CAJ | 학술논문

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.
목적 재소서모형체내탐토거서세포금속탄력단백매(macrophage metalloelastase,MME)분해섬용매원생성유생물학공능적혈관억소적도경,급기대종류생장화미혈관밀도(MVD)적억제작용.방법 구건중조질립pEGFP-C1-MME.급90지소서분별피하접충은정전염pEGFP-C1-MME(MME전염조)、공질립pEGFP-C1(공질립전염조)화미전염(미전염조)적CT-26세포,매조30지.응용방사성전표섬용매원급기시종기술분석체내혈관억소적생성정황.결과 각조종류표본진행SDS-PAGE전영,재MME전염조전영효중,단백상대분자질량위35×103화38×103적구역방사성명현고우공질립전염조화미전염조(P치균위0.00).Western인적검측발현삼조균검측출35×103화38×103편단표체,통과회도소묘분석발현상술2개편단재MME전염조표체강도위9.32±1.52화5.61±2.24,명현고우공질립조(2.47±0.23화0.67±0.12,P치균위0.00)화미전염조(1.21±0.69화0.86±0.44,P치균위0.00).MME전염조MVD평균치화혈관내피생장인자(VEGF)형광표체강도균명현저우공질립전염조화미전염조(P치균위0.00).MME전염조소서피하충식류평균체적소우공질립조화미전염조(P치균위0.00).결론 MME시여혈관억소생성밀절상관적간질금속단백매,동시구유억제결장종류혈관형성적작용.
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.