【作者】 杨月峰；

【导师】 吴祖泽； 王立生；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2009， 博士

【摘要】 前列腺癌(Prostate Cancer,PC)是西方国家常见的男性恶性肿瘤。近年来随着人口老龄化和饮食结构的改变,PC也逐渐成为影响我国老年男性生活质量和预期寿命的重大疾病。PC的常规治疗手段包括手术、激素、放化疗等,尽管这些疗法对于局灶型患者有效,但仍有部分病人复发,且具有明显毒副作用;而对于晚期、转移和激素非依赖型患者仍缺乏有效的治疗手段。因此,探索一种更加有效针对PC的治疗手段显得十分迫切。基因治疗能有效针对肿瘤发生、发展的各个关键环节,已逐渐成为PC研究的热点。腺病毒载体是目前PC治疗中应用最为广泛的载体,已有多个方案进入临床试验。溶瘤腺病毒,又称条件复制型腺病毒(conditionally-replicating adenoviruses, CRADs),能在肿瘤细胞中特异性复制并杀伤肿瘤细胞,同时介导治疗基因的高效表达,因此,溶瘤腺病毒与治疗基因的联合应用,较单纯的溶瘤治疗或单纯的基因治疗具有更好的疗效。细胞毒性T淋巴细胞(cytotoxicity T lymphocyte,CTL)是肿瘤细胞最终清除的效应细胞,而树突状细胞(Dendritic Cell,DC)的激活是诱导有效T淋巴细胞免疫所必需的。DC表面高表达CD40分子,而CD40与CD40配体(CD40 Ligand,CD40L)是特异性免疫系统的一对重要共刺激分子,在T淋巴细胞活化及效应性细胞因子产生过程中起着重要调节作用。有研究表明,将CD40L与肿瘤抗原融合表达,能通过CD40L的靶向作用将肿瘤抗原带到DC表面,进而有效激活机体抗肿瘤免疫反应。本课题将这种免疫基因治疗的最新成果与溶瘤治疗相结合,构建携带CD40L和前列腺特异性抗原(Prostate Specific antigen,PSA)融合蛋白基因(PSA-IZ-CD40L,PL)以及GM-CSF基因、且具有PC靶向复制能力的CRADs,进一步研究其体内外免疫激活及溶瘤功能。首先,根据溶瘤腺病毒制备系统要求,设计并构建了全长为712bp的PC特异性启动子PPTp,由前列腺特异性抗原增强子(prostate specific antigen enhancer,PSAe)、前列腺特异性膜抗原增强子(prostate specific membrane antigen enhancer,PSMAe)、和TARPp顺序连接而成。对PPTp的启动效率和特异性进行评价,结果表明,无论在质粒水平还是腺病毒水平,PPTp均能有效并特异地在前列腺癌细胞中启动报告基因的表达。其次,采用本室建立的能携带1-2个基因的CRADs制备体系,将融合蛋白基因PL和GM-CSF基因,以及由PPTp调控腺病毒复制必需基因的序列构建到腺病毒载体上,获得具有免疫和溶瘤双功能的腺病毒。(1)设计并构建了全长为1445bp的融合蛋白基因PL,并将其构建到穿梭质粒,得到pshuttle-cmv-PL;(2)利用现有溶瘤腺病毒制备辅助质粒TE-SV-IR-TP-55K,将端粒酶启动子TP替换为PPTp,得到TE-SV-IR-PPTp-55K;(3)MfeI酶切TE-SV-IR-PPTp-55K,得到含全部功能基因的片段PPT-E1A,将其正向克隆到pshuttle-cmv-PL上,得到穿梭质粒pshuttle-cmv-PL-PPT-E1A;(4)采用Adeasy系统,制备得到免疫溶瘤双功能腺病毒Ad-psh-PL-PPT;(5)Western-blot可检测到病毒感染的293细胞有50KD左右的目的基因PL的表达;ELISA可检测到病毒感染的293细胞培养上清中有GM-CSF的分泌;(6)氯化铯密度梯度离心法得到了纯度为1.2338,感染滴度为3.2×1010IU/ml的双功能腺病毒Ad-psh-PL-PPT。此外,还构建、制备、鉴定并纯化了对照病毒Ad-psh-cmv-PL。再次,在体外分别对双功能腺病毒的特异性复制和杀伤效应及免疫激活作用进行评价。(1)10IU/细胞双功能腺病毒Ad-psh-PL-PPT感染人前列腺癌细胞DU145后,96h可观察到细胞病变状态;将其收集后,反复冻融收集上清,再次感染DU145,仍可见病变状态出现。(2)双功能腺病毒以不同感染强度分别感染不同细胞系,MTT法和结晶紫法检测结果显示:Ad-psh-PL-PPT能特异并有效杀伤PC细胞。当感染复数大于10IU/细胞时,Ad-psh-PL-PPT对PC细胞系具有更强的杀伤作用(与Ad-GFP组相比,P<0.05);当感染复数达到250IU/细胞时,其对PC3、DU145、PC3M和LNCaP的杀伤效应分别达到60%、70%、80%和90%以上。双功能腺病毒Ad-psh-PL-PPT对非PC细胞没有明显杀伤作用。(3)双功能腺病毒靶向复制能力检测结果显示,25IU/细胞感染各细胞系24h、48h和72h后,随培养时间的延长,PC细胞系PC3M、DU145和PC3中产生有活性的病毒数逐渐升高,且明显高于对照细胞HepG2。(4)双功能腺病毒感染PC细胞系后,其培养上清能有效刺激人外周血单个核细胞的增殖能力,其中,感染PC3M和DU145细胞后,其促增殖作用明显高于Ad-GFP组(P <0.01)。(5)双功能腺病毒感染的PC细胞系与人外周血单个核细胞的共培养,能提高其对PC细胞系的杀伤作用,但这种杀伤作用呈现PSA依赖性,即对有PSA分泌的PC细胞系的杀伤效应明显高于其他细胞。最后,对双功能腺病毒的体内溶瘤及免疫激活作用进行探讨。成功建立了人前列腺癌细胞PC3的裸鼠移植瘤模型,1×109IU/50μl瘤内注射给药后,评价体内复制及溶瘤作用。结果显示,Ad-psh-PL-PPT治疗组的肿瘤中,病毒滴度明显高于Ad-GFP组;对肿瘤生长状态和生长曲线的观察,可见Ad-psh-PL-PPT给药组与Ad-GFP组相比,肿瘤的生长明显受到抑制。对于体内免疫功能的研究缺乏可靠的动物模型,本研究将CD40L基因替换为鼠源性CD40L基因,构建能同时表达融合蛋白PSA-IZ-mCD40L(PmL)和GM-CSF的重组质粒载体pUDK-PmL-IR-GM,在正常Babl/c小鼠体内对其免疫激活作用进行评价。(1)细胞水平鉴定其能表达融合蛋白PmL和GM-CSF;(2)肌肉注射免疫小鼠后,流式细胞结果表明,脾脏T淋巴细胞的CD4+T比例有升高的趋势;而CD4+T与CD8+T的比值明显升高(与PBS组相比,P <0.05)。(3)MTT检测结果显示:PSA有效刺激pUDK-PmL-IR-GM免疫小鼠T淋巴细胞的增殖能力(与阴性对照组比,P <0.05);同时,PSA对pUDK-PmL-IR-GM免疫小鼠T淋巴细胞的促增殖作用明显高于pUDK和pUDK-mCD40L-IR-GM免疫组(P <0.01)。(4)细胞杀伤结果显示:pUDK-PmL-IR-GM免疫能增强T淋巴细胞对LNCaP细胞的杀伤效应,在效靶比为5:1时,与pUDK免疫组相比具有统计学意义(P <0.05)。综上所述,本研究以本实验室建立的CRADs制备体系为平台,成功构建并制备具有PC靶向复制能力,携带PL基因和GM-CSF基因的CRADs。治疗前列腺癌的体外研究表明,其具有PC特异性的复制及溶瘤作用;具有一定的免疫激活作用。体内研究表明,具有一定的抗肿瘤效应;同时,其携带的目的基因能有效激活小鼠T淋巴细胞免疫功能。上述研究结果为进一步探讨其可药性提供了实验依据。更多还原

【Abstract】 Prostate cancer is most frequently diagnosed in male in western countries. Recently, It has been becoming a major disease which affects the living quality and anticipated life span of Chinese elder men because of some changes of their daily habits, such as increase consumption of animal fat, obesity and physical inactivity. Current standard therapeutic approaches including radical prostatectomy, irradiation, chemotherapy and hormonal therapy, shows effective in patients with organ-confined prostate cancer. However, a significant proportion of patients, who initially present with localized disease, relapse ultimately.The application of these therapeutic approaches are often limited by their significant side effects and effectless on metastasized and hormone-refractory disease. Thus, it is urgent to develop a novel therapeutic strategy to cure prostate cancer efficiently.Gene therapy which can target key points in any phase during prostate cancer genesis and development has become the hottest research in prostate cancer therapy. Adenovectors are the most popular vectors in gene therapy of prostate cancer. Some adenoviral therapeutic regimens have been approved in clinical trials. Oncolytic adenovirus, also termed conditionally-replicating adenoviruses (CRADs), can specifically replicate in tumor cells leading to their death finally. Another advantage of CRADs is its high efficiency for therapeutic gene delivery and expression. Therefore, the therapeutic effect of CRADs armed with effective genes is more effective than that of single CRADs or replication defective adenoviruses carrying therapeutic genes.The ultimate effector cells that mediate the rejection of solid tumors in preclinical animal models are the cytolytic T lymphocytes. And the activation of professional antigen-presenting cells (APC) is essential to induce effective T cell immunity. CD40 is highly expressed on dendritic cells(DC).CD40 and CD40 Ligand(CD40L) is a pair of important co-stimulatory molecules of specific immune system.And it plays an important roles in regulation of T lymphocyte activation and effector cytokines production. Latest researches show that fusion protein of CD40L and tumor antigen can activate immune response through binding of CD40L and CD40 on DC.Oncolytic therapy and immunotherapy are combined in this study. We developed a prostate cancer specific CRADs carrying PSA-IZ-CD40L gene(fusion protein gene of prostate cancer antigen and CD40L,PL)and GM-CSF gene, named Ad-psh-PL-PPT. Followed by evaluating its oncolytic and immuno activation effects both in vitro and in vivo.Firstly, according to our oncolytic adenovirus preparation system, we designed and constructed a 712bp length prostate specific promoter PPTp,which composed of prostate specific antigen enhancer(PSAe), prostate specific membrane antigen enhancer(PSMAe) and TARPp in proper order. Moreover we evaluated PPTp on plasmid and adenovirus level. Results show that the report genes, controlled by PPTp, could be expressed effectively and specifically in various prostate cancer cells.Secondly, recombinant adenovirus Ad-psh-PL-PPT was prepared using oncolytic adenovirus preparation system, which established by our laboratory. (1) pshuttle-cmv-PL was obtained by insert PL gene into pshuttle-cmv.(2)Recombinant plasmid TE-SV-PPT-IR-55K,carrying genes of PPTp,E1A,E1Bp,E1B55K and GM-CSF(PPT-E1A),was constructed by replace TP gene of TE-SV-IR-TP-55K with PPTp. (3)PPT-E1A was obtained by MfeI cutting, and then fragment was inserted into pshuttle-cmv in forward direction. Recombinant plasmid pshuttle-cmv-PL-PPT-E1A was obtained by screening. (4) Ad-psh-PL-PPT was prepared by Adeasy system..(5) The expression of fusion protein PL and GM-CSF was confirmed by Western-blot and ELISA post infected byAd-psh-PL-PPT. (4) Ad-psh-PL-PPT was purified by density gradient centrifugation of cesium chloride. The purity was 1.2338 ,and the infectious units(IU) was 3.2×1010 IU per milliliter(IU/ml).In order to evaluate the function of Ad-psh-cmv-PL-PPT objectively, we prepared control adenovirus, Ad-psh-cmv-PL.Thirdly, the specific replicate ability, oncolytic efficacy and immune activation of this recombinant adenovirus were detected in vitro. (1)Cytopathic effect(CPE) was observed 96h post infected by 10 IU/cell Ad-psh-PL-PPT in DU145.And CPE was detected again in DU145 after infected by supernatant of freeze thawing. (2)The cell viability of various cells was measured by MTT assay and crystal violet staining 6d post infect. Results showed that Ad-psh-PL-PPT could kill the PC cells effectively and specifically. When infect with more than 10IU/cell Ad-psh-PL-PPT could kill PC cells more effectively than Ad-GFP. And at the IU/cell of 250, 60% of PC3,70% of DU145,80% of PC3M,and 90% of LNCaP were killed, while it could not kill other cells, such as 7721,HepG2 and ECV304. (3) 24h,48h and 72h post infect by 25IU/cell Ad-psh-PL-PPT, We detected the replicate ability of Ad-psh-PL-PPT.. The results showed that Ad-psh-PL-PPT could replicate selectively in prostate cancer cell lines, such as PC3,PC3M and DU145. And the titers increased with the time extending. (4)We collected the cell supernatant of PC cells infected by Ad-psh-PL-PPT. Mononuclear cells isolated from peripheral blood(PB)was stimulated by supernatant, then the proliferation ability of mononuclear cells was identified. The outcome showed that supernatants from PC3M and DU145 could stimulate the proliferation of mononuclear cells more effectively (P<0.01,comparing with the supernatants from Ad-GFP infection) (5) Mononuclear cells co-cultured with PC cells infected by Ad-psh-PL-PPT could kill PC cells stronger.Finally, we evaluated the oncolytic effects and immune response in vivo. In order to evaluate the anti-tumor effect, we established the transplantation tumor model of prostate cancer by injected PC3 cells subcutaneouly in Babl/c nude mice. After tumor formed, 1×109 IU/50μl Ad-psh-PL-PPT was administered intratumorally. The result shows that adenovirus titers in tumors of Ad-psh-PL-PPT therapy was much higher than that of Ad-GFP therapy. We observed the basic states of mice and growth curve of tumors. The results showed that Ad-psh-PL-PPT could delay the growth of tumor.In order to evaluate the immune activation ability, we replace the CD40L gene of PL with mouse CD40L(mCD40L) generating a recombinant plasmid pUDK-PmL-IR-GM, carrying PmL (fusion protein of PSA and mCD40L) gene and GM-CSF gene.(1)We confirm the expression of PmL and GM-CSF by Western-blot and ELISA in 293 cells transfected by pUDK-PmL-IR-GM. (2)pUDK-PmL-IR-GM was administered into muscle of normal Babl/c mice every 10 days for four times. We isolated T lymphocyte from spleen of mice 10 days after the latest administration, then measured the subset of T lymphocyte. The results showed that CD4+T was increased after immuned by pUDK-PmL-IR-GM, and the ratio of CD4+T and CD8+T was elevated obviously comparing control group(P<0.05).(3)MTT results of T lymphocytes showed that PSA could stimulate the proliferation of T lymphocytes from PUDK-PmL-IR-GM group(P <0.05,comparing negative control).And the results also showed that PSA could stimulate the proliferation of T lymphocytes more effectively in PUDK-PmL-IR-GM group than in other groups(P <0.01).The results showed that pUDK-PmL-IR-GM could provoke the immune activation of T lymphocytes to kill LNCaP(P <0.05.in E/T ration of 5:1,comparing with pUDK group).In conclusion, we developed a CARDs armed with PL fusion gene and GM-CSF gene and evaluated its therapeutic effects on prostate cancer. In vitro, it could specifically replicate in prostate cancer cells leading to cell death.; it also could stimulate the proliferation and enhance the toxicol effect of mononuclearcells in human peripheral blood. In vivo, it could encourage the immune activation of T lymphocytes in normal mice and induce effective antitumor response in nude mice PC xenograft tumor model.更多还原