重组腺病毒介导的血管内皮生长因子165在大鼠骨髓基质细胞中的表达

Expression of recombinant adenovirus-mediated VEGF_(165) in bone marrow stromal cells of rats

目的利用AdEasy腺病毒载体系统构建携带血管内皮生长因子165(VEGF165)的重组腺病毒(Ad-VEGF165),并观察Ad-VEGF165转染大鼠骨髓基质细胞(bMSCs)后VEGF165的表达情况。方法将PCR获取的VEGF165目的基因插入到pAdTrack-CMV中,构建腺病毒穿梭质粒pAdTrack-VEGF165,经PmeI酶切线性化后,采用电穿孔法转化到含腺病毒骨架质粒pAdEasy-1的BJ5183大肠杆菌感受态细胞中,挑选同源重组菌落。提取质粒并用PacI酶切鉴定。线性化重组质粒pAdEasy-VEGF165转染293T细胞,包装成重组腺病毒颗粒,并扩增收集重组腺病毒,测定病毒滴度。体外培养大鼠骨髓基质细胞,Ad-VEGF165转染骨髓基质细胞,转染后在荧光显微镜下观察。转染的骨髓基质细胞采用RT-PCR和ELISA法检测VEGF165的表达水平。结果经酶切鉴定,基因测序及绿色荧光观察证实成功构建了携带VEGF165基因的重组腺病毒,并扩增出109pfu/ml的高滴度重组腺病毒。Ad-VEGF165转染骨髓基质细胞后,RT-PCR证明转染的骨髓基质细胞内有VEGF165mRNA表达。ELISA检测发现转染组上清液中VEGF165蛋白分泌量明显高于对照组(P<0.01)。结论pAdEasy-VEGF165转染对骨髓基质细胞的增殖能力无明显影响,而且骨髓基质细胞能够表达并分泌VEGF165,为研究骨组织工程血管化局部基因治疗奠定了基础。

Objective To construct recombinant adenovirus vector carrying vascular endothelial growth factor 165 （Ad-VEGF165）, amplify the adenovirus vector in 293T cells, transfect Ad-VEGF165, into the bone marrow stromal cells （bMSCs） of Wistar rats, and then to assay the expression of VEGF165. Methods VEGF165 obtained by PCR was digested and inserted into adenovirus shuttle plasmid pAdTrack CMV to generate recombinant plasmid pAdTrack-VEGF165, and then the Pine Llinearized plasmid pAdTrack VEGFl65 was electroporated into E.coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1. The identified recombinant plas mid pAdEasy-VEGF165 DNA was digested with Pac I and transfected into 293T cells to package adenovirus, followed by identification of the recombinant adenovirus by means of observation of the enhanced green fluorescence protein （EGFP） expression under fluorescent microscope. After amplified in 293T cells, the obtained adenovirus were transfected into 293T cells again, and EGFP expression was detected. Acl VEGF165 transfected bMSCs were cultured in vitro. The expression of VEGF165 in bMSCs after transfection was determined by observing the expression of EGFP and detected by RT PCR. ELISA method was applied to assay the secretion of VEGF165. Results Recombinant adenoviral VEGF165. was constructed successfully, which was confirmed by restriction enzyme digestion, gene sequencing and EGFP expression. EGFP expression could be observed under fluorescent microscope, and the expression of VEGF165 was confirmed by RT-PCR.EI.ISA analysis showed the quantity of expression of VEGF165 in transfeetion group was higher than that in control group （P〈0.01）. Conclusion Ad-VEGF165 has been successfully constructed, which may provide a basis for VEGF165 gene therapy. Exogenous VEGF165 can he expressed in bMSCs, which may offer the possibility of a novel approach to local gene therapy of bone tissue engineering.