CAJ | 학술논문

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。
목적：탐토선병독재체개도혈관내피생장인자( VEGF)전염제대간충질간세포( UCMSCs)적가행성,이급VEGF전염대UCMSCs공능적영향。방법구건VEGF165-EGFP기인중조선병독재체,분리화배양UCMSCs,휴증강형록색형광단백( EGFP)적선병독재체전염UCMSCs,도치형광현미경하관찰세포전염효과,류식세포의검측세포전염솔,학정최가병독감염복수( MOI)。장세포분위VEGF-EGFP전염조、EGFP공재조급대조조3조,의거최가MOI치전염병수집세포,채용단백인적법( Western blotting)검측VEGF급Akt표체변화；매련면역흡부시험( ELISA)검측VEGF단백표체정황。채용CCK-8법평개전염대UCMSCs증식적영향。결과선병독개도적VEGF165-EGFP기인능구성공전염UCMSCs,차VEGF기인재세포내능구전록화표체,병능분비도세포외。전염후48 h VEGF-EGFP전염조VEGF、Akt수평현저고우EGFP공재조화대조조(P<0．05)；전염후48 h채용ELISA법재VEGF-EGFP전염조세포배양상청중즉검측도VEGF적표체화분비,재전염후제4천체도고봉,차VEGF표체현저고우EGFP공재조급대조조(P<0．01),이후축점하강,병은정표체일정시간。 CCK-8법검측결과현시,개도VEGF기인전염적선병독대UCMSCs증식무명현영향。결론선병독개도VEGF기인능구성공전염UCMSCs,보지기원유생물학특성,병능지속고효표체VEGF,위통과VEGF기인연합UCMSCs치료획득당뇨병하지혈액순배중건적가행성제공이론의거。
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 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.