小鼠结肠癌中巨噬细胞金属弹力蛋白酶对血管抑素生成和肿瘤血管生长的影响  

Effects of macrophage metalloelastase on angiostatin generation and growth of tumor vessels in murine colon carcinoma

作　　者：徐张巍[1] 许建明[1] 石海[1] 梅俏[1] 鲍峻峻[1] 沈玉先[2] 

机构地区：[1]安徽医科大学第一附属医院消化科安徽省高校消化疾病重点实验室,合肥230032 [2]省部共建教育部重要遗传病基因资源利用重点实验室

出　　处：《中华消化杂志》2011年第4期258-263,共6页Chinese Journal of Digestion

基　　金：安徽省自然科学基金资助项目（01043904）

摘　　要：目的 在小鼠模型体内探讨巨噬细胞金属弹力蛋白酶（macrophage metalloelastase,MME）分解纤溶酶原生成有生物学功能的血管抑素的途径,及其对肿瘤生长和微血管密度（MVD）的抑制作用.方法 构建重组质粒pEGFP-C1-MME.给90只小鼠分别皮下接种稳定转染pEGFP-C1-MME（MME转染组）、空质粒pEGFP-C1（空质粒转染组）和未转染（未转染组）的CT-26细胞,每组30只.应用放射性碘标纤溶酶原及其示踪技术分析体内血管抑素的生成情况.结果 各组肿瘤标本进行SDS-PAGE电泳,在MME转染组电泳胶中,蛋白相对分子质量为35×103和38×103的区域放射性明显高于空质粒转染组和未转染组（P值均为0.00）.Western印迹检测发现三组均检测出35×103和38×103片段表达,通过灰度扫描分析发现上述2个片断在MME转染组表达强度为9.32±1.52和5.61±2.24,明显高于空质粒组（2.47±0.23和0.67±0.12,P值均为0.00）和未转染组（1.21±0.69和0.86±0.44,P值均为0.00）.MME转染组MVD平均值和血管内皮生长因子（VEGF）荧光表达强度均明显低于空质粒转染组和未转染组（P值均为0.00）.MME转染组小鼠皮下种植瘤平均体积小于空质粒组和未转染组（P值均为0.00）.结论 MME是与血管抑素生成密切相关的间质金属蛋白酶,同时具有抑制结肠肿瘤血管形成的作用.Objective To determine the pathway of macrophage metalloelastase （MME）generate active angiostatin by decomposing plasminogen and its effect on inhibiting growth of tumor and microvessel density （MVD） in vivo in mouse models. Methods The recombined plasmid pEGFPC1-MME was constructed. Thirty mice were subcutaneously inoculated with CT-26 cells that were stably transfected with pEGFP-C1-MME （MME-transfected group）, 30 with CT-26 cells transfected with empty vector pEGFP-C1 （vector-transfected group） and 30 with CT-26 cells （non-transfected group）. Radioiodination and radioisotope tracer were used to explore the pathway of angiostatin generation in vivo. Results SDS-PAGE electrophoresis analysis revealed that, in the PAGE gel contained the protein with molecular weights of 35 000 and 38 000, radioactivity in MME-transfected group was significantly higher than vector-transfected and non-transfected groups （P = 0. 00）.Western blotting analysis demonstrated two bands containing 35 000 and 38 000 fragments in three groups. Quantification of the protein signals by image analysis revealed that the levels of 35 000 and 38 000 fragments were obviously increased in MME-transfected group （9.32±1.52 and 5.61±2.24,respectively） than those in vector-transfected （2.47 ± 0.23 and 0. 67 ± 0. 12, respectively） and nontransfected （1.21±0. 69 and 0. 86 ± 0.44, respectively） groups （P= 0.00）. The average value of MVD and fluorescent express of vascular endothelial growth factor （VEGF） were lower in MMEtransfected group when compared with those in vector-transfected and non-transfected groups （P =0.00）. The average tumor size in MME-transfected group was small in comparison with vectortransfected and non-transfected groups （P= 0.00）. Conclusions MME is demonstrated to be one of matrix metalloproteinase that closely related with angiostatin production and has inhibitory effect on tumor growth in tumor-bearing mice.

关 键 词：结肠肿瘤 基质金属蛋白酶类 巨噬细胞 血管抑制素类 新生血管化 病理性 疾病模型 动物 

分 类 号：R735.35[医药卫生—肿瘤]