs）were transfected with rAAV-hVEGF165-internal ribosome entry site（IRES）-hBMP-7 （experimental group）and green fluorescent protein（GFP）labeled rAAV-IRES-GFP（control group）.The expressions of hVEGF165 and hBMP-7 were detected by ELISA assay at the 1st,2nd,3rd,7th,14th days and Western blot assay at the14th day after transfection.The expression consistencies of hVEGF165 and hBMP-7 were observed by immunofluorescence assay at the 14th day after transfection.The biological activity of hVEGF165 was assessed by angiopoiesis experiment of the 3rd passage human umbilical vein endothelial cells（HUVEC）.The biological activity of hBMP-7 was assessed by mineralization of BMSCs detected by ALP staining and alizarin red staining.Results With infecting time,the hVEGF165 and hBMP-7 expressions increased gradually in two groups,showing significant difference between two groups（P0.05）.The expressions of hVEGF165 and hBMP-7 were positive in experimental group and negative in control group,respectively.Immunofluorescence assay showed positive expressions of hVEGF165 and hBMP-7 in the exprimental group and negative expression in the control group,the expression of hVEGF165 and hBMP-7 had good consistencies.hVEGF165 secreted from BMSCs enhanced HUVEC migration,proliferation and tube formation in experimental group.There was significant difference in the number of blood vessel between two groups（P0.05）.The ALP staining showed more strongly stained granules in experimental group than in control group.There was significant difference in the number of the mineralized nodules between two groups（P0.05）.Conclusion The rAAV-hVEGF165-IRES-hBMP-

关 键 词：重组腺相关病毒载体 HVEGF165 HBMP-7 共表达 生物学活性 

分 类 号：R341[医药卫生—基础医学]