【作者】 郑少江；

【导师】 吴人亮；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 肿瘤血管生成是肿瘤生长与转移的必要条件,抗肿瘤血管生成研究已成为肿瘤研究的一个热点。bFGF是最重要的肿瘤血管生成因子之一,其生物活性主要是通过与高亲和性受体——FGFR-1结合而发挥作用。研究表明,FGFR-1特异表达于肿瘤新生血管的内皮细胞和许多肿瘤细胞中,是肿瘤血管生成的重要调节分子。作为配体结合区的FGFR-1胞外段和配体bFGF结合后发生二聚体化导致自身磷酸化,从而诱导肿瘤血管生成,进而促使肿瘤生长和转移。可见,封闭FGFR-1胞外段就可阻断bFGF/FGFR-1的信号传导通路,阻止肿瘤血管生成,从而抑制肿瘤生长和转移。已有研究证实利用抗FGFR-1单克隆抗体被动免疫治疗在动物模型中具有明显的抗肿瘤作用,但是这种被动免疫治疗方式存在许多不足。此外,研究还表明抗肿瘤血管生成治疗联合低剂量化疗可以提高抗肿瘤血管生成作用,而且没有明显毒副作用。因此,蛋白疫苗主动免疫治疗或者联合低剂量化疗的抗肿瘤血管生成策略展示了良好的临床应用前景。本研究设想利用肿瘤血管生成过程中特异表达的FGFR-1分子作为治疗的靶点,利用基因工程技术构建了鸡FGFR-1 (cFR)和小鼠自身FGFR-1 (mFR)胞外段重组蛋白,了解异种同源cFR蛋白是否能够打破小鼠自身免疫耐受,诱导肿瘤模型小鼠产生抗自身FGFR-1的抗体阻止血管生成,从而抑制小鼠肿瘤的生长,并初步探讨其作用机制,为抗肿瘤血管生成治疗提供新途径。为此,我们首先用cFR蛋白质作为疫苗,在CT26结肠腺癌和Meth A纤维肉瘤两个小鼠肿瘤模型中进行实验观察。实验结果发现,用cFR蛋白疫苗主动免疫治疗的小鼠能明显减缓肿瘤的生长并延长荷瘤小鼠的生存时间,且没有发现毒副作用。Western blot和ELISA检测都发现cFR免疫小鼠血清中产生的抗体不仅能识别cFR,而且能识别同种的重组蛋白质mFR。ELISPOT显示cFR免疫后小鼠脾脏中有较多特异性抗cFR或者抗mFR抗体的B淋巴细胞分泌。免疫荧光检测也证实在cFR免疫小鼠肿瘤组织内有内皮细胞性自身抗体沉积。在阻断CD4+T淋巴细胞亚群后,cFR免疫小鼠不能产生自身抗体。用cFR免疫小鼠血清中纯化的免疫球蛋白进行过继性免疫治疗也同样具有抑制肿瘤作用。抗CD31免疫组化结果显示,经cFR免疫治疗后小鼠的肿瘤组织内微血管计数明显减少。接着,我们用cFR蛋白疫苗联合小剂量吉西他滨在CT26肿瘤模型中进行抗肿瘤作用观察。结果发现两者单独治疗均有不同程度的抗肿瘤作用,而联合治疗效果更佳且未发现明显的毒副作用。免疫组化检测结果表明,联合治疗在肿瘤组织微血管密度、细胞调亡和增殖中具有协同作用。Western blot和ELISPOT检测发现联合治疗和单独免疫治疗均能诱导产生抗mFR的抗体,并可检测到分泌特异性抗mFR抗体的B淋巴细胞。综上所述,本研究成功构建了鸡和小鼠FGFR-1胞外段原核表达质粒,获得了纯化的重组蛋白质并制备成疫苗。cFR蛋白疫苗主动免疫治疗抗肿瘤的作用可能是疫苗诱导荷瘤小鼠体内产生了特异性抗自身FGFR-1抗体,自身抗体阻断了bFGF与FGFR-1间的信号传导通路,诱导血管内皮细胞调亡、从而抑制肿瘤血管生成的结果。CD4+T淋巴细胞在抗肿瘤特异性免疫反应中发挥重要作用。联合治疗不仅没有影响蛋白疫苗诱导产生特异性免疫反应,而且协同增强了其抗肿瘤作用,亦没有发现明显的毒副作用。这些结果将为抗血管生成的肿瘤治疗提供新的途径,值得进一步深入研究。更多还原

【Abstract】 It is generally believed that the growth and metastases of a tumor are angiogenesis-dependent and thus that anti-angiogenic therapy, which targets genetically stable endothelial cells as a strategy for cancer therapy, is highly warranted. At present, basic fibroblast growth factor (bFGF) has been shown to be one of the most important angiogenic growth factors for tumor angiogenesis. bFGF conducts its biological function through interaction with its high-affinity receptor, fibroblast growth factor receptor-1(FGFR-1), which is markedly expressed both in active endothelial cell and in many different forms of tumor and plays an important role in tumor angiogenesis and tumor growth. Thus, it is logical to consider using FGFR-1-mediated anti-angiogenesis as a target of immune therapy for tumor treatment, which could suppress angiogenesis and further inhibited tumor growth by block the bFGF/FGFR-1 signal transduction. Studies suggested that passive immunotherapy with a monoclonal antibody FGFR-1-mediated anti-angiogenesis could inhibit tumor growth, but the blocker may be involved some side-effects. Moreover, much recent evidence has also confirmed that xenogeneic homologous molecules can induce cross-reaction against self homologous molecules. Furthermore, other findings have also demonstrated that antiangiogenic therapy combined with chemotherapy could more effectively inhibited tumor growth without overt toxicity than either therapy alone. So the antiangiogenic therapy in solid tumors should be used as a new approach for cancer therapy with active immunity or combined with chemotherapy agents.To test this notion, in the first part, we recombined the extracellular domain of chicken FGFR-1(cFR) protein vaccine and a mouse FGFR-1 (mFR) protein vaccine as a homologous control. We found that immunotherapy with cFR vaccine was effective at both protective and therapeutic antitumor immunization in two mouse tumor models. The tumor volume was significantly smaller and the survival time was significantly longer in cFR-1-immunized group than those in control groups. Auto-antibodies against mouse FGFR-1 were found in sera of mice immunized with cFR in Western blotting analysis and ELISA assay. Anti-FGFR-1 antibody producing B cells (APBCs)were were observed in all cFR-immunized mice, which detectable by ELISPOT. The endothelial deposition of autoantibodies was found within tumor tissues from cFR-immunized mice, which were detected by immunofluorescent staining.The antitumor activity and production of autoantibodies against FGFR-1 could be abrogated by depletion of CD4+T lymphocytes. The antitumor activity was also induced by the adoptive transfer of the purified immunoglobulins was apparently inhibited within the tumors. The microvessel density, which stained with antibodies reactive to CD31, was also significantly less in the cFR-1-immunized mice than in controls.In the second part, we evaluated primarily the antitumor activities of low-dose gemcitabine combined with a recombinant cFR vaccine in a mouse colon adenocaicinoma model. We found that low-dose gemcitabine or cFR vaccine treatment resulted in the inhibition of tumor growth to a certain extent. Remarkably, the combination therapy results in apparent decreases in tumor volume, microvessel density and tumor cell proliferation, and an increase in apoptosis without obvious side-effects as compared with either therapy alone or normal control groups. Moreover, the low-dose gemcitabine did not inhibit the host cross-immune response, but it potentiated anti-tumor effects as was demonstrated in the synergistic indexes of tumor volume, MVD, apoptosis and proliferation, and the presence of both auto-antibodies and the APBCs in the cFR-1-immunized mice.Taken together, these findings demonstrates that the mechanism of antitumor effects may be related to the autoantibodies against mouse self-FGFR-1 induced by cFR1 immunizaton could block the bFGF/FGFR-1 signal transduction, may involve CD4+ T lymphocytes, and further inhibited tumor growth by mediated anti-angiogenesis. The combination therapy strategy effectively and synergistically suppressed tumor growth via inhibition of tumor angiogenesis without systemic toxicity, which the low-dose gemcitabine did not inhibit the hose immune response. Moreover, no overt toxicity was found in all of the mice. Our study may provide an alternative strategy for treatment of tumor angiogenesis and needs further investigation.更多还原