摘要
背景:研究表明,通过转染反义基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)基因抑制增殖期血管瘤组织中MMP-2的分泌将成为增殖期血管瘤治疗的重要手段。目的:观察反义MMP-2cDNA基因感染对人增殖期血管瘤裸小鼠移植瘤生长的影响。设计、时间及地点:随机分组设计,对比观察,实验于2003-08/2004-09在四川大学华西临床医学院完成。材料:BALB/c-nu/nu裸小鼠(简称为裸鼠)18只,体质量20g左右;取1名出生52d女性患儿左胸壁的海绵状血管瘤,病理诊断证实为增殖期血管瘤。方法:将手术切除的人增殖期血管瘤新鲜标本切分为5mm×4mm×3mm小块,于1h内移植于18只裸小鼠双侧腋部皮下,制备荷瘤裸小鼠血管瘤移植模型。将移植瘤块成活后(45d)15只裸鼠进行分组治疗,Ad-GFP组,Ad-aMMP-2组分别沿肿瘤长径多方向瘤内注射重组腺病毒液Ad-GFP和Ad-aMMP-2,对照组注射等量的PBS,每组5只。隔日瘤内注射1次,共注射4次。主要观察指标:①大体观察肿瘤体积的变化及各组裸鼠肿瘤块坏死面积的比较。②绿色荧光蛋白在各组裸鼠体内的表达。③肿瘤组织形态学变化(大体、苏木精-伊红染色及透射电镜观察)。④免疫组织化学检测MMP-2cDNA基因及微血管密度的表达。⑤流式细胞仪检测肿瘤细胞的生长周期和凋亡。结果:①Ad-aMMP-2能抑制体内血管瘤的生长,没有观察到明显的毒副作用。3组肿瘤均有不同程度的坏死,其中Ad-aMMP-2组面积坏死率明显高于对照组和Ad-GFP组(P<0.01)。②组织学切片可见Ad-GFP组绿色荧光蛋白基因的表达。③大体观察对照组和Ad-GFP组瘤组织大;Ad-aMMP-2组瘤组织相对较小。苏木精-伊红染色可见对照组和Ad-GFP组内皮细胞密集,细胞呈条索状或团块状排列;Ad-aMMP-2组肿瘤有多处出血和凝固性的片状坏死灶。透射电镜观察对组血管内皮细胞形态正常;Ad-GFP组瘤细胞大,核仁明显;Ad-aMMP-2组部分血管内皮细胞核内染色质固缩,聚积成团块状形成凋亡小体。④Ad-aMMP-2组MMP-2和微血管密度较对照组和Ad-GFP组明显下降(P<0.05)。⑤Ad-aMMP-2组G0/G1期百分率大于其他两组(P<0.05),增殖指数降低;Ad-aMMP-2组细胞凋亡率大于对照组和Ad-GFP组(P<0.05),凋亡指数明显增加。结论:通过反义MMP-2基因阻断人增殖期血管瘤的生长是可行的,其机制主要是通过抑制血管内皮细胞分泌MMP-2导致局部缺血起作用的。
BACKGROUND： Evidence exists that inhibition of matrix metanoproteinase-2（MMP-2） secretion in the proliferating hemangioma tissue by transfection of adenovirus-active MMP-2（Ad-aMMP-2） cDNA would become an important means for treatment of proliferating hemangioma. OBJECTIVE： To investigate the influences of Ad-aMMP-2 cDNA transfection on human proliferating hemangioma growth in nude m ice. DESIGN, TIME AND SETTING： A randomized, grouping, and controlled observation was performed in West China Hospital of Sichuan University between August 2003 and September 2004. MATERIALS： Eighteen BALB/c-nu/nu nude mice, weighing approximately 20 g, were included. Cavernous hemangioma specimen pathologically confirmed as proliferating hemangioma was resected from one 52-day-old female child patient. METHODS： The freshly resected human proliferating hemangioma specimen was sliced into small pieces with a size of 5 mm× 4 mm×3 mm and subcutaneously implanted into the back of 18 nude mice within 1 hour to develop mouse models of hemangioma Forty-five days after hemangioma implantation, 15 successful hemangioma nude mice were treated by intratumoral administration of adenovirus green fluorescent protein （Ad-GFP, n = 5, Ad-GFP group）, adenovirus-active MMP-2 （n = 5, Ad-aMMP-2 group）, or the same amount of phosphate buffered saline （PBS, n = 5, control group）. Intratumoral administration was performed once every other day, for a total of 4 times. MAIN OUTCOME MEASURES： Observation of tumor volume and comparison of tumor necrosis area among 3 groups; detection of GFP expression in nude mouse; gross, hematoxylin-eosin staining, and transmission electron microscope observation of tumor tissue morphology; determination of MMP-2 cDNA expression and microvascular density by immunohistochemistry; and detection of growth cycle and apoptosis of tumor cells by flow cytometry. RESULTS： ① Ad-aMMP-2 could inhibit hemangioma growth in vivo, without marked adverse reactions. Tumor necrosis of different degrees was found in each group, and tumor necrosis area was significantly greater in the Ad-aMMP-2 group than in the control and Ad-GFP groups （P 〈 0.01）. ② Histological sections displayed GFP gene expression in the Ad-GFP group. ③ Gross observation results revealed relatively large tumor tissue in the control and Ad-GFP groups and relatively small tumor tissue in the Ad-aMMP-2 group. Hematoxylin-eosin staining results showed that in the control and Ad-GFP groups, endothelial cells aggregated together in strip-shaped or lump-shaped appearance, and in the Ad-aMMP-2 group, there were many necrotic foci arranging in lamellar-shape appearance. Transmission electron microscope results revealed vascular endothelial cells with normal morphology in the control group and tumor cells with apparent nucleeli in the Ad-GFP group, while in the Ad-aMMP-2 group, some vascular endothelial cells exhibited chromatin pycnosis in the nucleus, forming apoptotic bodies. ④ MMP-2 expression and microvascular density were significantly reduced in the Ad-aMMP-2 group than in the Ad-GFP and control groups （P 〈 0.05）. ⑤ The percentage of tumor cells in G0/G1 phase was significantly higher （P 〈 0.05）, while the proliferating index was significantly decreased, in the Ad-aMMP-2 group than in the Ad-GFP and control groups, The Ad-aMMP-2 group exhibited higher apoptosis rate of tumor cells （P 〈 0.05）, as well as more markedly increasing apoptosis index, than the control and Ad-GFP groups. CONCLUSION： It is feasible to block human proliferating hemangioma growth by transfection of Ad-aMMP-2 cDNA. The included mechanisms are to inhibit vascular endothelial cells to secrete MMP-2, thereby leading to local ischemia.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第20期3821-3828,共8页
Journal of Clinical Rehabilitative Tissue Engineering Research
