rhHSP70联合冻融抗原修饰树突状细胞诱导的抗肝癌作用

发布时间：2018-02-01 16:53

  本文关键词： 重组人热休克蛋白70 树突状细胞 冻融抗原 肝癌　出处：《中国医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的 树突状细胞(Dendritic cells,DCs)是目前发现的功能最强大的抗原递呈细胞(Antigen presenting cells,APC),能高效能递呈抗原,且是唯一能激活初始型T淋巴细胞的抗原递呈细胞,具有独特的诱导初始免疫反应对抗肿瘤的能力。目前在DCs疫苗抗肿瘤实验研究和临床研究方面已取得了令人振奋的进展,显示了广泛的应用前景。但肿瘤疫苗研制中仍然存在着一个主要问题:大多数肿瘤抗原的免疫原性很弱,不能正常的被呈递以激活细胞毒性T淋巴细胞(cytotoxictlymphocytes,CTLs),为了提高其免疫原性,必须加入佐剂。研究已表明HSP70具有佐剂样作用,HSP70可以促进DCs成熟,刺激多种细胞因子的分泌而增强免疫功能。本研究对DCs被重组人热休克蛋白70(recombinant human heat shock protein70,rhHSP70)和肝癌冻融抗原共同修饰前后的表型变化、形态变化及诱导CTLs产生的体外杀伤肝癌细胞作用进行了观察,以其明确rhHSP70联合肝癌冻融抗原共同修饰DCs后,体外可诱导产生针对肝癌细胞的高效杀伤作用,并初步探讨了rhHSP70增强DCs疫苗抗肿瘤能力的机制,为设计以DCs为基础的针对肝癌的生物治疗的高效瘤苗提供实验依据和理论基础。 方法 分离健康人外周血获得单个核细胞,经粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF),白细胞介素4(interleukin-4,IL-4)诱导生成DCs,负载冻融抗原的同时加入rhHSP70,分为四组:①A组:DCs+冻融抗原(Ag);②B组:DCs+rhHSP70;③C组:DCs+Ag+rhHSP70;④D组:单独DCs。流式细胞术(flow cytometry,FCM)检测各组DCs表型(CD80,CD83,CD86)变化,倒置显微镜和扫描电镜观察各组DCs形态特点。不同分组修饰的DCs激活淋巴细胞生成CTLs,四甲基偶氮唑蓝(MTT)法检测DCs刺激淋巴细胞增殖能力以及CTLs对肝癌细胞的体外杀伤活性,荧光染色观察各组肝癌细胞凋亡差异。 结果 rhHSP70,冻融抗原及二者联合修饰的DCs表型较对照组相比差异显著(P＜0.05)。CD80:A组为80.2±2.5%,B组为79.6±2.6%,C组为81.6±2.3%,D组为21.5±1.4%。CD83:A组为71.3±2.9%,B组为68.3±2.4%,C组为73.4±2.1%,D组为33.4±2.4%。CD86:A组为74.2±3.6%,B组为75.3±4.8%,C组为82.5±5.0%,D组为48.2±2.8%。 光镜及扫描电镜下可见rhHSP70,冻融抗原及二者联合修饰的DCs较对照组树枝状突起明显增加,表现出成熟DCs的形态特征,而对照组表现出相对不成熟DCs的形态特征。 不同分组修饰的DCs刺激淋巴细胞增殖能力不同,结果以增殖指数(proliferation index,PI,增殖指数=实验组OD值/对照组OD值)表示。其中A组为2.10±0.05,B组为2.06±0.05,C组为2.60±0.06,D组为1.20±0.05,PI以C组为最高,与其他两试验组(A,B组)相比差异显著(P＜0.05),其次为A组,但A组与B组比较差异无统计学意义(P＞0.05)。 不同分组DCs呈递肿瘤抗原诱导的CTL产生杀伤率不同,杀伤百分率=(1-实验组的OD值/对照组的OD值)×100%。在效靶比(效应细胞:靶细胞,即CTLs:肝癌细胞)为10:1,20:1,50:1时,A组杀伤率分别为40.27±4.89%,47.13±8.05%,51.64±6.73%,B组杀伤率分别为13.58±4.77%,12.13±4.26%,15.57±5.55%,C组杀伤率分别为61.09±7.89%,64.71±3.90%,74.59±3.34%,D组杀伤率分别为11.94±4.11%,13.90±4.11%,13.52±4.61%,在三种效靶比下杀伤率以C组为最高,与其他两试验组(A组,B组)相比差异显著(P＜0.05),更远强于对照组(P＜0.05);其次为A组;B组与D组比较差异无统计学意义(P＞0.05)。 结论 rhHSP70联合肝癌冻融抗原修饰DCs,能够促进DCs的成熟,增强DCs刺激淋巴细胞增殖的能力,诱导的CTLs在体外对肝癌细胞能产生高效杀伤。rhHSP70增强DCs抗肿瘤能力的机制可能与其促进DCs成熟有关。
[Abstract]:objective
Dendritic cells (Dendritic cells, DCs) are the most potent antigen-presenting cells (Antigen, presenting, cells, APC) can efficiently present antigen, and only can activate T lymphocyte antigen presenting cells with distinct initial induction of immune response against the tumor at present. In the DCs vaccine against tumor experimental research and clinical research has made exciting progress, and has wide application prospect in development of tumor vaccine. But there are still a major problem: most of the immunogenicity of tumor antigen is very weak, can not be normal presentation to activate cytotoxic T lymphocytes (cytotoxictlymphocytes, CTLs), in order to improve its immunogenicity, must add adjuvant. Studies have shown that HSP70 can be used as an adjuvant, HSP70 can promote DCs maturation and stimulate a variety of cytokine secretion and immune enhancement The study of DCs function. The recombinant human heat shock protein 70 (recombinant human heat shock protein70, rhHSP70) and phenotypic changes before and after antigen modified freeze thawing liver morphological changes and in vitro cytotoxicity induced by CTLs producing hepatocellular carcinoma cells were observed in the rhHSP70 combined with liver frozen thawed antigen co modified after DCs in vitro, can be induced for efficient killing effect of hepatoma cells, and to explore the mechanism of rhHSP70 enhanced antitumor DCs vaccine ability, provide experimental and theoretical basis for the design of DCs based on biological treatment of hepatocellular carcinoma, tumor vaccine.
Method
The separation of human peripheral blood mononuclear cells by granulocyte macrophage colony stimulating factor (granulocyte-macrophage colony-stimulating, factor, GM-CSF), interleukin 4 (interleukin-4, IL-4) induced the formation of DCs load, frozen thawed antigen and joined the rhHSP70, divided into four groups: group A: DCs+ (frozen thawed antigen Ag); B group: DCs+rhHSP70; group C: DCs+Ag+rhHSP70; D group: single DCs. flow cytometry (flow cytometry, FCM) were detected in DCs (CD80, CD83, CD86 phenotype changes), inverted microscope and scanning electron microscope DCs. Morphological characteristics of different groups of modified DCs activated lymphocytes to generate CTLs four, methyl thiazolyl tetrazolium (MTT) method was used to detect the killing activity of DCs to stimulate lymphocyte proliferation and CTLs on hepatocellular carcinoma cells in vitro, observe the apoptosis of hepatocellular carcinoma cell differences in fluorescence staining.
Result
RhHSP70, frozen thawed antigen and combination of the two modified DCs phenotype was significantly higher than the control group (P < 0.05) of.CD80:A group is 80.2 + 2.5%, 79.6 + 2.6% B group, C group is 81.6 + 2.3%, D group is 21.5 + 71.3 + 2.9% 1.4%.CD83:A group, B group is 68.3 + 2.4%. The C group is 73.4 + 2.1%, D group is 33.4 + 2.4%.CD86:A group is 74.2 + 3.6%, 75.3 + 4.8% B group, C group is 82.5 + 5%, D + 2.8%. group was 48.2
Under light microscope and scanning electron microscope, rhHSP70, frozen thawed antigen and two co modified DCs increased significantly compared with the control group, showing the morphological characteristics of mature DCs, while the control group showed relatively immature DCs morphological characteristics.
Different groups of modified DCs stimulated lymphocyte proliferation results in different proliferation index (proliferation index, PI, proliferation index = experimental group control group od / OD). The A group is 2.10 + 0.05, 2.06 + 0.05 B group, C group is 2.60 + 0.06, D group is 1.20 + 0.05, PI in the C group was the highest, and the other two experimental groups (A, B) compared with significant difference (P < 0.05), followed by the A group, but no significant difference between A group and B group (P > 0.05).
Different groups of DCs induced tumor antigens CTL killing rate, killing percentage (OD / OD = 1- experimental group control group) * 100%. (in effect target ratio of effector cells and target cells, i.e. CTLs: cells) of 10:1,20:1,50:1, A group killing rate was 40.27 + 4.89%, 47.13 + 8.05% 51.64, + 6.73%, B group killing rate were 13.58 + 4.77%, 12.13 + 4.26%, 15.57 + 5.55%, C group killing rate were 61.09 + 7.89%, 64.71 + 3.90%, 74.59 + 3.34%, D group killing rate were 11.94 + 4.11%, 13.90 + 4.11%, 13.52 + 4.61%, in the three target than the killing rate of C group is highest, and the other two experimental groups (A group, B group) compared with significant difference (P < 0.05), far higher than in the control group (P < 0.05); followed by A group; B group and D group had no significant difference (P > 0.05).
conclusion
RhHSP70 combined with liver cancer freeze-thaw antigen modified DCs can promote the maturation of DCs and enhance the ability of DCs to stimulate lymphocyte proliferation. The mechanism that induced CTLs can produce high effective killing.RhHSP70 and enhance DCs antitumor ability in vitro can be related to the promotion of DCs maturation.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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