【作者】 石海；

【导师】 许建明；

【作者基本信息】 安徽医科大学 ， 老年医学， 2005， 博士

【摘要】 目的 巨噬细胞金属弹力酶(mouse macrophage metalloelastase,MME)是基质金属蛋白酶(matrix metalloproteinase,MMP)家族成员之一,也称MMP-12。与其它MMP成员不同,MME可以分解纤溶酶原,产生具有抑制血管内皮细胞增殖作用的血管抑素(angiostatin),进而抑制体内肿瘤细胞的生长,在抗肿瘤血管生成中具有重要作用。本研究主要探讨小鼠巨噬细胞金属弹力酶对小鼠原位结肠癌生长、微血管生成及血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响。 方法 PCR扩增编码MME基因结构域Ⅰ和Ⅱ的cDNA片段,克隆入pGEM-T载体中,限制性内切酶、DNA序列分析鉴定目的基因后,定向亚克隆到真核细胞表达载体pcDNA3.1(+)中,并进行双酶切及PCR鉴定。再将构建的真核细胞表达载体pcDNA3.1-MME稳定转染小鼠CT-26结肠癌细胞。采用RT-PCR、免疫细胞化学和Western blot方法,鉴定MME mRNA和重组蛋白在CT-26细胞中的表达。采用体外分解Ⅰ型胶原蛋白和明胶酶谱方法,鉴定MME重组蛋白的酶活性。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,采用免疫组织化学、原位杂交及Western blot方法检测肿瘤组织中微血管密度(microvessel density,MVD)和VEGF的表达。 结果 以重组质粒pUC9-MME cDNA为模板进行PCR扩增,PCR产物经纯化后,在1%琼脂糖凝胶电泳中可见一条清晰的条带,位于750bp和1000bp之间,与预期的840bp目的基因片段长度相符。pGEM-T-MME用BamHI和XbaI双酶切,产生2个大小分别为3.0kb和832bp左右的条带,与预期结果基本一致。pGEM-T-MME核苷酸序列正向测序和反向测序结果表明,与小鼠MME cDNA序列的碱基符合率为99.63%。pcDNA3.1(+)-MME重组质粒用BamHI和XbaI双酶切,可见2条分别更多还原

【Abstract】 Objective Mouse macrophage metalloelastase (MME), a member of the matrix metalloproteinase family, is believed to play an important role in the generation of angiostatin, an internal fragment of plasminogen, which shows inhibition of tumor angiogenesis. The current study was designed to determine the correlation between MME and vascular endothelial growth factor (VEGF) expression involved in growth and 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 are 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), immunohistochemical staining (IHC) and Western blot to detect expressions of MME and VEGF. The microvessel counting was used to assess angiogenesis of murine colon tumors.Results pUC9-MME cDNA was subjected to 35 cycles of PCR amplification and produced a 840bp MME cDNA fragment coding for domains I and II of MME. The pGEM-T-MME plasmid was digested into two fragments by 1% agarose gel electrophoresis with BamH I and Xba I . They were consistent with theoretic values 3.0kb and 832bp. indicating that PCR products have been cloned into pGEM-T vector. The sequence of the insert was 99.63% identical to that in GeneBank. The recombinant pcDNA3.1-MME plasmid was separated into two bands(about 5.4kb and 832bp,respectively) in a 1% agarose gel using BamW I and Xba I , suggesting that MME gene fragment has been cloned into pcDNA3.1 vector correctly. CT-26 transfectants were selected by G418 and MME mRNA expression was analyzed by RT-PCR. The 840bp amplification products expected for MME were identified in MME-transfected clones. In contrast. MME mRNA was not detected in pcDNA3.1-transfected clone and nontransfected cells. To detect the presence of MME protein, immunocytochemistry was performed. Positive immunostaining for MME protein was present in the cytoplasm of MME-transfected cells, but not in those of pcDNA3.1-transfected and nontransfected cells. MME protein was also confirmed by Western blot analysis in CT-26 cells. A 30 kDa band was detected in MME-transfected cells, but not in pcDNA3.1-transfected cells and nontransfected cells. The protein with 30 kDa molecular mass corresponded to the domains I and II of MME. To determine if the recombinant MME expressed in CT-26 cells was capable of enzymatic activity in vitro. 500ug of insoluble type I collagen was incubated with the supernatants of transfected or nontransfected cells lysed by sonication. MME-transfected cells exhibited soluble cleavage of type I collagen by MME. In contrast, degraded soluble products of type I collagen were not seen in pcDNA3.1-transfected and nontransfected cells. Gelatin zymography was also performed. A 22 kDa lytic band was identified in MME-transfected cells, whereas no zone of lysis was indicated in the controls (pcDNA3.1-transfected and nontransfected cells). The 22 kDa band corresponded to the final active form of MME after cleavage with loss of domain I (8kDa). Mice carrying pcDNA3.1-transfected and nontransfected cells formed large primary tumors with volumes of 1151.07±35.91mm3 and 1201.13± 42.15mm3, respectively. In contrast, mice implanted with MME-transfected cells formed smaller tumors with volumes of 384.83 ±4.76mm3.Growth of MME-transfected tumors was significantly inhibited compared with control tumors (MME-transfected group versus pcDNA3.1 -transfected group, PO.001; and versus nontransfected group, PO.001). There is on significant difference between control groups, />=0.374. In addition, 2 of 30 mice (6.7%)developed liver metastases in control groups, and no livermetastasis was found in MME-transfected group. 18 of 30 mice (60.0%) developed peritoneum metastases in control groups, and 4 of 15 mice (26.7%) with peritoneum metastases were found in MME-transfected group. P=0.035. Tumors derived from MME-transfected cells demonstrated a lower microvessel density (8.48 ± 0.53) compared with control tumors derived from pcDNA3.1-transfected (21.87±0.47) and nontransfected (22.56 ± 0.71) cells (MME-transfected group versus pcDNA3.1-transfected group, P<0.001; and versus nontransfected group, PO.001). There is no significant difference between control groups, P=0A23. VEGF mRNA expression by ISH was significantly lower in MME-transfected tumors than that in pcDNA3.1-transfected and nontransfected tumors (MME-transfected group versus pcDNA3.1-transfected group, /)=0.028; and versus nontransfected group. P=0.003). There is no significant difference between control groups (P=0A09). VEGF protein expression by IHC was significantly lower in MME-transfected tumors than that in pcDNA3.1-transfected and nontransfected tumors (MME-transfected group versus pcDNA3.1-transfected group, />=0.025; and versus nontransfected group. .P=0.008). There is no significant difference between control groups (P=0.624). Western blot analysis of VEGF was also performed in the tumor tissue samples to quantify difference in protein level. A strong VEGF protein band was present in pcDNA3.1-transfected and nontransfected tumor tissues, whereas in MME-transfected tumor tissues a weaker band was observed. Quantification of the protein signals by image analysis revealed that the VEGF protein levels were 2.6-fold (7.83 + 1.14 versus 3. O4±0. 52) and 2.2-fold (6.57 ±0.76 versus 3.04+0.52) increased in pcDNA3.1-transfected and nontransfected tumor tissues, respectively, compared with MME-transfected tumor tissues. PO.01. Conclusion Our data show that the recombinant mammalian cell expression vector pcDNA3.1-MME is successfully constructed. The MME gene transfected into murine colon cancer cells can effectively suppresses 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更多还原