摘要
目的探讨腺病毒载体介导血管内皮生长因子(VEGF)转染脐带间充质干细胞(UCMSCs)的可行性,以及VEGF转染对UCMSCs功能的影响。方法构建VEGF165-EGFP基因重组腺病毒载体,分离和培养UCMSCs,携增强型绿色荧光蛋白(EGFP)的腺病毒载体转染UCMSCs,倒置荧光显微镜下观察细胞转染效果,流式细胞仪检测细胞转染率,确定最佳病毒感染复数(MOI)。将细胞分为VEGF-EGFP转染组、EGFP空载组及对照组3组,依据最佳MOI值转染并收集细胞,采用蛋白印迹法(Western blotting)检测VEGF及Akt表达变化;酶联免疫吸附试验(ELISA)检测VEGF蛋白表达情况。采用CCK-8法评价转染对UCMSCs增殖的影响。结果腺病毒介导的VEGF165-EGFP基因能够成功转染UCMSCs,且VEGF基因在细胞内能够转录和表达,并能分泌到细胞外。转染后48 h VEGF-EGFP转染组VEGF、Akt水平显著高于EGFP空载组和对照组(P<0.05);转染后48 h采用ELISA法在VEGF-EGFP转染组细胞培养上清中即检测到VEGF的表达和分泌,在转染后第4天达到高峰,且VEGF表达显著高于EGFP空载组及对照组(P<0.01),以后逐渐下降,并稳定表达一定时间。CCK-8法检测结果显示,介导VEGF基因转染的腺病毒对UCMSCs增殖无明显影响。结论腺病毒介导VEGF基因能够成功转染UCMSCs,保持其原有生物学特性,并能持续高效表达VEGF,为通过VEGF基因联合UCMSCs治疗获得糖尿病下肢血液循环重建的可行性提供理论依据。
Objective To investigate the feasibility of transfection of adenovirus-mediated vector vascular endo-thelial growth factor （ VEGF） into umbilical cord mesenchymal stem cells （ UCMSCs） and the effect of VEGF transfection on UCMSCs function. Methods VEGF165 - EGFP genetic recombination of adenovirus vectors were constructed, and UCMSCs were isolated and cultured, and then adenovirus vector-enhanced green fluorescent protein （ EGFP） transfected UCMSCs. The transfection effect was observed under inverted fluorescence microscope, and transfection effectiveness was detected by flow cytometer so to confirm the best multiplicity of infection （ MOI） . Cells were divided into VEGF-EGFP transfection group, EGFP group and control group, and the cells were transfected and collected based on the best MOI. Expressions of VEGF and protein kinase B （ PKB also named Akt） signaling protein were detected by Western blotting;the expression of VEGF protein was detected by enzyme linked immunosorbent assay （ ELISA） . The proliferation effect of transfected UCMSCs was evaluated with CCK 8 method. Results Adenovirus-mediated VEGF165-EGFP gene success-fully transfected UCMSCs, and VEGF gene was transcribed and expressed in cells, and then was secreted out of cells. Levels of VEGF and Akt transfection secretion in VEGF-EGFP group 48 hours after transfection were significantly higher than those in EGFP group and control group; the expression and secretion of VEGF could be detected in VEGF-EGFP transfection group by ELISA 48 hours after transfection, and the peak appeared 4thd after transfection, at the same time the expressions in VEGF group were significantly higher than those in EGFP group and control group （ P〈0. 01 ） , and then the levels gradually decreased, but can maintain stable expression for several days. Results of CCK 8 method showed＆amp;nbsp;that VEGF gene transfection had no significant effect on the UCMSCs proliferation. Conclusion Adenovirus-mediated VEGF-EGFP gene may successfully transfect UCMSCs, maintain UCMSCs original biological characteristics, and signifi-cantly and efficiently express VEGF, which can provide theoretical basis for the feasibility of VEGF gene combined with UCMSCs in treatment of diabetic lower limb blood circulation reconstruction.
出处
《解放军医药杂志》
CAS
2014年第5期5-9,共5页
Medical & Pharmaceutical Journal of Chinese People’s Liberation Army
基金
河北省自然科学基金课题(H2013505067)
关键词
血管内皮生长因子类
脐带间质干细胞
腺病毒载体
转染
Vascular endothelial growth factor
Umbilical cord mesenchymal stem cell
Adenovirus vector
Transfection
