反义基质金属蛋白酶2基因对人增殖期血管瘤内皮细胞的影响

Effect of adenovirus-active matrix metanoproteinase-2 transfection on vascular endothelial cells in human proliferating hemangioma

目的:观察Ad-aMMP-2cDNA转染原代培养的增殖期血管瘤内皮细胞后,对体外培养的血管内皮细胞生物学特性的影响。方法:实验于2003-10/2004-06在四川大学华西临床医学院肿瘤实验室,四川大学基础与法医学院电镜室完成。选用1名出生52d女性患儿左胸壁的海绵状血管瘤(病理诊断为增殖期血管瘤)作组织块原代培养,分离、纯化并传代培养至4代后,经形态学观察、免疫组织化学染色和电镜观察鉴定确认为增殖期血管瘤内皮细胞。将血管瘤内皮细胞分3组:①M199组:继续用无血清M199培养24h。②Ad-GFP组:用10μLAd-GFP(MOI=100)转染每孔的细胞,1h后追加无血清M199培养24h。③Ad-aMMP-2组:用10μLAd-aMMP-2cDNA(MOI=100)转染每孔的细胞,1h后追加无血清M199培养24h。观察Ad-aMMP-2组血管内皮细胞转染后24,48,120h基质金属蛋白酶2mRNA的表达。逆转录-聚合酶链反应、免疫印迹技术和明胶酶谱试验的方法检测在核酸水平和蛋白水平基质金属蛋白酶2基因的表达。结果:①组织块法原代培养的增殖期血管瘤内皮细胞:获取的第1代血管瘤内皮细胞纯度达70%,椎虫蓝染色显示成活率在95%以上。②逆转录-聚合酶链反应定量检测mRNA:Ad-aMMP-2组血管瘤内皮细胞转染Ad-aMMP-2后24h,与同时段的Ad-GFP组比较,基质金属蛋白酶2mRNA表达水平均开始下降0.327±0.034,0.531±0.158;48h后下降的趋势更加明显0.189±0.028,0.505±0.083,(P<0.05);120h后基质金属蛋白酶2mRNA的表达显著下降0.106±0.014,0.510±0.106,(P<0.01)。③明胶酶谱试验和免疫印迹技术检测表明:Ad-GFP组血管瘤内皮细胞表达基质金属蛋白酶2与M199组无明显差异,而Ad-aMMP-2组血管瘤内皮细胞表达基质金属蛋白酶2明显低于Ad-GFP组和M199组。结论:①用组织块法原代培养增殖期血管瘤内皮细胞,能获得纯度较高的血管瘤内皮细胞。②在体外实验中,反义基质金属蛋白酶-2cDNA可以有效抑制血管瘤内皮细胞分泌基质金属蛋白酶2。

AIM： To investigate the effect of adenovirus-active matrix metanoproteinase-2 （Ad-aMMP-2） transfection on the biological characteristics of vascular endothelial cells （VEC）, which are cultured in vitro in human proliferating hemangioma. METHODS： The experiment was carded out in the Laboratory of Tumor in West China Clinical College and the Department of Electron Microscope of Basic and Forensic College, Sichuan University between October 2003 and June 2004. The tissues from a 52-day newbom gid patient with cavemous hemangioma on the left chest wall were primarily cultured. VEC of identified proliferating hemangioma were gained by divergence, purification and serial subcultivation to 4^th passage, and were used for identification of morphology, immunohistochemical staining and electron microscope. Then VECs were divided into three groups： （1）M199 group： Cells were culture with M199 without serum for 24 hours.（2） Ad-GFP group： Ad-GFP （10 μL, MOI=100） was used to transfer VEC, one hour later M199 without serum was added for 24-hour culture.（3）Ad-aMMP-2 group： Ad-aMMP-2 （10 μL, MOI=100） was used to transfer VEC, one hour later M199 without serum was added for 24-hour culture, Reverse transcription polymerase chain reaction （RT-PCR） was performed to determine the expression of endogenous MMP-2mRNA after 24, 48, 120 hours of Ad-aMMP-2 transfection; Westem Blotting and Gelatin Zymography analyses were performed to determine the level of endogenous MMP-2 expression at nucleic acid and protein levels respectively. RESULTS： （1）VEC originating from human proliferating hemangioma by tissue explant primary culturing： VEC reached 70% purity coefficient when serial subcultivation to 1. The survival rate was detected more than 95% by trypanblau dye. （2）uantitative detection of mRNA by RT-PCR： The endogenous MMP-2mRNA expression was felt off in the Ad-aMMP-2 group and Ad-GFP group after VEC were transfected for 24 hours （0.327±0.034, 0.531 ±0.158）, obvious decreased 48 hours later （0.189±0.028, 0.505±0.083, P 〈 0.05）, and significantly declined 120 hours later （0.106±0.014, 0.510±0.106, P 〈 0.01）.（3）Outcomes of Westem Blotting and Gelatin Zymography： The MMP-2mRNA expression was obviously lower in the Ad-aMMP-2 group compared with the other two groups. There was insignificant difference between Ad-GFP and M199 group. CONCLUSION：（1）We can gain VEC of high purification from human proliferating hemangioma by tissue explant primary culturing.（2）In vitro, Ad-aMMP-2cDNA can inhibit MMP-2 secretion of VEC effectively.