血管内皮生长因子165基因对真皮替代物血管化影响的实验研究

Experimental study on the effect of vascular endothelial growth factor 165 gene on vascularization of dermal substitute

目的研究血管内皮生长因子165（VEGF165）琏因对真皮替代物体内血管化的影响。一方法采用含量体积分数10％FBS的M199培养基（简称完全培养基）培养人脐静脉内皮细胞（HUVEC）。（1）按随机数字表法将HUVEC分为3组，每组6孔：未转染组，不行转染；空载质粒组，转染pIRES2-增幔型绿色荧光坐h（EGFP）质粒；VEGF质粒组，转染pIRES2-EGFP-血管内皮生长因子（VEGF）质粒。转染后24h，倒置相差荧光显微镜下观察各组细胞中绿色荧光蛋白（GFP）表达情况，流式细胞仪榆测各组细胞pIGFP的表达率；未转染组行相同检测。以新霉素筛选出空载质粒组和VEGF质粒绀稳转细胞进行实验。分别采用实时荧光定髓PCR法和蛋白质印迹法、ELIISA法枪测3组细胞中、VEGF、165mRNA和蛋白表达以及细胞培养上清液中VEGF165的蛋白含量。（2）按随机数字表法将48只雄性裸鼠分为4组，每组12只：生理盐水组，背部两侧（部位下同）埋植经生理盐水培养2d的真皮替代物；培养基组，埋植经完全培养基培养2d的真皮替代物；未转染细胞组，埋植经含未转染HLjVEC的完全培养琏培养2d的真皮棒代物；转染细胞绀，埋植经含VEGF质粒稳转HUVEC的完食培养堪培养2d的真皮替代物。埋植术后3、7、14和21d取出各组真皮替代物，行免疫组织化学染色，观察其上微血管和HUVEC分布，技数术后14d微血管数量；蛋白质印迹法检查真皮替代物中VEGF165蛋白表达。各项检测及观察均重复进行3次，数抓采用单因素方差分析和bD法处理。结果（1）转染后24h，仅红2个转染组细胞中观察到明显绿色荧光。细胞中GFP表达率：未转染绀为0，守载质粒组（85．2±3．2）％，VEGF质粒组（93．1±2．4）％。末转染组、审载质粒组、VEGF质粒组细胞中VEGF 165mRNA相对表达啦分别为1、I．05±0．09、3．02±0．13（F=5．28，P〈0．05），VEGF165蛋白相对表达量分别为0．78±0．16、0．76±0．13、I．92±0．18（F=7．62，P〈0．05）；细胞㈠占液中VEGF165堡白含量分别为（62．4±2．7）、（73．1±3．8）、（117．5±3．1）pg／mL（F=15．08，P〈0．05）。VEGF、质粒组各项指标水甲均显著高于其余2组，P值均小于0．05。、（2）术后14d起转染细胞组真皮替代物中HUVEC明显多于其他3组。术后4d，各组真皮替代物中每200倍视野中微血管数量：生理盐水组（4．2±1，1）个、培养基组（5．2±1．1）个、未转染细胞纰（6．6±0．9）个、转染细胞绀（13．8±0．8）个，4组间比较，著芹有统训学意义，F=17．96，P〈0．01；转染细胞组微血管数明显多于其余3组（P值均小于0．05）。真皮替代物上VEGF165蛋白相对表达量比值：术后7d起，转染细胞组显著高于牛理盐水组（P〈0．05）；术后14、21d术转染细胞组（1．652±0．086、2．152±0．062）与转染细胞组（2．403±0．091、2．879±0．047）均显著高于生理盐水组（1．299±0．027、1．362±0．103，P值均小于0．05），且转染细胞组明娃高于未转染细胞组（P值均小于0．05）。各组真皮铪代物中VEGF165蛋白含量均随术后时间延长而增加。结论VEGF、165基因转染使HUVEC持续高表达VEGF165蛋白，可有效促进真皮棒代物体内血管化。

Objective To investigate the effect of vascular endothelial growth factor 165 （VEGF 165 ） geue on vascularization of dermal substitute in vivo. Methods Human umbilical vein endothelial cells （HUVECs） were cultured in M199 medium containing FBS in the volume fraction of 10% （briefly called complete medium）. （1） HUVECs were divided into non-transfection group （without transfection） , empty vector group [ transfected with pIRES2-enhanced green fluorescent protein （EGFP） plasmid ], and VEGF plasmid group （ transfected with pIRES2-EGFP-VEGF plasmid） according to the random number table, with 6 wells in each group. At post transfection hour （PTH） 24, the expression of green fluorescent protein （GFP） in each group was observed under inverted phase contrast fluorescence microscope, and the expres- sion rate of GFP was detected with flow cytometer. Cells in non-transfection group were tested with the same mehods as listed above. The ceils in stable transfection in empty vector group and VEGF plasmid group were sifted by neomycin. The mRNA and protein expression levels of VEGF 165 in cells and the protein amount of VEGF 165 in the supernatant of cell culture medium in 3 groups were respectively determined by real-time fluorescent quantitation PCR, Western blotting, and enzyme-linked immunosorbent assay. （2） Forty-eight male nude mice were divided into 4 groups according to the random number table, with 12 mice in each group. Mice in saline group were subcutaneously implanted with dermal substitutes which had been cultured in saline for 2 days on both sides of back （the same site below） ; mice in medium group were subcutaneously implanted with dermal substitutes which had been cultured in complete medium for 2 days; mice in non- transfected cells group were subcutaneously implanted with dermal substitutes that had been cultured in com- plete medium with non-transfected HUVECs for 2 days; mice in transfected cells group were subcutaneously implanted with dermal substitutes that had been cultured in complete medium with HUVECs stably transfect- ed with VEGF plasmid for 2 days. The dermal substitutes in every group were taken out on post operation day （POD） 3, 7, 14, and 21. Distributions of microvessels and HUVECs in dermal substitutes were observed by immunohistochemical staining, and the microvessel number was counted on POD 14; the expression level of VEGF 165 protein in dermal substitutes was determined by Western blotting. The experiments were all done in triplicate. Data were processed with one-way analysis of variance and LSD method. Results （ 1 ） Obvious green fluorescence was only observed in the two groups with transfected ceils at PTH 24. Expression rates of GFP in the cells of non-transfection group, empty vector group, and VEGF plasmid group were re- spectively 0, （85.2 ± 3.2） % , and （93.1 ± 2.4） %. In the non-transfection group, empty vector group, and VEGF plasmid group, the relative expression amounts of VEGF 165 mRNA were respectively 1, 1.05 ± 0.09, and 3.02 ± 0.13 （ F = 5.28, P 〈 0.05） ; the relative expression amounts of VEGF 165 protein were respectively 0.78 ±0.16, 0.76 ±0.13, and 1.92 ±0.18 （ F =7.62, P 〈0.05） ; the protein quantity of VEGF 165 in cell supernatant was respectively （62.4 ±2.7）, （73. 1 ±3.8）, （117.5 ±3. 1） pg/mL （F = 15.08, P 〈 0.05）. The mRNA and protein levels of VEGF 165 and VEGF 165 protein amount in su- pernatant were significantly higher in VEGF plasmid group than in the other two groups, with P values all be- low 0.05. （2） The number of HUVECs in dermal substitutes of transfected cells group was significantly higher than that of the other three groups on POD 14. The numbers of microvessels of dermal substitutes on POD 14 in saline group, medium group, non-transfected cells group, transfected cells group were respective- ly 4.2±1.1, 5.2±1.1, 6.6±0.9, 13.8±0.8 per 200 times visual field （ F =17.96,P 〈0.01）. The microvessel number in transfected ceils group was significantly higher than that of the other three groups, with P values all below 0.05. The relative expression ratio of VEGF 165 protein of dermal substitutes in transfected cells group was significantly higher than that in saline group as of POD 7. On POD 14 and 21, the relative expression ratios of VEGF 165 proteins in non-transfected cells group （ 1. 652 ± 0. 086, 2. 152 ± 0. 062 ） and transfected cells group （2. 403 ± O. 091, 2. 879 ± 0. 047 ） were significantly higher than those of saline group （ 1. 299 ± 0. 027, 1. 362 ± O. 103 ）, with P values all below O. 05. And the index level of trans- fected cells group was significantly higher than that in non-transfected cells group （with P values below 0.05）. The VEGF 165 protein content in dermal substitutes increased with time extension in all groups. Conclusions Transfection of VEGF 165 gene in HUVEC could effectively facilitate vascularization of der- mal substitutes in vivo by high expression of VEGF 165 protein.