【作者】 易韬；

【导师】 魏于全；

【作者基本信息】 四川大学 ， 肿瘤学， 2004， 博士

【摘要】 目的:基质金属蛋白酶—2（matrix metalloproteinase-2/MMP-2）是一种分子量为72KD的明胶酶,在降解细胞外基质中起重要作用,高表达于多种肿瘤,并与病情进展及预后密切相关。MMP-2可作为肿瘤免疫治疗的靶标。本研究在拟本室既往工作的基础上,利用基因工程技术,在真核体系转染异种细胞疫苗的同时,以小鼠为模型动物观察其作为外源免疫原所激发的体液与细胞免疫反应以及可能的抗肿瘤免疫效应并初步观察其安全性。 方法:用Lipofectamine reagent将鸡MMP-2、鼠MMP-2和空质粒转染到CT26和LL/2细胞内,经过G418筛选转染的肿瘤细胞。用TrizolTM试剂从筛选的单克隆细胞中提取总RNA,做RT-PCR扩增MMP-2,确认实验用的高表答细胞株。用MTT（四甲基偶氮唑盐3-（4,5-dimetylthiazol-2-yl）-2,5-diphenyltetrazolium bromide）比色法（MTT colorimetric assay）检测转染后的细胞活性,再在小鼠体内做疫苗的致瘤性实验,保护性实验。ELISA检测免疫后鼠血清中MMP-2水平,Western blot检测抗体对抗原的识别能力,⁵¹Cr释放法检测脾T细胞及抗体对肿瘤细胞的特异杀伤活性;同时观察重组疫苗对EGFR阳性表达肿瘤的保护及治疗效应T细胞与抗体过继治疗 结果:用G418筛选转染的肿瘤细胞,得到数个CT26和LL/2的单克隆表达株,经过RT-PCR筛选出MMP-2的高表达株,用MTT法检测出转染前后的细胞增殖活性没发生改变,可用于后续动物实验研究。更多还原

【Abstract】 matrix metalloproteinase-2 (MMP-2), a 72kD gelatine A, plays an important role in the processes involving degradation of the extracellular matrix (ECM).Matrix metalloproteinase-2 (MMP-2) is overexpressed in human cancers and facilitates tumor growth and metastasis. Breaking of immune tolerance of MMP-2 should be a useful approach to cancer therapy by active immunity. To demonstrate the feasibility, we used transfectants with plasmid DNA encoding chicken homologous MMP-2 or corresponding mouse MMP-2 to evaluate tumor cells with MMP-2 as a tumor vaccine. Autoantibodies against mouse MMP-2 were identified by Western blot analysis and ELISA. The anti-tumor activity and production of autoantibodies against MMP-2 could be abrogated by depletion of CD4+ T lymphocytes. The CD8+-dependent MHC I restricted CTL response to MMP-2 positive tumor cells was also observed. All the induced antibody and cellular immune responses could cross-react with MMP-2 from mice or chichen. In addition, antitumor activity acquired with adoptive purified antibody and CD8+ T lymphocytes. We found that immunotherapy with c-MMP-2 was effective at both cellular and humoral antitumor immunity in several tumor models in mice. Taken together, these findings suggested the antitumor immunity induced by chicken MMP-2 may be provoked in a cross-reaction between the xenogeneic homologous and self-mmp-2, and may provide insight into treatment of MMP-2-positive tumors through the induction of the autoimmune response against MMP-2. Theobservations may provide a vaccine strategy for cancer therapy through the induction of autoimmunity and CTL in a cross reaction with transfectants.更多还原