人脐血来源树突状细胞诱导CTL对BGC823肿瘤细胞的抑制作用

发布时间：2018-03-15 11:17

本文选题：脐血　切入点：树突状细胞　出处：《郑州大学》2011年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的近年来,随着人口老龄化及人类生存环境的恶化,恶性肿瘤的发病率和病死率均呈现明显升高的趋势。目前临床肿瘤的治疗普遍还是采用手术、放疗和化疗等传统方法。但这些方法难以清除体内的微小肿瘤病灶且对机体损伤较大,在杀伤癌细胞的同时,往往也伴随着正常组织细胞的损伤。随着人们对肿瘤发生发展机制的进一步认识和肿瘤免疫分子生物学和生物工程技术的进展,肿瘤生物治疗迅速发展,成为肿瘤治疗的第四种治疗模式。树突状细胞(dendritic cells, DCs)因具有抗原呈递能力和活化初始T细胞的能力而成为肿瘤免疫治疗的研究热点。但DCs在肿瘤患者外周血和组织中含量较少,体外扩增也较难达到理想的治疗用剂量。因此,探讨从健康产妇脐血分离诱生DCs的方法,研究其体内外的抗肿瘤效应及机制,有助于为脐血来源的DCs及细胞毒性T细胞的临床应用打下基础。方法采集健康足月分娩产妇脐血50-80ml,分离出人脐血单个核细胞(Human Umbilicus blood mononuclear cell, HUBMC),将培养瓶静置2小时,取贴壁生长细胞,在含15%胎牛血清1640培养液中加入GM-CSF、IL-4和SCF,培养诱生人脐血树突状细胞(Human Umbilicus blood dendritic cell, HUBDC)。取对数生长期的BGC823肿瘤细胞,用反复冻融法取得肿瘤细胞提取物作为肿瘤抗原,负载HUBDCs,促使HUBDCs成熟。于普通光学显微镜下观察HE染色后的HUBDCs形态,并用流式细胞仪对其进行表型鉴定。将HUBDCs中的非贴壁细胞采用尼龙毛柱法分离出T细胞并鉴定其纯度。观测负载抗原成熟的HUBDCs对T细胞的活化增殖能力(混合淋巴细胞反应)。采用MTT比色法检测活化的T细胞对BGC823靶细胞的杀伤。动物实验部分,首先建立荷瘤动物模型,将BGC823细胞注射于裸鼠并观察成瘤情况,记录肿瘤大小。将荷瘤裸鼠随机分成四组,治疗方案如下：A组：体外负载抗原的HUBDCs和未激活T细胞；B组：体外经HUBDCs诱导活化的特异性T细胞(CTL); C组：负载BGC823抗原的HUBDCs; D组：未经HUBDCs致敏的新分离T细胞。观察裸鼠体内肿瘤的生长情况并分别记录。结果用细胞因子GM-CSF、SCF和IL-4配伍能诱导人脐血贴壁单个核细胞向DC分化,加入肿瘤抗原提取物能促使HUBDCs成熟,HE染色光镜下观察HUBDCs形态符合典型毛刺状、树枝状DCs形态,流式分析仪检测细胞表型：MHC-Ⅰ、MHC-Ⅱ、CD86(协同刺激分子)、CD54(黏附分子)、CD11c均较诱导前有显著升高。其中,CD86表达率为40.59%±3.27%,CD54为59.21%±6.32%,CD11c表达率为67.01%±5.17%,MHC-Ⅰ和MHC-Ⅱ分别为：42.37%±10.11%和56.31%±6.76%,与诱导前相比,差异具有统计学意义P0.01。同种混合淋巴细胞反应显示：HUBDCs体外可以使抗原特异性T细胞活化增殖为效应T细胞(cytotoxic T lymphocytes, CTL),且CTL对靶细胞BGC823可以产生特异性的抑制及杀伤。在动物实验中,成功建立人胃癌BGC823荷瘤裸鼠模型。按实验方案分别对各组治疗后,注射负载抗原HUBDCs +新分离T细胞的A组和注射特异性CTL的B组都有明显抑瘤效应,至30天时A组肿瘤大小为：211mm3B组为：153mm3,B组抑瘤效应更加显著；C组单独注射负载抗原的HUBDCs, D组注射未致敏新分离的T细胞均无明显抑瘤效果,至30天时C组,D组肿瘤大小分别为：1093 mm3和1022mm3。C组D组肿瘤细胞仍快速生长。结论 1)利用细胞因子能从人脐血单个核细胞(HUBMC)中诱导出具有典型形态、表型和具有活化T细胞功能的HUBDCs; 2)成熟人脐血DC与T细胞共同移植,能显著增强荷瘤裸鼠的抗肿瘤能力。
[Abstract]:Background and purpose of research
In recent years, with the population aging and deterioration of the living environment of mankind, the incidence and mortality rate of malignant tumors showed a significant increasing trend. The current clinical treatment of tumors generally or with surgery, radiotherapy and chemotherapy and other traditional methods. But it is difficult to remove small tumor lesions in vivo and the damage to the body is larger in these methods, killer cancer cells at the same time, often accompanied by normal tissue damage. With the progress of the mechanism of the occurrence and development of the further understanding of molecular biology and tumor immunity and tumor biological engineering technology, biological treatment of tumor development, become the fourth method for cancer treatment. Dendritic cells (dendritic cells, DCs) research hotspot in tumor immunity the treatment has become the antigen-presenting ability and the ability to activate naive T cells. But the DCs in the peripheral blood and tissues containing less In vitro expansion is also difficult to achieve an ideal therapeutic dose. Therefore, exploring the method of inducing DCs from umbilical cord blood of healthy women, studying its anti-tumor effect and mechanism in vivo and in vitro, is helpful to lay a foundation for the clinical application of cord blood derived DCs and cytotoxic T cells.
Method
Collected from healthy full-term delivery maternal cord blood 50-80ml, isolated from human umbilical cord blood mononuclear cells (Human Umbilicus blood mononuclear cell, HUBMC), the flask standing 2 hours, adherent cells in GM-CSF medium containing 15% fetal bovine serum IL-4 and SCF, 1640 training, training induced human umbilical cord blood dendritic cells (Human Umbilicus blood dendritic cell, HUBDC). BGC823 cells in logarithmic growth phase, the load of HUBDCs by freeze-thaw method to obtain tumor cell extracts as tumor antigen, HUBDCs, to mature. In the ordinary optical microscope after HE staining, the morphology of the HUBDCs, and the phenotype was identified by flow cytometry. The HUBDCs in non adherent cells by nylon fiber isolated T cells and identify the purity. Observe the load on T cell activation and proliferation of mature HUBDCs antigen (mixed lymphocyte reaction) by MTT. Colorimetric detection of activated T BGC823 cells to kill target cells. Animal experiment, first to establish tumor bearing animal model, BGC823 cells were injected into nude mice and the tumor growth was observed and recorded. The size of tumor bearing nude mice were randomly divided into four groups, treatment group were as follows: A: in vitro antigen loaded HUBDCs and not the activation of T cells; group B: in vitro induced by HUBDCs activation of specific T cells (CTL); group C: load BGC823 antigen HUBDCs; D group: without HUBDCs sensitized isolated T cells. Tumor growth of and were recorded.
Result
Cell factor GM-CSF, and SCF and IL-4 can induce human umbilical cord blood adherent mononuclear cells to differentiate into DC, with tumor antigen extracts can promote HUBDCs maturation and HE staining of HUBDCs with typical morphology of burr, dendritic DCs morphology, flow cytometry analyzer to detect cell surface type: MHC- 1, MHC- 2, CD86 (costimulatory molecules), CD54 (CD11c, adhesion molecules) were induced significantly increased. Among them, the expression rate of CD86 was 40.59% + 3.27%, CD54 = 59.21% + 6.32%, the expression rate of CD11c was 67.01% + 5.17%, MHC- I and MHC- II respectively: 42.37% + 10.11% and 56.31% + 6.76%, compared with the induction the difference has statistical significance P0.01. showed that allogeneic mixed lymphocyte reaction: HUBDCs in vitro can make antigen-specific T cell activation and proliferation of effector T cells (cytotoxic T lymphocytes, CTL), and CTL BGC823 on target cells to produce specific inhibit and kill Injury. In animal experiments, establishment of human gastric cancer BGC823 in nude mice model. According to the experimental scheme in each group after treatment, B group were injected with antigen loaded HUBDCs + T cells isolated A injection group and the specific CTL has obvious anti-tumor effect, and 30 days in A group, tumor size: 211mm3B group 153mm3, group B: the antitumor effect of C group was more significant; a single injection of antigen loaded HUBDCs, D group were injected with unsensitized new isolated T cells had no obvious inhibitory effect on tumor growth, to 30 days in C group, D group, tumor size was 1093 mm3 and 1022mm3.C group D group of tumor cells is still fast growth.
conclusion
1) the use of cytokines to induce a typical form, phenotype and HUBDCs that has the function of activating T cells from human umbilical cord blood mononuclear cells (HUBMC).
2) the combined transplantation of DC and T cells in mature human umbilical cord blood can significantly enhance the anti-tumor ability of nude mice.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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