【作者】 鲍传庆；

【导师】 钱海鑫；

【作者基本信息】 苏州大学 ， 普外科， 2010， 博士

【摘要】 大肠癌包括结肠癌和直肠癌,是最常见的消化道恶性肿瘤之一。在世界范围内,大肠癌发病率居恶性肿瘤第4位,已占全身恶性肿瘤的12%-15%,每年有102万新发病例和53万死亡病例。自上世纪70年代以来,我国大肠癌发病率和死亡率有不断上升趋势(以结肠癌上升为主),并且青年期(<30岁)大肠癌发病率高是我国大肠癌的一个显著临床特点。因大肠癌早期症状不明显,多数患者就诊时已发生区域或远处转移,单纯手术治疗不能显著改善患者预后,因此综合治疗成为目前治疗大肠癌的重要方法。生物治疗作为大肠癌综合治疗的组成部分,即可以独立运用,又可与手术及放、化疗结合,具有疗效高、特异性强、副作用小等优点,已成为研究的热点之一。树突状细胞(DC)是目前已知功能最强大的专职抗原递呈细胞,是机体T细胞特异性免疫应答的直接启动和调控者。它最大的特点是能够显著刺激初始型T细胞的增殖,在免疫反应的诱导中具有独特的地位。DC在外周组织中摄取抗原,加工分解成抗原肽与MHC-I/II类分子结合,然后移行到周围淋巴器官通过MHC分子递呈抗原给T淋巴细胞,激发特异性抗肿瘤免疫反应。与其他抗原递呈细胞相比,DC表达MHC-I/II类分子、共刺激分子和粘附分子的水平更高,因此在刺激T细胞增殖,抗肿瘤免疫中起重要作用。早在1995年国外学者就开展了DC治疗肿瘤的I期临床试验,以DC为载体开发的DC肿瘤疫苗在前列腺癌、恶性黑色素瘤、结肠癌、乳腺癌、卵巢癌、多发性骨髓瘤、脑胶质瘤、肾细胞癌等恶性肿瘤患者治疗中取得了显著的疗效。DC肿瘤疫苗用于结直肠癌的研究也表明其安全有效,不仅可降低结直肠癌患者癌胚抗原(CEA)水平,甚至可使肺部转移病灶消失。以DC为载体开发的DC肿瘤疫苗已成为现今最先进、最有希望的肿瘤免疫治疗方法之一。由于大肠癌是一种免疫原性较弱的肿瘤,且肿瘤细胞缺乏共刺激分子或某些粘附分子,并可通过抗原调变、下调MHC分子表达等机制介导肿瘤免疫逃逸,从而影响免疫治疗效果。另有研究表明,肿瘤患者体内DC数量减少及功能缺陷,使其无法有效递呈肿瘤抗原,亦是肿瘤免疫逃逸的主要原因之一。因此,提高肿瘤患者体内DC的数量并维持其强大的抗原识别、呈递功能是实施抗肿瘤免疫治疗的关键措施之一。正因为此,应用足够数量和功能正常的DC细胞进行细胞免疫治疗或基于DC细胞的抗肿瘤疫苗研究工作越来越受到重视。对肿瘤患者提供外源性DC细胞进行细胞免疫治疗的方法现已经历了以下阶段的发展:早期多采用已知肿瘤相关抗原肽或肿瘤细胞mRNA修饰致敏DC,但疗效不佳。随着研究的深入,改用肿瘤全抗原致敏DC的方法,即,将肿瘤细胞与DC融合或用肿瘤细胞裂解物负载DC制成杂交疫苗,用于胃癌、肝细胞癌及大肠癌治疗的临床研究取得较好的抗肿瘤效果。而最近研究发现,DC表面表达多种热休克蛋白(HSP)受体(如CD91,CD40,TLR2/4或LOX1),HSP可作用于DC或单核细胞,刺激其产生细胞因子(IL-12,TNFa,IL-6等),增强机体非特异性免疫反应。2004年免疫学权威杂志《Immunity》报道,与多肽负载的DC相比,HSP70-抗原肽复合物负载的DC在体外激发CD8+ T的能力远远高于前者(1000倍)。提示如果将HSP与DC进行有机结合,发挥二者各自的优势,将会产生更强的免疫激发作用。有鉴于此,本研究对大肠癌患者热休克凋亡自体的大肠癌细胞抗原制备、自身的树突状细胞体外诱导、以及抗原负载方法等进行了初步探讨,并比较了分别负载热休克诱导凋亡大肠癌细胞株和肿瘤细胞裂解物两种不同抗原的DC对大肠癌细胞的杀伤效果,并探讨了热休克凋亡的人自体大肠癌细胞致敏自体的树突状细胞疫苗对大肠癌患者术后免疫功能的影响,为制备大肠癌细胞肿瘤疫苗研究提供技术基础。研究共分为三个部分:第一部分自体大肠癌细胞负载树突状细胞的方法研究目的:建立自体热休克凋亡大肠癌(包括结肠、直肠癌)细胞抗原制备、树突状细胞体外诱导、以及抗原负载方法,为制备树突状细胞肿瘤疫苗提供技术基础。方法:采用酶消化法从手术切除的14例大肠癌新鲜组织获得单细胞悬液,热休克处理后用桦脂酸诱导其凋亡制备成细胞抗原;采集外周静脉血,分离单个核细胞,经GM-CSF与IL-4体外诱导成未成熟树突状细胞,负载细胞抗原后制备成DC肿瘤疫苗;苔盼蓝染色进行细胞总数及活细胞计数,计算细胞活率;用FITC-Annexin-Ⅴ和PI标记细胞,流式细胞仪检测,计算细胞凋亡率。软琼脂克隆法检测细胞体外成瘤能力;按《中华人民共和国药典》2005版第三部规定的方法进行内毒素检测。结果: (1)肿瘤细胞抗原得率:(11.81±0.65)×106/g组织;平均凋亡率:(93.16±2.31)%;(2)imDC平均得率为(9.75±0.82)×106(/121.64)×106个PBMC,细胞活率>95%;imDC表型分析:CD11c+CD14-、CD11c+HLA-DR+、CD11c+CD80+、CD11c+CD83+、CD11c+CD86+表达率分别为(87.58±2.56)%、(87.97±0.98)%、(2.21±0.69)%、(4.85±1.22)%、(5.02±0.95)%;(3)DC平均得率为(6.76±0.98)×106/(9.75±0.82)×106个imDC,细胞活率>95%,DC表型:CD11c+CD14-、CD11c+HLA-DR+、CD11c+CD80+、CD11c+CD83+、CD11c+CD86+表达率分别为( 93.45±1.25 ) %、(89.79±1.35)%、(87.85±1.62)%、(70.74±6.45)%、(95.54±2.18)%。内毒素检测均合格(≤5 IU/mL)。结论:本方法稳定、安全、可靠,可制备出成熟DC。第二部分两种不同方式负载大肠癌细胞株的树突状细胞体外刺激淋巴细胞抗肿瘤活性的比较目的:比较树突状细胞以两种不同方式负载大肠癌细胞株体外刺激淋巴细胞的抗瘤活性。方法:分离30例大肠癌患者外周血单核细胞体外诱导DC,分别负载热休克诱导凋亡大肠癌细胞株和肿瘤细胞裂解物,以此刺激淋巴细胞作为效应细胞,大肠癌细胞株为靶细胞,MTT法测定效应细胞对靶细胞的杀伤作用。结果:负载热休克大肠癌细胞的DC与负载肿瘤细胞裂解物的DC都显示对靶细胞的杀伤活性,但前者的杀伤率显著高于后者(P<0.05)。结论:负载热休克肿瘤细胞的DC是一个更为有效的负载方式。第三部分自体DC治疗对大肠癌患者术后免疫功能的影响目的:探讨热休克凋亡的人自体大肠癌细胞致敏的树突状细胞疫苗对大肠癌术后免疫功能的影响。方法:从大肠癌患者外周血单个核细胞中诱导DC,并用重组人粒细胞-巨噬细胞集落刺激因子和白介素-4刺激活化,经热休克凋亡自体大肠癌细胞致敏制备DC疫苗。将28例大肠癌术后患者随机分为DC疫苗治疗组14例,化疗对照组14例。对两组病例治疗前后免疫功能、临床疗效进行观察比较。结果:DC疫苗组治疗后外周血CD3+、CD4+/CD8+及NK细胞比率较治疗前明显升高(P<0.05),且明显高于对照组化疗后的CD3+、CD4+/CD8+及NK细胞比率(P<0.05);DC疫苗治疗后患者血清IL-2、IL-12、IFN-γ水平较治疗前明显升高(P<0.05),且明显高于对照组(P<0.05),生存时间明显延长。结论:大肠癌术后行热休克凋亡自体大肠癌细胞致敏的DC疫苗治疗,可提高患者的细胞免疫水平。更多还原

【Abstract】 Colorectal cancer,including colon and rectal cancer, is one of the most common malignant tumors in the digestive tract. In the worldwide scale, incidence rate of colorectal cancer ranks No.4 that has occupied 12%-15% of the whole body malignant tumor and there are 1,020,000 new cases and 530,000 death cases each year. Since the 70s of last century, China’s morbidity and mortality of colorectal cancer have showed a rising trend (by colon cancer rise primarily), and the high incidence of colorectal cancer in adolescent (<30 years old) is a significant clinical feature in our country. The incipient symptoms of colon cancer are not obvious,so the regional or distant metastasis has occurred when most of patients come for treatment. Surgical treatment can not significantly improve the prognosis of patients alone, so currently the combined therapy is an important method. As an integral part of comprehensive treatment, biological treatment of colorectal cancer can be used independently, or combined with surgery, radiotherapy and chemotherapy,which has the merits of high efficacy, strong specificity, few side effects,etc. Now it has become one of the research hot spots.Dendritic cells (DC) is known as the most powerful professional antigen-presenting cells,and DC is the direct initiator and regulator of body’s T cell-specific immune responses. The biggest feature of DC is the ability to stimulate the proliferation of naive T cells significantly which has a unique position in the immune response induction. DC uptake antigen in peripheral tissue, process and decompose it to antigenic peptides, which combined with MHC-I/II molecules, migrate to peripheral lymphoid organs, present antigen to T lymphocytes through the MHC molecules, then stimulate specific anti-tumor immune response. Compared to other antigen-presenting cells, DC expresses higher levels of MHC-I/II molecules, costimulatory molecules and adhesion molecules, therefore it plays an important role in stimulating T cell proliferations and anti-tumor immunity. As early as 1995, foreign scholars began to carry out the phase I clinical trials of cancer therapeutics with DC. DC vaccine has achieved remarkable effects in prostate cancer, malignant melanoma, colon cancer, breast cancer, ovarian cancer, multiple myeloma, brain glioma, renal cell carcinoma and other malignant tumors . Study of DC tumor vaccine for colorectal cancer therapy also shows that the safty and clinical effect. It can reduce carcinoembryonic antigen (CEA) levels of colorectal cancer patients, and even makes lung metastases disappeared. DC cancer vaccines have been one of the most advanced and promising cancer immunotherapy methods nowaday,.The colorectal cancer is one kind of weak immunogenicity tumor. The tumor cells lack costimulatory molecules or some adhesion molecules, and can arise tumor immune escape mediated by antigenic modulation,also can reduce expression of MHC molecule, thus affect the immune therapeutic effect. Another study shows that the reduction of DC and functional defects in cancer patients, so that it can not present tumor antigen effectively,which is also one of the main mechanism of tumor immune escape. Therefore, raising the number of DC in vivo of cancer patients, maintaining its strong antigen recognition and presenting function is one of the key measures to implement the anti-tumor immunotherapy. Because of this, application of sufficient quantity and functional DC for treatment or DC-based anti-tumor vaccine research is becoming increasingly important.Immunotherapy approaches for cancer patients with exogenous DC have developed through the following stages: earlier using the known tumor-associated antigen peptides or mRNA of tumor cells to modificate and allergize DC, but the effects are poor. With deeper study, switching over to use tumor entire antigen to sensitize DC. That is, use the integration of tumor cells with DC or DC loaded with tumor cell lysate to produce hybrid vaccine, which obtain a better anti-tumor effects in gastric cancer, hepatocellular carcinoma and colorectal cancer treatment. The recent study found that DC express a variety of heat shock protein (HSP) receptors (such as CD91, CD40, TLR2 / 4 or LOX1) in the surface. HSP can act on DC or monocytes to stimulate the production of cytokines (IL-12, TNF-α, IL-6, etc.) and enhance the non-specific immune response. In 2004,《Immunity》reported that DC loaded with HSP70-peptide complexes can stimulate CD8+ T much higher (1000 times) compared to DC loaded with peptide, which indicated that integrating HSP with DC to play their respective strengths will produce a stronger immune stimulating effect.In view of this, this study is to explore the methods both for preparation of auto-heat shocked apoptotic colorectal cancer cells as tumor antigens from fresh surgery tumor tissue and the induction of dendritic cell as well as the antigen loading system in vitro,and to compare the lymphocytic antitumor activity stimulated in vitro by dendritic cell (DC) loaded with colorectal cancer cell strain in two different ways and to investigate the effects of dendritic cell (DC) loaded with autologous heat shock-induced apoptotic colorectal cancer cell on the immune function of the postoperative patients with colorectal carcinoma.This study provides a technical basis for the DC vaccines preparation of colorectal cancer.Study is divided into the following three parts:Part I Study on the method of Dendritic Cells Loaded with Auto Colorectal Cancer CellsObjective: To establish the methods both for preparation of auto-heat shocked apoptotic colorectal cancer (colon carcinoma, rectal carcinoma) cells as tumor antigens from fresh surgery tumor tissue and in vitro dendritic cell induction as well as the antigen loading system through which dendritic cell based tumor vaccine could be developed.Methods: Single colorectal cancer cell suspension was made from 14 fresh colorectal tissue by using enzyme digestion assay. The cell suspension was heated and induced into apoptosis with adding BA (Betulinic Acid). Monocyte derived dendritic cells was prepared from peripheral blood mononuclear cells (PBMC) of the patients with GM-CSF and IL-4 cytokine combination culture. Co-culture of imDC and apoptotic colorectal cancer cells was harvested and its phenotype was analyzed by FACS. Results: (1)The yield rate of single colorectal cancer cell from fresh colorectal cancer tissue was (11.81±0.65)×106/g and average apoptotic rate was (93.16±2.31)%;(2)im DC average yield rate from PBMC was(9.75±0.82)×106/(121.64)×106,and the percentage of live cells was more than 95%. The cell percentages of CD11c+CD14- , CD11c+HLA+DR+ , CD11c+CD80+ , CD11c+CD83+ , CD11c+CD86+ among total population were ( 87.58±2.56 ) % , (87.97±0.98)%,(2.21±0.69)%,(4.85±1.22)%, (5.02±0.95)%, respectively.(3)The average yield rate of DC from co-culture of imDC and tumor cells was(6.76±0.98)×106/(9.73±0.84)×106 with 95% alive cells. The cell percentage of CD11c+CD14-, CD11c+HLA+DR+, CD11c+CD80+, CD11c+CD83+, CD11c+CD86+ were (93.45±1.25)%, (89.79±1.35)%, (87.85±1.62)%, (70.74±6.45)%, (95.54±2.18)%, respectively.Conclusions: This method is steady, safe and credible by which potential DC could be developed.Part IIThe comparison of lymphocytic antitumor activity stimulated in vitro by dendritic cells (DC) loaded with colorectal cancer cell strain in two different waysObjective: To compare the lymphocytic antitumor activity stimulated in vitro by dendritic cells (DC) loaded with colorectal cancer cell strain in two different ways. Methods: DC isolated from peripheral blood mononuclear cell of 30 colorectal cancer peoples were loaded with heat shock-induced apoptotic colorectal cancer cell strain and tumor cell lysates separately. Using lymphocytes stimulated in vitro by DC as effector cells and colorectal cancer cell strain as target cells. The cell-killing effect of effector cells on target cells was determined by MTT method.Results: DC loaded with heat shock-induced apoptotic colorectal cancer cell strain and tumor cell lysates both showed the killing activity on target cells , but the killing rate of the former was significantly higher than the latter (P <0.05).Conclusions: The method of loading with heat shock-induced apoptotic colorectal cancer cell strain was more effective. Part III Effects of Dendritic Cells on the Immune Function of Postoperative Patients with Colorectal CancerObjective: To investigate the effects of dendritic cells (DC) induceded by autologous heat shock-induced apoptotic colorectal cancer cell on the immune function of the postoperative patients with colorectal carcinoma.Methods: DC isolated from peripheral blood mononuclear cell of the patients with colorectal cancer were cultured and proliferated in vitro by using rhGM-CSF and rhIL-4,and then were pulsed with autologous heat shock-induced apoptotic colorectal cancer cell itself. 28 postoperative patients with colorectal carcinoma were randomly divided into 2 groups. 14 cases were treated with dendritic cell vaccine. The other 14 cases received only chemotherapy and were established as control group. Immune function, clinical effects in two groups of patients were compared and observed.Results: The levels of CD3+, CD4+/CD8+ and NK cells in the DC vaccine group significantly increased after vaccination (all P values < 0.05), while those of the control group had no significant changes. The levels of IL-2, IL-12 and IFN-γin the DC vaccine group significantly increased and life time was delayed significantly after the vaccination. (all P values < 0.05).Conclusions: The autologous heat shock-induced apoptotic colorectal cancer cell pulsed DC vaccine can improve the immune function of the postoperative patients with colorectal carcinoma. This method results in fewer side effects and should be recommended.更多还原