【作者】 颜汝平；

【导师】 王剑松；

【作者基本信息】 昆明医学院 ， 泌尿外科， 2011， 博士

【摘要】 1.探讨肿瘤相关抗原(Tumor associated antigen,TAA)在膀胱癌组织、癌旁组织和多器官正常组织中的表达情况。2.探讨建立人免疫重建荷人膀胱癌的复合动物模型的方法,并对该动物模型进行鉴定。3.探讨负载膀胱癌抗原成分DC疫苗的制备和体外对同源T淋巴细胞的活化功能,及其诱导T淋巴细胞的特异性杀伤膀胱癌细胞的作用。4.探讨负载膀胱癌抗原的DC疫苗体内诱导的对小鼠膀胱原位移植瘤特异性抗膀胱癌效应。1.检测人膀胱移行细胞癌(bladder transitional cell carcinoma, BTCC)组织、癌旁组织和多器官正常组织中TAA的表达：使用免疫组化Elivision法检测组织芯片中BTCC组织、癌旁组织和多器官正常组织中与抗人膀胱癌单克隆抗体BDI-1结合的TAA的表达,分析比较膀胱移行细胞癌不同病理分级、癌及癌旁组织中TAA的阳性表达率、表达程度。2.建立人免疫重建荷人膀胱癌的复合动物模型和鉴定：密度梯度离心法分离人外周血单个核细胞(human peripheral blood mononuclear cell,hu-PBMC),腹腔注射hu-PBMC建立人免疫重建Balb/c裸小鼠动物模型,流式细胞仪检测小鼠外周血中人CD3+T和CD19+B淋巴细胞的表型、ELISA检测小鼠外周血人IgG含量和免疫组化法检测小鼠肝脾组织中人CD3+T和CD19+B淋巴细胞的浸润,并与未进行免疫重建的小鼠比较。建立人免疫重建荷人膀胱癌皮下移植瘤Balb/c裸小鼠复合动物模型,绘制肿瘤生长曲线,计算小鼠生存率,和单纯荷瘤组小鼠进行比较。3.负载EJ细胞裂解物抗原DC疫苗的构建及体外诱导的对膀胱癌细胞的特异性杀伤作用：冻融法制备EJ细胞裂解物抗原成分,BCA蛋白定量法测定裂解物蛋白含量；密度梯度离心法分离hu-PBMC,体外培养液中加入rhGM-CSF,rhIL-4,TNF-α诱导分化出DC,并对其进行表型鉴定；EJ细胞裂解物抗原致敏DC制备负载膀胱癌肿瘤抗原的DC疫苗；免疫磁珠分离法从人免疫重建Balb/c裸小鼠脾脏组织中分离CD3+T淋巴细胞,检测DC疫苗刺激自身T淋巴细胞增殖的能力和诱导的特异性细胞毒性T淋巴细胞(CTL)对EJ细胞的杀伤作用。4.DC疫苗体内诱导的特异性抗膀胱癌效应和活体成像监测：携带绿色荧光蛋白(green fluorescent protein,GFP)基因的慢病毒感染EJ细胞,将GFP基因转染至EJ细胞,建立稳定、高效表达GFP的细胞株；人免疫重建小鼠膀胱穿刺灌注表达GFP的EJ细胞,建立人化免疫荷人膀胱癌原位移植瘤可视复合动物模型；腹腔注射DC疫苗后活体成像动态观察肿瘤的生长情况,计算小鼠存活率；ELISA检测小鼠外周血中人IFN-γ含量,FCM检测脾脏和肿瘤组织中T淋巴细胞、成熟DC的浸润情况。1.人BTCC组织、癌旁组织和多器官正常组织中TAA的表达：与BDI-1特异结合的TAA在不同病理分级BTCC组织中的阳性表达程度无统计学差异(P>0.05)；BTCC组织中TAA阳性表达明显高于癌旁组织,差异有统计学意义(P<0.05)；正常器官组织如：食道、宫颈、胃、结肠、小肠、前列腺、胰腺、肺脏、胸腺、乳腺中检测到TAA的表达。2.人免疫重建荷人膀胱癌的复合动物模型的建立和鉴定：建立的人免疫重建Balb/c裸小鼠动物模型外周血和脾脏组织中均检测到人CD3+T、CD19+B淋巴细胞,人免疫重建组小鼠外周血中检测到的人IgG明显高于对照组(P<0.05),免疫重建后第12周仍能维持较高水平；EJ细胞接种到人免疫重建小鼠皮下的成瘤率为]00%；人免疫重建荷瘤小鼠的肿瘤生长曲线、小鼠生存率和单纯荷瘤组一致(P>0.05)。3.负载EJ细胞裂解物抗原DC疫苗的构建及体外诱导的对膀胱癌细胞的特异性杀伤作用：冻融法获取的EJ细胞裂解物抗原蛋白浓度约为1ng/m1(103个EJ细胞)；hu-PBMC在细胞因子rhGM-CSF、rhIL-4存在的条件下可诱导分化成DC, DC在TNF-α的刺激下分化为成熟DC,表型分子CD83、CD80的增高有统计学差异(P<0.05)；负载EJ抗原的DC疫苗体外可使同源T淋巴细胞活化,刺激指数增加,诱导的CTL对EJ细胞的杀伤率为62.58±6.13%,和对照组比较有统计学差异(P<0.05)4.DC疫苗诱导的体内特异性抗膀胱癌效应和活体成像监测：成功建立了稳定表达GFP的EJ细胞株(EJ-GFP),和EJ细胞相比,细胞生长曲线、FCM检测、皮下移植瘤生长曲线、荷瘤小鼠的生存时间无差别；接种EJ-GFP建立的人膀胱癌皮下移植瘤和原位移植瘤均在活体成像系统下检测到荧光信号,DC疫苗腹腔注射后活体成像观察到实验组小鼠膀胱肿瘤生长缓慢,小鼠生存时间延长,和对照组比较有统计学意义(P<0.05)；实验组小鼠脾脏和肿瘤组织中均检测到CD3+、CD4+、CD8+T淋巴细胞和成熟DC浸润,外周血IFN-γ含量高于对照组(P<0.05)1.人BTCC组织、癌旁组织和多器官正常组织中TAA的表达检测：1)与BDI-1结合的TAA主要在膀胱癌细胞的胞膜上表达,膀胱癌组织中的阳性表达程度要高于癌旁组织。2)与BDI-1结合的TAA在膀胱尿路上皮癌中的阳性表达程度和肿瘤的病理分级无关,在某些正常器官组织中有阳性表达。3)组织芯片检测TAA的表达高效、快捷,是一种检测TAA的有用工具。2.人免疫重建荷人膀胱癌的复合动物模型的建立和鉴定：1)经腹腔注射hu-PBMC能够建立人免疫重建Balb/c裸小鼠模型,移植的人T、B淋巴细胞在小鼠体内能够正常迁移和较长时间存活,并且能保持一定的生物学特性(如分泌人IgG等),为人淋巴细胞在小鼠体内发挥有效的免疫应答提供了保证；2)建立的Balb/c裸小鼠人免疫重建及膀胱癌皮下移植瘤复合模型,有助于进行人的膀胱癌免疫治疗研究；3)本实验中,人CD3+T、CD19+B淋巴细胞在肝脏组织中极少见到,提示人淋巴细胞在Balb/c小鼠体内的迁移并不是简单的随血流或淋巴循环随机分布,而是有选择性地定居在小鼠次级淋巴器官。3.负载EJ细胞裂解物抗原DC疫苗的构建及体外诱导的对膀胱癌细胞的特异性杀伤作用：1)从健康人外周血中分离出的PBMC,体外经rhGM-CSF、rhIL-4和TNF-α联合诱导分化,可成功获取成熟DC；2)冻融法获得的EJ细胞裂解物含有人膀胱癌的抗原成分,DC负载裂解物中的抗原成分后可成功制备肿瘤疫苗；3)负载膀胱癌冻融抗原的DC,体外可使同源的人T淋巴细胞活化增殖,诱导活化的CTL对EJ细胞有杀伤作用。4.DC疫苗体内诱导的特异性抗膀胱癌效应和活体成像监测：1)以携带GFP基因的慢病毒为载体,能成功建立在体外稳定、长期、高效表达GFP的EJ-GFP细胞株。该株细胞接种Balb/c裸小鼠,能成功建立人免疫重建荷人膀胱癌的皮下和原位移植瘤可视动物模型。2)腹腔注射负载EJ细胞裂解物抗原的DC疫苗,能诱导免疫细胞的抗膀胱癌效应,抑制人膀胱癌原位移植瘤生长,延长动物的生存时间。可能与肿瘤组织中CD4+、CD8+T淋巴细胞、成熟DC的浸润和外周血IFN-y含量增高有关。3)活体荧光成像系统可以无创、实时、动态地观察裸小鼠膀胱原位移植瘤的生长情况,客观评价DC疫苗在动物体内诱导的特异性抗肿瘤作用。更多还原

【Abstract】 1. To investigate the expression of tumor associated antigen(TAA) in tissues of bladder cancer, adjacent tissues and normal tissues of multiple organ.2. To investigate a method of establishing human bladder cancer-bearing composite animal model with reconstituted human immune system, and to evaluate the model.3. To investigate the way to prepare DC vaccine loaded with bladder cancer antigen and the activation of homologous T lymphocytes in vitro, further to observe the specific killing effect of bladder cancer cells induced by T lymphocytes.4. To investigate the specific antitumor effects against orthotopic transplantation tumor of mice model with human bladder cancer in vivo, induced by DC vaccine loaded with bladder cancer antigen. Methods1. Expression detection of TAA in human bladder transitional cell carcinoma (BTCC) tissues, adjacent tissues and normal tissues of multiple organs: Expression of TAA combined with antihuman bladder carcinoma monoclonal antibody BDI-1 in BTCC, adjacent tissues and normal tissues of multiple organs were identified by tissue microarray technology using immunohistochemical Elivision method. The positive expression rate and positive intensity of TAA were analyzed and compared among three different pathological grading of BTCC, between the bladder cancer tissue and adjacent tissues.2. Establishment and identification of human bladder cancer-bearing composite animal model with reconstituted human immune system:Human peripheral blood mononuclear cells(hu-PBMCs) were isolated by density gradient centrifugation. Balb/c-nu mice model with reconstituted human immune system was established by intraperitoneal injection of hu-PBMC. Phenotype of human CD3+T lymphocytes and CD19+B lymphocytes in murine peripheral blood were detected by Flow Cytometry. The level of human IgG in Balb/c-nu mice blood-serum was measured by Enzyme-linked Immunosorbent Assay(ELISA). Immunohistochemistry was used to detect infiltration of human CD3+T lymphocytes and CD19+B lymphocytes in the liver and spleen of mice. The experimental results of immune reconstitution group were compared with non-humanized group. Balb/c-nu mice composit model with reconstituted human immune system and bearing subcutaneous transplantation tumor of human bladder cancer was established. Tumor growth curve was drawn and survival rates of mice were calculated. The experimental group was compared with tumor group without reconstituted human immune system.3. Construction of DC vaccine loaded with EJ cell lysate antigen and the effect of anti-bladder cancer induced by DC vaccine in vitro:EJ cell lysate antigen components were obtained by freeze-thaw method. The cell lysate protein content was determined by BCA protein quantitative method. Hu-PBMCs were isolated by density gradient centrifugation and were cultured in vitro. The dendritic cells(DCs) were induced by rhGM-CSF, rhIL-4, TNF-αfrom hu-PBMCs cultured in vitro. The phenotypes of the cultured DCs were identified by FCM. EJ cell lysate antigen pulsed DC. DC vaccine loaded with EJ cell lysate antigen was generated. CD3÷T lymphocytes in spleen tissue in Balb/c-nu mouse with reconstituted human immune system were isolated by T-immunomegnetic beads. The proliferation ability of autologous T cells and CTL’s cytotoxicity towards EJ cells induced by DC vaccine was detected.4. The specific effects of anti-bladder cancer induced by DC vaccine loaded with tumor antigen in vivo and monitored by fluorescence imaging system:EJ cells were infected by lentivirus carrying green fluorescent protein(GFP) gene. GFP gene was transfected into EJ cells. The stable highly expressing GFP cell lines were established. EJ cells expressing GFP were perfused into bladder of Balb/c-nu mice with reconstituted human immune system by puncturing method. Human bladder cancer-bearing composite and visual animal model with reconstituted human immune system was established. After DC vaccines were injected intraperitoneally in mice, the growth of tumor was dynamically observed by fluorescence imaging system in vivo. The survival rates of mice were calculated in the experimental group and control group. The level of IFN-γin peripheral blood of Balb/c-nu mice was measured by ELISA. The infiltration of human T lymphocytes and mature DC in the spleen and tumor tissues of mice was detected by FCM.Results1. Expression of TAA in human BTCC tissues, adjacent tissues and normal tissues of multiple organs:The positive staining was in the membrane of cells. In the G1、G2、G3 pathological grades of BTCC, the positive expression intensity of TAA combined with BDI-1 had no statistical difference(P>0.05). Compared with adjacent tissues, the positive expression intensity of TAA in BTCC was significant higher. The difference was statistically significant(P<0.05). In tissues of normal organs, the positive expression of TAA was observed in esophagus, cervix, gastric, colon, small intestine, prostate, pancreas, lung, thymus and breast tissues.2. Establishment and identification of human bladder cancer-bearing composite animal model with reconstituted human immune system:Human CD3+T lymphocytes and CD19+B lymphocytes cells were detected in peripheral blood and spleen of Balb/c-nu mice with reconstituted human immune system. The level of human IgG in mice with reconstituted human immune system was significant higher comparing with control group(P<0.05). The level of human IgG was also higher at the twelveth week after human immune system was reconstituted in Balb/c-nu mice. EJ cells were implanted subcutaneously in the mice with reconstituted human immune system and tumor formation rate was 100%. Tumor growth curve and survival rates in the experimental group was the same as control group(P>0.05).3. Construction of DC vaccine loaded with EJ cell lysate antigen and the effect of anti-bladder cancer induced by DC vaccine in vitro:The EJ cell lysate protein content was about lng/ml(103 EJ cells). The dendritic cells(DCs) were induced by rhGM-CSF, rhIL-4 from hu-PBMC. Tumor necrosis factor-a(TNF-α) promoted immature DCs transformed into mature DCs. The expressions of CD83 and CD80 on mature dendritic cells were statistically significant(P<0.05). T lymphocytes were activated by DC vaccine loaded with EJ cell lysate antigen. The stimulate index was higher, and the killing rate of CTL against EJ cells was 62.58±6.13%. Compared with control group, these difference were statistically significant(P<0.05).4. The specific effects of anti-bladder cancer induced by DC vaccine loaded with tumor antigen in vivo and monitored by fluorescence imaging system:The stable highly expressing GFP cell lines were established using lentivirus vectors. Compared with EJ cells, no obvious difference was found in cell growth curve, FCM detection, growth curve of subcutaneous transplantation tumor and survival time of mice(P>0.05). Fluorescence signals were detected in EJ-GFP cell subcutaneous transplantation or orthotopic transplantation tumor by fluorescence imaging system in vivo. Orthotopic transplantation tumor grew slowly in mice after DC vaccine intraperitoneal injection. Survival time of mice in experimental group was prolong. Compared with control group, the difference was statistically significant(P<0.05). CD3+、CD4+、CD8+T lymphocytes and mature DC were detected in mice spleen and tumor tissues. The level of IFN-y was higher in mice peripheral blood. Compared with control group, the difference was statistically significant(P<0.05).Conclusion1. Expression of TAA in human BTCC tissues, adjacent tissues and normal tissues of multiple organs:1) The positive staining of TAA combined with BDI-1 is mainly in the membrane of cells. The positive expression intensity of TAA combined with BDI-1 in BTCC tissues is higher than that in adjacent tissue.2) In the three different pathological grades of BTCC, the positive expression intensity of TAA combined with BDI-1 has no statistical difference. In normal tissues of multiple organs, the positive expression of TAA is observed.3) It is an efficient and rapid method to detect TAA by tissue microarray technology. Tissue microarray technology is an useful tool for the detection of TAA.2. Establishment and identification of human bladder cancer-bearing composite animal model with reconstituted human immune system:1) Balb/c-nu mice model with reconstituted human immune system can be established by intraperitoneal injection of hu-PBMC. The human T and B lymphocytes can migrate normally and live for a long time in mice body, and can maintain certain biological activities (such as secreting human IgG, etc), which provides guarantee for human lymphocytes to play an effective immune response in mice body. 2) Balb/c-nu mice composite model with human bladder cancer-bearing and reconstituted human immune system is useful for the immunotherapy research of human bladder urothelial carcinoma.3) In this research, human CD3+T lymphocytes and CD19+B lymphocytes are rare in mice liver. Distribution and migration of human lymphocytes is not random. Human lymphocytes settle in subprime lymphopid organs of mice selectively.3. Construction of DC vaccine loaded with EJ cell lysate antigen and the effect of anti-bladder cancer induced by DC vaccine in vitro:1) The mature DCs can be induced by rhGM-CSF, rhIL-4 and TNF-αfrom hu-PBMCs.2) EJ cell lysate contains human bladder cancer antigen composition. DC vaccine can be constructed by loading EJ cell lysate antigen composition.3) The proliferation ability of autologous T cells and CTL’s cytotoxicity towards EJ cells can be induced by DC loading with EJ cell lysate.4. The specific effects of anti-bladder cancer induced by DC vaccine loaded with tumor antigen in vivo and monitored by fluorescence imaging system:1) The EJ cell lines expressing GFP(EJ-GFP) can be established in vitro by lentivirus vectors carrying GFP gene. The expression of GFP is stable, highly and long time. The Balb/c-nu mice with reconstituted human immune system are inoculated with the cell lines. The visual model with hunan bladder urothelial cancer subcutaneous transplantation or orthotopic transplantation tumor and reconstituted human immune system can be established successfully.2) DC vaccine loaded EJ cells lysate antigen composition can induce the specific effects of anti-bladder cancer by intraperitoneal injection in vivo. DC vaccine can inhibit tumor growth and obviously prolonged the exist time of animals bearing tumor. The immune response may be related with the infiltration of human CD4+, CD8+T lymphocytes and mature DC in the tumor tissues, or IFN-γcontent increasing in peripheral blood of Balb/c-nu mice.3) The orthotopic transplantation tumor growth of mice can be monitored by fluorescence imaging system noninvasively and real time dynamically. The specific effects of anti-bladder cancer induced by DC vaccine can be evaluated objectively.更多还原