【作者】 龚伟；

【导师】 冯作化； 张桂梅；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 过去的二十年在细胞和分子免疫的进展使我们进一步认识了肿瘤-宿主之间相互作用，这为肿瘤免疫治疗的发展创造了极好的机会。迄今为止，在调节免疫系统方面已经设计出一些非常有效肿瘤免疫治疗方法并取得了非常好的治疗效果。尽管这些方法也取得了令人激动的效果，但完全清除肿瘤的目的却是很难达到。实体肿瘤免疫治疗后肿瘤复发和转移却是导致患者致命最终和最后的结果。现在已经发现在肿瘤微环境中肿瘤细胞也有一些免疫分子的表达，这却成为肿瘤进一步进展和更有利于肿瘤逃逸以及抑制宿主肿瘤免疫的因素。但仍然不清楚的是在免疫治疗过程中哪些因子具有双刃剑的作用，又能够抑制肿瘤侵袭转移，而在免疫治疗后这些因子的下调是否会成为促进肿瘤侵袭转移的因素?IFN-γ是由活化的淋巴细胞分泌的一种重要的细胞因子，并且已经发现它的一些抗肿瘤活性机制，包括通过介导FAS依赖和非FAS依赖细胞凋亡途径改变肿瘤细胞的特性而更有利于宿主免疫识别和免疫攻击。IFN-γ也被发现能够通过下调肿瘤内生抗原促进肿瘤免疫逃逸，以及上调肿瘤细胞表面负性免疫调节分子B7-H1的表达诱导负性免疫调节。但是在肿瘤微环境中增加IFN-γ的水平仍然是一种关键而且与众不同的有效免疫治疗方法。然而，尽管在肿瘤生长方面IFN-γ正性和负性效果有大量广泛的研究报道，却很少有关于IFN-γ影响肿瘤侵袭转移行为方面的的报道。在我们的研究中发现了在免疫治疗结束后肿瘤细胞侵袭转移潜力的增强，其可能的机制是免疫治疗结束后微环境中的IFN-γ水平降低增强了肿瘤细胞对ECM分子的反应性，并加强活化肿瘤细胞表面的整合素ανβ3信号通路，而且这种免疫治疗后的缺陷可以利用作用肿瘤细胞整合素分子ανβ3的重组蛋白（CH50多肽）补救，达到抑制肿瘤细胞生长和侵袭的作用。本实验研究内容主要分为以下五部分：第一部分：研究肿瘤免疫治疗后残存肿瘤细胞的侵袭转移能力。免疫治疗并不能完全清除肿瘤细胞，而且我们不清楚残存肿瘤的侵袭转移能力又发生什么改变?体内转染4-1BBL／sPD-1质粒，RT-PCR和Western blot结果显示，转染48 h后，小鼠肌肉注射部位均有4-1BBL和sPD-1 mRNA和蛋白质表达。体内转染p4-1BBL／psPD-1质粒进行基因治疗，能够显著抑制小鼠腿部肿瘤生长，肿瘤生长速率显著低于空白对照组和pcDNA3．1组。动物实验结果表明，经过p4-1BBL／psPD-1质粒治疗的肿瘤组织块其肝移植瘤明显大于空白对照组和pcDNA3．1组的肝移植瘤，肝表面可见较多的转移结节。第二部分：研究免疫治疗结束后残存的肿瘤细胞侵袭转移能力的变化及其影响因素。IFN-γ是肿瘤免疫治疗中非常重要的细胞因子，IFN-γ具有明显抑制小鼠腿部肿瘤生长的作用。采用IFN-γELISA试剂盒检测，结果显示p4-1BBL／psPD-1组治疗结束时和治疗结束后14天IFN-γ浓度降低10倍。IFN-γ治疗后的移植瘤停用IFN-γ治疗，肝移植瘤生长速度加快，瘤体明显增大，而且肝表面可见多个转移结节。体外经IFN-γ处理的B16细胞和H22细胞分别接种于C57BL／6和BALB／c小鼠的肝脏，16天后解剖小鼠，IFN-γ撤除组（接种IFN-γ处理的瘤细胞后，动物不再进行免疫治疗组），肿瘤生长明显增强，瘤体明显增大。第三部分：研究旨在观察IFN-γ处理和撤除后肿瘤细胞生物学特性的改变，并探讨引起这些生物学特性改变可能的机制。IFN-γ处理后浓度降低s速度与细胞体外增殖速度成正比。而且IFN-γ撤除后B16细胞在抵抗MMC诱导凋亡、粘附以及恢复粘附能力、侵袭能力等方面均明显高于对照组细胞。同时应用RealTime-PCR、western-blot检测相关基因变化情况：与增殖、抵抗凋亡正相关的基因如c-Myc、Bcl-2和Bcl-xL表达均明显高于对照组细胞：而负相关基因如p21^WAF1、p27^Kip和Bax均明显低于对照组细胞；Western blot检测ERK1／2、JNK磷酸化，IFN-γ撤除后B16细胞的ERK1／2、JNK磷酸化水平增高。尾静脉接种IFN-γ处理B16细胞于小鼠，5、24小时小鼠肺部肿瘤荧光数目明显高于对照组；14天后IFN-γ撤除组肺部转移结节数目明显多于对照组，并且小鼠的存活时间明显短于对照组。第四部分：探讨IFN-γ撤除后B16细胞侵袭潜能增强可能的机制。采用Real-time PCR，RT-PCR和Western blot检测αν、β3、CDC2、MMP2、MMP9mRNA和蛋白表达。结果显示：IFN-γ处理后的B16细胞与ECM作用后，αν、β3、CDC2、MMP2、MMP9上调，Western blot检测IFN-γ撤除后的B16细胞与ECM作用后FAK磷酸化水平增强，并且时间持续更久。荧光显微镜检测细胞微丝F-actin聚合能力增强。Zymography assay结果显示，活化型MMP2、MMP9蛋白水平增高。第五部分：探讨防止肿瘤免疫治疗后残存肿瘤细胞侵袭潜能增强的可能措施。在体内转染pCH510 12 h、24 h、48 h和72 h后，通过RT-PCR检测到小鼠肝脏细胞表达的CH50的mRNA，同时在体内转染pCH510 72 h后应用Western-blot检测到小鼠肝脏组织和血清中表达的CH50蛋白。小鼠尾静脉肿瘤肺转移模型结果显示，IFN-γ撤除后的B16细胞组的小鼠肺部肿瘤转移结节最多。体外IFN-γ撤除后联合CH50处理B16细胞能有效地抑制肿瘤细胞转移至肺部，同时体内表达CH50也同样能抑制肿瘤的转移。在小鼠肿瘤移植模型中我们观察到，CH50无论在免疫治疗过程中和免疫治疗后都能极大地抑制移植瘤的生长和侵袭。更多还原

【Abstract】 Progress in molecular and cellular immunology during the past two decades hasadvanced our understanding of tumor-host interactions and opened extraordinaryopportunities for the development of antitumor immunotherapy. So far, there aremany available approaches designed to modulate the immune system for a betterefficacy in tumor immunotherapy. Although the exciting efficacy has been obtained byusing these approaches, the complete tumor eradication occurs infrequently. Tumorrecurrence and metastasis are frequently the final and fatal step in the progression ofsolid malignancies in patients after immunotherapy. It has been found that a variety ofmolecular alterations occur in tumors so that they become more progressive and betterequipped to evade or inhibit host defenses. However, it is still unclear whichfactor（s） is responsible for the inhibition of tumor metastasis during immunotherapyand what happens if such factor（s） is downregulated after immunotherapy.Interferonγ（IFN-γ） is an important cytokine produced by the activatedlymphocytes and contributes to antitumor activity via several mechanisms, includingphenotypic or functional modification of neoplastic cells, rendering them moreamenable to immune recognition and attack via Fas-dependent and Fas-independentpathways. Although IFN-γ, has also been found to promote tumor immune evasionby downregulating cellular level of an endogenous tumor antigen and induce thenegative immune regulation by upregulating B7-H1 expression, the increase ofIFN-γlevel is still crucial for the therapeutic efficacy of different immunotherapeuticapproaches. However, despite the extensive studies on the positive and negative effectsof IFN-γon tumor growth, little is known about the influence of IFN-γon the metastatic behavior of tumor cells.In this study, we found that the metastatic potential of tumor cells was increasedafter the termination of immunotherapy. Underlying mechanisms involve the increasedactivity of integrin.Theαvβ3 signaling pathway and the increased responses of tumorcells to ECM molecules after the removal of IFN-γ. This pitfall of immunotherapytermination could be remedied by the administration of a recombinant polypeptide offibronectin, which counteracts the increasedαvβ3 signaling of tumor cells. Thesefindings suggest that the termination of immunotherapy, especially the drop of IFN-γ,may increase the risk of tumor metastasis and that targetingαvβ3 signaling.Thisexperiments are subdivided into four parts.Part 1: To explore the invasive behavior of tumor cells after immunotherapy.Although the exciting efficacy has been obtained by using tumor immunotherapy. Thecomplete tumor eradication occurs infrequently. Plasmid p4-1BBL was injected intomuscle of mice. The expressed 4-1BBL and sPD-1 were identified by RT-PCR andWestern blot Gene therapy with p4-1BBL/psPD-1 inhibits the tumor growth but theresidual tumor exhibits an enhanced capability of invasive growth. The donor mice（n=8 per group） were inoculated by subcutaneous injection of B16 cells, and genetherapy with p4-1BBL/psPD-1 was performed. Small pieces of tumor tissues afterimmunotherapy were transplanted to recipient mice. The transplanted tumors weremeasured and other tumor nodes in the liver of recipient mice were counted. The resultshowed that the transplanted tumors from p4-1BBL/psPD-1-treated mice grew fasterthan those from control mice, concomitant with the appearance of more satellitenodules around the main tumor.Part 2: To explore which factor（s） is responsible for the enhanced of the residualtumor metastasis after immunotherapy and what happens if such factor（s） is changed inmicroenvironment. Interferonγ（IFN-γ） is an important cytokine produced by theactivated lymphocytes and contributes to antitumor activity. IFN-γexpression in tumormicroenvironment was 10 fold lower several days after the termination of P4-1BBLtransfaction. Gene therapy with pIFN-γinhibits the tumor growth but the residualtumor exhibits an enhanced capability of invasive growth. The donor mice （n=8 pergroup） were inoculated by subcutaneous injection of B16 cells, and gene therapy with pIFN-γwas performed. Small pieces of tumor tissues after immunotherapy weretransplanted to recipient mice. The transplanted tumors were measured and othertumor nodes in the liver of recipient mice were counted. The result showed that thetransplanted tumors from pIFN-γ-treated mice grew faster than those from controlmice, concomitant with the appearance of more satellite nodules around the maintumor. B16 cells and H22 cells were treated with IFN-γ（20 ng/ml） in vitro for 48 h,and then injected into the liver of C57BL/6 and BALB/c mice （n=8 per group）respectively. The mice were sacrificed d14 later. The result showed that the injectedinto the liver tumors from IFN-γtreatment mice grew faster than those from controlmice, concomitant with the appearance of more satellite nodules around the maintumor.Part 3: This study was designed to investigate the tumor cells biologicalphenotype after the treatment of the IFN-γand to analyze the possible mechanism.Decrease or removal of IFN-γresulted in the increase of tumor cell proliferation.Similarly, and the cells were more resistant to MMC-induced apoptosis. The resultsshowed that the expression of c-Myc, Bcl-2, and Bcl-xL was down-regulated and thatof p21^WAF1, p27^Kip1, and Bax was up-regulated in the presence of IFN-γ, but theexpression pattern of these genes was totally reversed after the decrease or removal ofIFN-γ. And the activation of ERK and JNK in tumor cells was intensified in therecovered tumor cells. Adhesion assay showed that the continuous presence of IFN-γslightly suppressed the adhesive ability of tumor cells to fibrinogen, fibronectin andlaminin, whereas the adhesion of tumor cells to these molecules was strongly increased24 h after the removal of IFN-γ. The increased adhesive ability of tumor cells lastedfor several days after the removal of IFN-γ. After the injection of CSFE-labeled B16cells into mice via tail vein, the fluorescent spots in lung tissues, both 5 h and 24 hafter tumor cell injection, were significantly increased in the treatment/recover group.The results showed that the metastatic tumor nodes in lung were significantlyincreased and the survival of mice was shortened if B16 cells were pretreated withIFN-γbefore inoculation.Part 4: To elucidate the molecular mechanism involved in the increased invasivegrowth and metastasis of tumor cells after IFN-γ. The results showed that the activation of FAK in tumor cells was intensified in the recovered tumor cells,indicating thatαvβ3 signalling pathway is indeed intensified. The expression of cdc2gene, a down-stream gene ofαvβ3 signalling pathway, was up-regulated in thepresence of matrigel, and further up-regulated in the recovered cells. The productionand activation of MMP-2 and MMP-9, and the polymerization of actin in response toECM molecules were also increased in the recovered B16 cells.Part 5: This study was designed to explore possible method of therapy that theresidual tumor metastasis were enhanced after immunotherapy. The CH50 mRNAs of12 h, 24 h, 48 h and 72 h on hepatic cells of the mouse after in vivo transfection ofpCH510 by i.v. injection were detected by RT-PCR. 72 h after in vivotransfection of pCH510 by i.v. injection, the expressed CH50 in serum or livertissue was detected by Western blot. The results showed that the in vitropretreatment with IFN-γsignificantly increased the lung metastasis of tumor cells,which was abolished by in vivo expressed CH50. The in vitro pretreatment of tumorcells with IFN-γtogether with CH50 also suppressed the tumor metastasis to lung sothat the in vivo expressed CH50 effectively inhibited the lung metastasis of tumor cells.We then investigated the effect of CH50 in immunotherapy/transplantation model.The treatment with in vivo expression of CH50 either during immunotherapy （in donormice） or after immunotherapy （in recipient mice） significantly inhibited the invasivegrowth of transplanted tumor.更多还原