巨噬细胞金属弹力酶基因转染CT-26细胞对小鼠原位结肠癌生长及微血管生成的影响

Effect of mouse macrophage metalloelastase gene transfer into murine CT-26 colon cancer ceils on orthotopic tumor growth and angiogenesis

目的探讨小鼠巨噬细胞金属弹力酶(MME)对小鼠原位结肠癌生长、微血管生成及血管内皮生长因子(VEGF)表达的影响。方法扩增编码MME基因结构域Ⅰ和Ⅱ的cDNA片段,构建真核细胞表达载体pcDNA3．1-MME并转染小鼠CT-26结肠癌细胞。建立MME转染组及对照组小鼠原位结肠癌种植模型,观察MME对原发性结肠癌生长的影响,采用免疫组化及原位杂交方法检测肿瘤组织中VEGF的表达和微血管密度(MVD)。结果重组MME蛋白在CT-26细胞内成功表达并具有酶活性。MME转染组原发结肠癌的体积明显小于对照组(P＜0．001),结肠癌组织中的MVD值明显低于对照组(P＜0．01)。VEGF mRNA和蛋白水平在MME转染组中均显著低于对照组(P＜0．05)。结论转染入小鼠结肠癌细胞的MME基因通过抑制新生血管的生成,从而起到抑制原位结肠癌生长的作用。MME和VEGF都与肿瘤的血管生成密切相关,它们之间的平衡可能调控着肿瘤中新生血管的生成。

Objective To determine the correlation between mouse macrophage metalloelastase （MME） and vascular endothelial growth factor （VEGF） expression involved in angiogenesis of colon cancer. Methods A cDNA fragment coding for domains Ⅰ and Ⅱ of MME was transfected into murine CT-26 colon cancer cells that were MME deficient. The enzymatic activity of recombinant MME was confirmed by cleavage of native substrate in vitro. An orthotopic implantation model was established by using MME-transfected cells and control cells. Tumor samples were subjected to in situ hybridization （ISH） and immunohistochemical staining （IHC） to detect expressions of MME and VEGF. The microvessel counting was used to assess angiogenesis of murine colon tumors. Results It was demonstrated that the tumor growth was significantly inhibited in MME-transfected group compared with pcDNA3.1-transfected and nontransfected groups（P 〈 0. 001）. It was also found that,compared with pcDNA3.1-transfected and nontransfected groups, the microvessel formation in MME-transfected group was significantly reduced（P〈0. 001）. The expression of VEGF mRNA and protein was significantly lower in MME-transfected group than those in the controls, as demonstrated by ISH（MME transfected group versus pcDNA3.1-transfected group, P = 0. 028; and versus nontransfected group, P = 0. 003） and by IHC （MME-transfected group versus pcDNA3.1-transfected group, P = 0. 025; and versus non- transfected group, P = 0. 008）. Conclusions The MME gene transfected into murine colon cancer cells can effectively suppress the growth of orthotopic tumors by inhibition of vascularity. Both MME and VEGF gene expression is highly associated with the vascularity of tumors, which may depend on a balance between MME and VEGF expression.