【作者】 汪琼；

【导师】 袁守军；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药理学， 2012， 博士

【摘要】 肿瘤细胞要想在机体存活必须能够逃避免疫系统的监视和免疫细胞的清除。待肿瘤细胞存活并生长到一定的数目时会在局部形成组织块，那么肿瘤块内的微环境为其增殖、侵袭和转移等过程提供的“保护伞”，使得微环境外的免疫细胞无法突破这个“深度免疫抑制的黑洞区”攻击癌细胞，这个时候使用免疫药物治疗癌症往往事倍功半。而手术治疗癌症一方面切除大部分瘤组织，减轻肿瘤负荷，另一方面机械地快速地破坏已经形成“免疫抑制黑洞”的肿瘤微环境，可使残灶内部的肿瘤细胞、免疫抑制细胞暴露，这样微环境外活化的免疫细胞才有机会直接接触肿瘤细胞，产生吞噬、细胞毒作用。基于此原理并结合临床实体癌治疗原则，我们成功建立了一种新型的肿瘤药效学研究模型---小鼠术后残瘤模型来评价和筛选抗肿瘤生物反应调节药物，并在此模型上筛选到一个很有潜力的抗肿瘤生物反应调节剂T肽。T肽是以天然四肽Tuftsin为母体进行结构改造获得的一种新衍生物，它不仅保留了Tuftsin的促吞噬和抗肿瘤的生物活性，而且大大提高了抗酶解能力，延长了体内半衰期。对T肽抗癌作用的药效学研究中，我们采用了三种肿瘤小鼠模型评价药效。T肽对传统的皮下移植瘤小鼠模型的抑瘤率约在40%左右，而在手术荷瘤小鼠模型上T肽对术后残瘤生长的抑制效果显著，抑制率达到48.9%-84.3%，并且未观察到毒副作用，小鼠生存期较细胞毒性药物环磷酰胺明显延长。本实验室随后建立了细胞免疫缺陷裸鼠手术单瘤模型和联合免疫缺陷的SCID鼠手术单瘤模型，T肽对多种皮下移植瘤残瘤都有良好的抑制作用。在小鼠实验性转移模型中，T肽延缓恶性肿瘤细胞的血液转移发生，减轻侵袭肿瘤的严重程度。结合T肽的药物结构特性和细胞生物学功能，采用RT-PCR、Realtime-PCR、免疫组化、ELISA、western blot等检测手段开展抗癌作用机理的研究。巨噬细胞是T肽的特异靶细胞之一。体外，T肽能够提高巨噬细胞的增殖能力。通过流式检测Ana-1细胞的周期，进一步确证T肽能够促进Ana-1巨噬细胞的增殖，调节其G1期向S期的进程。细胞周期中G1/S期的调控点是细胞内外信号经过传递，整合汇集到细胞核，对细胞的增殖进行调控的关键点。调控细胞周期的众多重要蛋白中， P21WAF1是G1/S期转移的负向调节因子。Western blot的结果表明随着T肽给药的时间延长和给药剂量的增加，P21WAF1的蛋白量明显下调，其作用底物蛋白CDK2的磷酸化水平相应增加。T肽影响巨噬细胞增殖的活性与其激活ERK/JNK/MAPK信号转导通路密切相关。另一方面，T肽能够结合巨噬细胞表面特异性抗体，在短时间内激活NF-κB信号通路，活化巨噬细胞。巨噬细胞被活化后其吞噬功能、细胞毒功能明显增强，同时介导M1表型极化，分泌Th1促炎性因子TNF-α、IL-12、NO、IFNα/β以及趋化因子CXCL9、CXCL11等等。在体外用IL-4诱导M2极化的Ana-1巨噬细胞模型中，T肽同样能够促使M2巨噬细胞中Th1细胞因子的表达增加，随着T肽给药浓度的增加，M2巨噬细胞代表基因Arg-1mRNA明显下调，M1巨噬细胞代表基因iNOS、IL-12转录水平和蛋白水平表达上调。以上实验结果提示T肽能够逆转M2巨噬细胞的表型。肿瘤微环境中占主导地位的肿瘤相关巨噬细胞TAM大多数为M2巨噬细胞。从T肽治疗的小鼠术后残瘤组织中原代分离出TAM，同样方法检测，发现T肽给药组TAM和Tuftsin给药组TAM与术后荷瘤组TAM相比，除了IL-1β，其它Th1和Th2细胞因子的转录能力被显著抑制。NF-κB通路介导T肽对静止期巨噬细胞的M1表型极化，而TAM细胞为一群特殊的NF-κB信号通路缺陷的巨噬细胞，T肽很有可能不能通过此通路激活TAM，从而逆转TAM的功能和表型。TAM中的Th2细胞因子表达同样被抑制，说明T肽还是能够通过其它途径影响TAM的炎症因子表达。2006年Tuftsin受体NRP-1的发现和五肽TKPRR在VEGF165/VEGFR2/NRP-1中的作用，提示Tuftsin可以与VEGF165竞争结合NRP-1，阻断VEGF165与VEGFR2结合，抑制肿瘤血管生成。作为Tuftsin的聚合类似物，T肽减少MCF-7裸鼠术后残瘤组织和M2巨噬细胞中VEGF和MMP-9两种因子的蛋白合成，抑制肿瘤的侵袭和转移。对NRP-1受体，T肽显著促进巨噬细胞中NRP-1的表达，对血管内皮细胞的NRP-1表达没有影响。NRP-1的表达增加可能与T肽促进巨噬细胞的增殖和活化能力密切相关，也可能参与了T肽间接抑制肿瘤的生长过程。本研究还初步探讨了T肽对PD-1/PD-L1信号通路的影响。在肿瘤微环境中，肿瘤细胞与其间质中的抗原递呈细胞可以通过负性协同共刺激分子PD-L1和PD-1分子相互作用诱导T细胞凋亡，抑制CD4和CD8T细胞的增殖和活化，最终导致肿瘤免疫逃逸的发生。体外实验和体内实验结果均证实T肽对肿瘤细胞、巨噬细胞表面的多种B7家族共刺激因子的表达产生影响，例如4-1BB、PD-L2和OX40分子的表达上调，PD-L1/PD-1分子下调，提示T肽可以阻滞PD-1/PD-L1程序性死亡通路，从而促进T淋巴细胞的活化，调控机体对肿瘤的免疫监视能力。总而言之，T肽通过诱导机体外周的未被活化的巨噬细胞发生M1表型极化，释放Th1细胞因子，增强细胞毒功能和抗原递呈功能，发挥抗癌效应。Th1细胞因子IL-12和IFN-γ同时还能活化T淋巴细胞和NK细胞，由此改变机体整体免疫状态，巩固机体的抗癌作用。对T肽抗癌作用和机理的研究，不仅为T肽的药物报批奠定基础，同时也为研发更加高效低毒的Tuftsin衍生物或其它BRM类药物提供新的动物模型和新的临床应用。更多还原

【Abstract】 Tumor cells survived in the body must be able to escape immune surveillance andkilling by immune cells. When the tumor cells grow to a certain size in local place, tumormicroenvironment is formed. Tumor microenvironment support tumor for proliferation andmetastasis and immune cells out of the tumor microenvironment can’t break through thisbarrier to attack cancer cells. So biological response modifiers treatment with patients cannot make effort expectably under this circumstances.Surgery is helpful to reduce tumorburden after resecting most of tumor tissue. On the other hand, tumor microenvironment isdamage quickly by surgery and internal immunosuppression cells and tumor cellsexposured so that the outside activated immune cells have change to identificate and killtumour cells. Based on this principle and combined with clinical cancer therapy principle,we successfully established a novel murine pharmacodynamics model with a postsurgeryresidual tumor. And in this model we find T peptide as a potential immunopotentiator.T peptide is a new derivative of Tuftsin. It not only retains phagocytosis andanti-tumor biological activities, but also greatly improves the enzyme-resistant ability toextend its half-life in vivo. We adopted three tumor model evaluation anti-cancer activity ofT peptide. Compaired with the traditional xenograft tumor mouse model, TP displayedbetter effects on murine model with a pseudosurgery and the inhibition rate reached above80%.T peptide almost no observed toxicity to mice during experiments. And the mice weresignificantly longer life than cyclophosphamide group mice. T peptide also had highinhibition rate of anti-tumor effects on nu/nu and SCID mice operational tumor model. Onmice transfer model, T peptide delayed invasion and transformation by preventingmalignant cells spread to the blood circulation.Combined with the structure characteristics of peptide T drugs and cell biologyfunction, RT-PCR, realtime-PCR, immunohistochemical, ELISA and western blot assayswas tested to study the mechanism of anti-cancer action of TP. Macrophages are one ofspecific target cells for TP. In vitro, T peptide can improve macrophage proliferation, butthis effect was slight and no dose-response relationship. Through detection cell cycle, itconfirmed that T peptide can promote macrophage cell to proliferation by adjusting the G1phase to S phase process. The cell cycle in the regulation of G1/S point is internal andexternal signal transmission through cell, integrate together to the nucleus, so theproliferation of cells can be controlled in the key point. The cell cycle regulation proteinP21WAF1is a negative adjustment factor in G1/S phase. The results showed that T peptidemarkedly decreased P21WAF1expression with CDK2phosphorylation level increased. Tpeptide impacted macrophage proliferation via ERK/JNK/MAPK signal transductionpathways.On the other hand, T peptide activated macrophage through NF-κB signaling pathwaysin a short time. Macrophage phagocytosis was activated and its cytotoxic function obviously enhanced after TP treated. Activated macrophage by TP had been polarized toM1phenotype. M1macrophage secretes Th1proinflammatory cytokines and chemokinessuch as TNF-α,IL-12, NO, IFN α/β,CXCL9and CXCL11. In vitro, T peptide can alsotrigger M2macrophages polarized by IL-4to release Th1cytokines expression of cytokines.Marker gene of M2macrophages like Arg-1was obviously down-regulated and theexpression of M1macrophages marker gene rised, such as iNOS and IL-12.The experimentresults suggested that T peptide could be reverse phenotype of M2macrophage. Tumorassociated macrophage in tumor microenvironment appears M2phenotype. In TAM frompostoperative residual tumor tissue of T peptide group and Tuftsin group, the transcriptionsof Th1and Th2cytikines were decreased except in IL-1β. T peptide activated macrophagethrough NF-κB signaling pathways. NF-κB signaling pathways can not work in TAM resultthat TP could not swith TAM phenotype from M1to M2.The suppression of Th2cytokinesalso prompted that T peptide influenced TAM through other pathways.It is reported in2006that NRP-1is a receptor of Tuftsin. Tuftsin can block VEGFR2phosphorylation by competition with VEGF165via NRP-1and inhibited tumorangiogenesis. As a new Tuftsin polymerization analogues, T peptide inhibited VEGF andMMP-9in tumor and M2macopahe. To NRP-1receptor, T peptide significantly improvedmacrophages to express NRP-1.But the expression of NRP-1in HUVEC had not changed.This activity of T peptide may be involved in macrophage proliferation and activation Tpeptideas as well as anticancer activity. T peptides block PD-1/PD-L1signaling pathwaysin tumor and macrophage.4-1BB, PD-L2and OX40molecular expression were higher thancontrol and PD-L1as well as PD-1protein decreased. The results confirmed that T peptidemodulated immunity of macrophage and T cells.In conclusion, we successfully established a novel murine model with a postsurgeryresidual tumors for anticancer pharmacodynamics evaluation. And we researched theanticancer effects of TP on this novel murine model in vivo and in vitro. On the basis of thenew model, we studied the mechanisms of TP on macrophages, TAMs.The results and theknown low toxicity are clues that TP might be a promising candidate drug for the treatmentof clinical patients with postsurgery residual tumors or micrometastasis.In short, T peptide induced macrophage to M1phenotype polarization. Activemacrophage by TP released Th1cytokines, strengthen antibody-dependent cell-mediatedcytotoxicity function. IL-12and IFN-γ can also activate T lymphocytes and NK cells. Theall results are clues that TP might be a promising candidate drug for the treatment ofclinical patients with postsurgery residual tumors or micrometastasis.更多还原