【作者】 刘琼；

【导师】 王红阳；

【作者基本信息】 第二军医大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 一、信号调节蛋白α在树突状细胞活化中的调节作用研究背景和目的信号调节蛋白α(SIRPα)是SIRP家族中最主要的成员。一方面,SIRPα可被多种有丝分裂原活化而发生磷酸化,通过其ITIM结构域与SHP-1和SHP-2结合,传递抑制性信号。另一方面,SIRPα还可通过与CD47配体相互作用抑制下游通路活化,传递负向信号。SIRPα在天然免疫的调节中发挥着重要作用,在巨噬细胞中LPS可通过转录抑制和蛋白降解途径诱导SIRPα表达下调,SIRPα通过屏蔽SHP-2作用抑制MAPK、IKKs、NF-κB和IRF3激活,减少炎性因子和IFNβ释放。有研究显示树突状细胞(DC)中的SIRPα还能通过与T细胞表面的CD47结合,双向负调控DC和T细胞的功能,一方面抑制T细胞表达IL-12R,并使成体CD4+和CD8+T细胞对IL-12反应性降低;另一方面,抑制不成熟DC的表型和功能成熟,并抑制其细胞因子的释放。DC是目前发现的功能最强的专职性抗原递呈细胞(antigen-presenting cells,APC),能摄取和加工递呈抗原,具有强大的激活CD8+、CTL及CD4+T辅助细胞的能力,控制着体内免疫反应的过程,在免疫应答中处于中心地位,因而成为肿瘤免疫反应的中心环节。目前普遍认为,免疫逃逸是肿瘤产生的重要因素。由于SIRPα对DC的功能成熟有着很大影响,并可通过结合CD47直接抑制T细胞功能,因此在肿瘤免疫逃逸中可能起重要作用。本研究旨在通过对DC中的SIRPα进行miRNA干扰,阻断SIRPα负向信号通路,进一步揭示SIRPα对DC和T细胞功能的影响,明确其在肿瘤免疫治疗中的作用并分析其可能的分子机制。实验方法1.构建慢病毒质粒:LV-microRNA-SIRPα、LV -GFP、LV-SIRPα;慢病毒包装和滴度检测。2.骨髓来源DC分离培养。3.体外观察慢病毒干扰SIRPα的DC(a)成熟和迁移表型的改变—PE标记的MHCⅡ、CD80、CD86、CCR7、CCR5流式细胞计数;(b)生存能力的改变—PI染色流式细胞计数;(c)分泌细胞因子功能的改变—IL12、IL-6、TNFαELISA检测。4.体外观察干扰SIRPα的DC对T细胞功能调节:(a)T细胞的增殖能力的改变—H3增殖实验;(b)T细胞分泌IFN-γ水平的改变—ELISA检测。5.体内观察干扰SIRPα的DC迁移和增殖能力的改变—CFSE标记,流式检测代谢。6.体内观察干扰SIRPα的DC对T细胞功能调节(a)T细胞增殖能力的改变—CFSE标记,流式检测代谢;(b)T细胞杀伤功能的改变—非放射性乳酸脱氢酶检测;(c)T细胞分泌IFN-γ水平的改变—ELISPOT。7.体内观察干扰SIRPα的DC的肿瘤免疫功能的改变—在治疗和预防模型中应用SIRPα干扰的DC疫苗,观察对肿瘤大小和小鼠生存时间的影响。8.讨论SIRPα对DC功能调节的机制:(a)生存能力相关PI3K-AKT通路的改变—Western-blot;(b)分泌功能相关JAK-STAT通路的改变—Western-blot;(c)成熟迁移相关NF-κB通路的改变—Western-blot。9.讨论SIRPα的上、下游信号蛋白在DC功能调节中的作用:TLR-4、SHP-1、SHP-2、P85—免疫共沉淀IP、Western-blot。实验结果1. LPS诱导DC活化过程中,SIRPα表达下调,细胞生存相关的AKT磷酸化水平升高、Bcl-2表达增高。SIRPα-silened DC在加入polybrene(8ug/ml)条件下培养12hr后,抗凋亡能力显著强于对照组。2. SIRPα-silenced DC成熟表型:MHCⅡ、CD80和CD86表达增强,迁移相关表型CCR7表达增强、CCR5表达降低。3. SIRPα-silenced DC分泌细胞因子IL-12、IL-6增强。4. SIRPα-silenced DC体外刺激T细胞增殖和分泌IFNγ能力增强。5. SIRPα-silenced DC体内刺激T细胞分泌IFNγ能力增强。6.降低SIRPα表达的DC免疫na?ve C57小鼠,对免疫原性强的5×105 EG7细胞皮下种植有100%免疫预防能力,肿瘤注射两周后能观察的小鼠脾CD8+T分泌IFNγ能力增强。7.降低SIRPα表达的DC免疫na?ve C57小鼠,对免疫原性弱的1×105 B16F10皮下种植有>50%免疫预防能力。结论本实验通过慢病毒干扰技术有效抑制DC中SIRPα的表达,并通过体内体外实验证实SIRPα在DC生存、成熟和抗原递呈中起着重要的抑制作用。此外,我们还通过小鼠肿瘤预防模型进一步证实SIRPα在肿瘤免疫中发挥抑制性调节作用。以上提示我们,干扰DC中SIRPα制备的DC疫苗可能成为肿瘤免疫治疗中重要的组成部分。二、射频消融术联合热休克DC疫苗抑制肿瘤复发研究背景和目的射频消融(RFA)是肝脏肿瘤获得根治的微创治疗方法之一,其原理主要是通过射频产生的热量,使局部肿瘤消融。但治疗后常见消融灶内部或边缘活性肿瘤细胞残留或局部复发,所以,射频消融术联合其它治疗方案势在必行。射频消融后肿瘤细胞坏死释放大量肿瘤抗原,可在射频区域能检测到微弱的免疫反应,这可能与消融区外周肿瘤组织产生的大量热休克蛋白对DC识别、摄取和加工肿瘤抗原的激活效应相关。如果联合治疗方案能使射频术后产生强大而持久的特异性肿瘤免疫反应,将对肿瘤射频消融术预后有很大改善。DC是目前发现的功能最强的专职性抗原递呈细胞(antigen-presenting cells,APC),能摄取和加工递呈抗原,具有强大的激活CD8+、CTL及CD4+T辅助细胞的能力,控制着体内免疫反应的过程,在免疫应答中处于中心地位,因而成为肿瘤免疫反应的中心环节。目前,DC疫苗已成为临床免疫治疗的重要手段,联合DC疫苗免疫治疗增强射频消融术后局部和全身特异性肿瘤免疫,是改善射频术预后的可行方案。本研究设计采用体外热休克肿瘤细胞制备肿瘤抗原,模拟体内射频术后肿瘤组织释放的抗原,负载DC制备热休克DC疫苗。通过热休克DC疫苗注射使荷瘤鼠产生初次免疫和免疫记忆,当接受射频术时,大量原位肿瘤抗原引发强烈持久的再次特异性肿瘤免疫应答,有效防止射频术后复发。本研究旨在探索物理治疗联合生物治疗肝脏肿瘤的应用价值和可行性,并为建立新的肿瘤综合治疗模式提供有价值的线索和理论支持。实验方法1. DEN诱导C57原发性肝癌—DEN喂饮8个月。2.皮下C57 B16F10肿瘤模型的建立。3.观察小鼠原发性肝癌和皮下B16F10肿瘤射频后组织热休克反应(a)HSP-70、gp96的表达—免疫组化;(b)HMGB1的表达—免疫组化。4.观察体外肿瘤细胞43度热休克是否能模拟体内射频后热休克状态:(a)HSP-70、gp96的表达—Western-blot;(b)HMGB1的表达—免疫组化—细胞免疫荧光。5.利用体外肿瘤热休克制备热休克抗原,DC负载热休克抗原制备DC疫苗。6.热休克DC疫苗联合射频消融治疗,观察其对皮下B16F10肿瘤复发的影响:设立实验组:43度培养的B16F10肿瘤抗原制备DC疫苗联合射频消融治疗对照组(a)37度培养的B16F10肿瘤抗原制备DC疫苗联合射频消融治疗;(b)负载PBS的DC疫苗联合射频消融治疗;(c)43度培养的EL4肿瘤抗原制备DC疫苗联合射频消融治疗。7.探讨热休克DC疫苗联合射频消融治疗抑制肿瘤复发的机制:(a)诱导特异性T细胞增生;(b)诱导特异性T细胞分泌IFNγ;(c)诱导特异性T细胞对肿瘤细胞杀伤。实验结果1.射频后肿瘤组织(原发性肝癌和皮下B16F10肿瘤)HSP70、gp96表达升高,HMGB1出现核转位。2. 43度加热后细胞HSP70、gp96表达升高,HMGB1出现核转位,且对DC成熟和活化能力增强。3. 43度B16F10加热制备肿瘤抗原,用其负载DC制备的疫苗能有效防止肿瘤射频术后复发。4.实验组引流淋巴结T细胞增殖能力高于对照组。同时T细胞分泌IFNγ水平高于对照组。5.实验组引流淋巴结T细胞对肿瘤细胞特异性杀伤能力高于对照组。6.实验组联合治疗后肿瘤局部淋巴细胞浸润增加,肿瘤细胞凋亡增多。实验组全脾淋巴细胞移联合植射频术治疗能有效抑制射频后肿瘤复发,该效应主要由特异性CD8+T细胞介导。结论体外肿瘤细胞热休克(43度热休克)制备的肿瘤抗原能很好模拟体内射频消融术后释放的热休克抗原,以此制备的DC疫苗联合射频治疗能有效降低射频术后肿瘤复发。RFA联合免疫疗法治疗HCC可能代表了一种诱发原位强力免疫应答的相对简单的治疗模式,也是一种新颖、有效、可行的肿瘤综合治疗策略,具有潜在的应用价值和临床意义。更多还原

【Abstract】 Part I Signal Regulatory ProteinαSets a Threshold for Dendritic Cell ActivationPurpose: Signal regulatory protein (SIRP)a, also known as SHPS-1 or SIRPα, is a transmembrane protein that binds to the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region. Due to its high expression in dendritic cells and macrophages, SIRPαwas often thought to have an important role in the immune system. In innate immune responses, SIRPαhad been proved to play a critically negative role in macrophage activation. However, whether or how SIRPαregulate the adaptive immune response is not very clear. Here we firstly purposed to delineate potential regulations of SIRPαin DCs, and then discussed its potential functions in adaptive immunity against tumor.Experimental Design: To examine SIRPαregulation of the adaptive immune responses, lentiviral-mediated, microRNA-based RNA interference of SIRPαin DC was designed. After infected by lentivirus, in vitro and in vivo growth and survival of DCs were evaluated firstly. Secondly, maturation and cytokine production of DCs regulated by SIRPαwere detected. Thirdly, T cell responses and anti-tumor immune effects were evaluated. Finally, the molecular mechanisms.of SIRPαin regulation of DC function were discussed.Results: SIRPαwas found to suppress the survival, phenotypic and functional maturation of immature dendritic cells (DCs) and to inhibit cytokine production by mature DCs. Moreover, SIRPαsilenced-DCs showed potent antitumor effects by activation of antigen-specific cytotoxic T lymphocytes.Conclusions: This study indicates that SIRPαis a critical regulator of DC function, and inhibiting SIRPαis necessary to evoke potent adaptive immunity and induce effective antitumor responses in an antigen-specific manner. Part II Abrogation of Local Cancer Recurrence after Radiofrequency Ablation by Dendritic Cell-based Hyperthermic Tumor Vaccine in MicePurpose: Radiofrequency thermal ablation (RFA) has become a viable treatment option for small solid focal malignancies. However, aggressive local and distant recurrence indicate a need to combine it with other treatment modalities. Here we investigated whether proactive priming with tumor heat-shocked tumor lysate-pulsed dendritic cells (HT-DC), which mimics RFA effects in vivo, can prevent local cancer recurrence after RFA treatment.Experimental Design: Poorly immunogenic melanoma tumors were established in syngeneic mice and sequentially treated with DC cancer vaccines and RFA. Local cancer recurrence and survival were monitored. In vitro and in vivo induction of antitumor responses of RFA as single modality and in combination with DC pre-vaccination were evaluated by immunohistochemistry, IFN-γrelease, and CTL assay.Results: The combination treatment with HT-DC and RFA showed potent anti-tumor effects compared with either treatment modality alone, with >90% of tumor recurrence abrogated in the combination group. However, pre-vaccination with un-heated tumor lysate-pulsed DC failed to prevent late tumor recurrence, suggesting a need for hyperthermia-induced DC activation and cryptic antigen presentation. The spleens of mice treated with HT-DC plus RFA contained tumor-specific cytotoxic and IFN-γ-secreting T ells whereas the spleens of control groups did not. Moreover, adoptive transfer of splenocytes from successfully treated tumor-free mice protected naive animals from a tumor recurrence after RFA, and this was mediated mainly by CD8+ T cells.Conclusions: The optimal priming for the DC vaccination before RFA is important for boosting antigen-specific T cell responses and this combined modalities may provide a potent therapeutic strategy for cancer treatment.更多还原