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目的构建携带hVEGF165基因的重组腺病毒载体pAdxsi-增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)-hVEGF165,体外转染大鼠BMSCs,观察转染后hVEGF165的表达情况以及对BMSCs生长增殖的影响,为进行血管再生的基因治疗奠定实验基础。方法将hVEGF165从原始质粒切下连接到pShuttle-巨细胞病毒-EGFP重组穿梭载体,并转移至pAdxsi载体上,获得重组腺病毒载体质粒pAdxsi-EGFP-hVEGF165,进行酶切鉴定及基因测序。采用PacI限制性内切酶线性化pAdxsi-EGFP-hVEGF165,并转染人胚肾细胞HEK293,收获重组腺病毒,测定病毒滴度。贴壁法培养Wistar大鼠BMSCs,体外转染携带EGFP基因的重组腺病毒(pAdxsi-EGFP),荧光倒置相差显微镜及流式细胞术确定最佳病毒感染复数(multiplicities of infection,MOI)。依据最佳MOI转染pAdxsi-EGFP-hVEGF165至BMSCs,采用Western blot、RT-PCR及ELISA法检测转染后hVEGF165的表达;MTT法评价转染后BMSCs生长增殖情况。结果酶切鉴定及基因测序提示重组腺病毒载体质粒含有hVEGF165 cDNA;经多轮感染、扩增后,病毒滴度可达1×1010 pfu/mL。荧光倒置相差显微镜观察转染pAdxsi-EGFP后MOI为150 pfu/cell时转染效果最佳,流式细胞仪检测转染效率约88%。Western blot和RT-PCR检测示,pAdxsi-EGFP-hVEGF165转染BMSCs 48 h后在蛋白质和基因水平均可有效表达hVEGF165;ELISA检测示其含量在7 d达高峰,在20 d时仍有表达,1、3、5、7、9、11、13、15、20 d各时间点hVEGF165含量均显著高于EGFP基因转染组及未转染组(P<0.05)。MTT结果显示,各时间点转染pAdxsi-EGFP-hVEGF165的BMSCs与未转染组细胞的吸光度(A)值比较差异无统计学意义(P>0.05)。结论 BMSCs是一种较理想的基因载体细胞,成功制备的重组腺病毒载体质粒可在MOI值为150时将hVEGF165基因有效转染至BMSCs;转染后hVEGF165表达水平较高、持续时间较长,且对BMSCs的生长增殖无明显影响。
Objective To construct the recombinant adenovirus vector pAdxsi-enhanced green fluorescent protein (EGFP) -hVEGF165 carrying hVEGF165 gene and transfect rat BMSCs in vitro to observe the effect of hVEGF165 on the proliferation and proliferation of BMSCs , Which laid the experimental foundation for the gene therapy of angiogenesis. Methods hVEGF165 was excised from the original plasmid and ligated into pShuttle-CMV-EGFP shuttle vector and transferred into pAdxsi vector to obtain the recombinant adenovirus plasmid pAdxsi-EGFP-hVEGF165. The recombinant plasmid was identified by restriction enzyme digestion and gene sequencing. The pAdxsi-EGFP-hVEGF165 was linearized with PacI restriction endonuclease and transfected into human embryonic kidney cell line HEK293. The recombinant adenovirus was harvested and the virus titer was determined. Wistar rat BMSCs were cultured by adherent method. Recombinant adenovirus carrying EGFP gene (pAdxsi-EGFP) was transfected in vitro. Optimal multiplicities of infection (MOI) were determined by fluorescence inverted phase contrast microscope and flow cytometry. The transfection of pAdxsi-EGFP-hVEGF165 to BMSCs was performed according to the optimal MOI. The expression of hVEGF165 was detected by Western blot, RT-PCR and ELISA. The proliferation and proliferation of BMSCs were evaluated by MTT assay. Results Restriction endonuclease digestion and gene sequencing suggested that the recombinant adenoviral vector plasmid contained hVEGF165 cDNA. After multiple rounds of infection, the titer of virus could reach 1 × 1010 pfu / mL. Fluorescence inverted phase contrast microscopy showed that the transfection efficiency was best when the MOI was 150 pfu / cell after transfection with pAdxsi-EGFP, and the transfection efficiency was about 88% by flow cytometry. The results of Western blot and RT-PCR showed that hVEGF165 was efficiently expressed at the protein and gene level after transfected with pAdxsi-EGFP-hVEGF165 for 48 h, and reached the peak at 7 d by ELISA. The content of hVEGF165 at each time point was significantly higher than that of EGFP gene transfected group and non-transfected group at 1, 3, 5, 7, 9, 11, 13, 15 and 20 d (P <0.05). MTT results showed that there was no significant difference in absorbance (A) between BMSCs transfected with pAdxsi-EGFP-hVEGF165 and untransfected cells at each time point (P> 0.05). Conclusions BMSCs are a kind of ideal gene carrier cells. The successfully prepared recombinant adenoviral vector plasmid can effectively transfect hVEGF165 gene to BMSCs when the MOI value is 150. The hVEGF165 expression level is high and lasts longer, And had no significant effect on the growth and proliferation of BMSCs.