可溶性人PD-1蛋白对肝癌患者树突状细胞功能的影响

发布时间：2019-05-22 06:59

【摘要】： 原发性肝癌(Hepatocellular carcinoma,HCC)是一种严重威胁人类健康和生命安全的恶性肿瘤,为全球常见肿瘤的第5位,其死亡率居恶性肿瘤的第3位[1],我国肝癌死亡率在城市仅次于肺癌,在农村则次于胃癌,其发病率逐年增高,每年全球HCC死亡率高达50多万人,而新发病人数则达到60多万人,其中约50%发生于我国[2]。肝癌恶性程度高、预后差、病死率高,多数患者在经确诊时,已属于中晚期[3],由于患者的肝功差,且多伴有转移,仅10%-20%的患者可行姑息性手术切除,切术后5年生存率仅为25%-50%。因此,新的能够有效抑制肝癌细胞生长的方法将是目前研究的关键。随着对肿瘤免疫学的深入研究,证明了机体对肿瘤存在特异性免疫反应,这为肿瘤治疗指明了新方向,使免疫疗法成为肿瘤治疗的重要手段之一,其中以DC瘤苗为基础的生物免疫治疗成为近年来人们研究的热点,目前部分DC瘤苗已进入Ⅰ、Ⅱ期临床试验[4]。树突状细胞(DC)作为目前已知的专职且功能最为强大的抗原递呈细胞(APC),参与抗原的捕捉、加工、处理和递呈,是淋巴细胞活化、增生、发挥效应的始动环节,主要参与细胞免疫和T细胞依赖的体液免疫,在抗肿瘤免疫应答中起关键作用[5]。体外和动物实验表明,以适当形式的肿瘤抗原负载DC制备瘤苗,可激活能够识别、杀伤肿瘤细胞的特异性T细胞,并可产生免疫记忆效应。大量的临床试验研究也表明DC瘤苗抗肿瘤免疫治疗具有良好的应用前景[6]。在人体的抗肿瘤免疫机制中,T细胞介导的细胞免疫为主要方式。初始T细胞的活化需要两个信号,即由T细胞抗原受体(TCR)与DC上的抗原肽-MHC分子复合物结合提供的抗原特异性信号,又需要DC上的B7共刺激分子与T细胞上的CD28分子结合提供的共刺激信号[7]。只有双信号共同刺激才能有效激活T细胞反应,若缺乏第二信号刺激,将使T细胞处于克隆无能或无应答状态[8],因此B7/CD28共刺激信号对T细胞的活化起着至关重要的作用。近年来,PD-L1/PD-1作为B7/CD28协同刺激分子超家族的一个重要成员,其作用已被越来越多人重视[9]。PD-L1作为B7家族的一个新成员,主要表达于造血细胞,如静息的T细胞、B细胞、单核细胞、树突状细胞[10],其受体PD-1主要表达在活化的T细胞、B细胞、髓系细胞和胸腺细胞[11]。DC上的PD-L1与其受体结合后,可产生抑制信号,并诱导抗原特异性T细胞凋亡、无反应性和衰竭,对机体的免疫应答起负性调控作用[12]。基础研究表明,肿瘤微环境中存在的VEGF、IL-10、TGF-β等细胞因子可诱导树突状细胞PD-L1表达上调,使得肿瘤患者的DC成熟和功能障碍,激活T淋巴细胞能力降低,不能产生有效的抗肿瘤免疫反应,使肿瘤细胞发生免疫逃逸[13]。研究发现,PD-L1是参与肿瘤免疫逃逸过程的一个重要分子,因此封闭PD-L1/PD-1信号传导通路已成为肿瘤免疫治疗的一个重要策略。目的:本实验观察了应用shPD-1蛋白封闭PD-L1/PD-1信号传导通路,对肝癌患者DC刺激自体T淋巴细胞增殖、分泌细胞因子和刺激CTL杀伤能力的影响,进而探讨通过阻断负性调节信号PD-L1/PD-1来增强肝癌患者DC的免疫刺激能力,为DC瘤苗在临床中的应用提供新的思路。方法:以Ficoll密度梯度离心法从肿瘤患者外周血中分离出外周血单个核细胞( PBMC)后,再以粘附法获得单核细胞(Mo),应用重组人粒细胞/巨噬细胞集落刺激因子( rhGM-CSF)、白细胞介素-4( rhIL-4)、α-肿瘤坏死因子(rhTNF-α)体外诱导培养DCs;观察给予或不给予shPD-1蛋白对DC的影响。应用流式细胞术检测DC免疫表型,MTT法检测DC刺激自体T淋巴细胞的增殖能力, ELISA法测定上清液中IL -12p70的水平,MTT法检测效应淋巴细胞对靶细胞的杀伤活性。结果:封闭DC表面PD-L1分子后,DC刺激T细胞增殖,分泌IL -12p70能力显著提高,促进CTL杀伤反应的能力显著增强(P0.05)。结论:可溶性人PD-1蛋白(shPD-1)封闭DC表面PD-L1分子后能显著提高DC活化T细胞的能力,促进DC诱导的抗肿瘤免疫反应。
[Abstract]:Primary liver cancer (HCC) is a malignant tumor which is a serious threat to human health and life safety. It is the fifth of the global common tumors. The mortality rate of HCC is the third[1] of the malignant tumor. The mortality of the liver cancer in China is the second to the lung cancer in the city. The incidence of HCC is increasing year by year, and the annual global HCC mortality rate is as high as more than half a million people, while the number of new patients reaches more than 600,000, of which about 50% is in China[2]. The malignant degree of the liver cancer is high, the prognosis is poor, the case fatality rate is high, the majority of the patients are in the middle and late stage[3] at the time of the diagnosis, due to the poor liver function of the patient and with the transfer, only 10% to 20% of the patients with the feasible palliative operation, and the 5-year survival rate after the resection is only 25% -50%. Therefore, the new method to effectively inhibit the growth of the liver cancer cells will be the key to the present study. With the further study of the tumor immunology, the specific immune response of the body to the tumor is proved, which indicates a new direction for the treatment of the tumor, so that the immunotherapy is one of the important means of the treatment of the tumor, Among them, bio-immunotherapy based on DC tumor vaccine has become the hot spot of people's research in recent years. At present, some of the DC tumor vaccine has entered the first and second phase clinical trials[4]. Dendritic cells (DC) are the most powerful antigen-presenting cells (APC), which are currently known and have the most powerful functions, and are involved in the capture, processing, treatment and presentation of the antigen, and are the starting steps of lymphocyte activation, proliferation and effect. Humoral immunity, which is mainly involved in cellular immune and T-cell-dependent, plays a key role in anti-tumor immune response[5]. In vitro and in animal experiments, it is shown that the tumor vaccine can be prepared by the tumor antigen loaded DC in an appropriate form, and the specific T cell capable of identifying and killing the tumor cells can be activated, and the immune memory effect can be generated. A large number of clinical trial studies have also shown that the anti-tumor immunotherapy of the DC tumor vaccine has a good application prospect[6]. In the anti-tumor immune mechanism of human body, T cell-mediated cell immunity is the main mode. The activation of the initial T cell requires two signals, the antigen-specific signal provided by the T-cell antigen receptor (TCR) and the antigen-peptide-MHC molecule complex on the DC, and the co-stimulatory signal[7] provided by the B7 costimulatory molecule on the DC and the CD28 molecule on the T-cell. Only two-signal co-stimulation can effectively activate the T-cell response. If the second signal stimulation is absent, the T-cells will be in a clone-incompetent or non-response state[8], so that the B7/ CD28 co-stimulatory signal plays a critical role in the activation of T-cells. In recent years, PD-L1/ PD-1, as an important member of the B7/ CD28 co-stimulatory molecule superfamily, has been given more and more attention to[9]. PD-L1, as a new member of the B7 family, is mainly expressed in hematopoietic cells, such as resting T cells, B cells, monocytes, dendritic cells[10], and the receptor PD-1 is mainly expressed in activated T cells, B cells, myeloid cells and thymus cells[11]. After the PD-L1 on the DC is combined with the receptor, the inhibition signal can be generated, and the antigen-specific T-cell apoptosis, no reactivity and failure can be induced, and the immune response of the organism is negatively regulated[12]. The basic research shows that the expression of VEGF, IL-10 and TGF-1 in the tumor microenvironment can induce the up-regulation of the expression of PD-L1 in the dendritic cells, so that the DC maturation and dysfunction of the tumor patients and the ability of activating T-lymphocytes can be reduced, and the effective anti-tumor immune response can not be generated, The tumor cells are immune to escape[13]. It is found that PD-L1 is an important molecule involved in the immune escape process of the tumor, so the closed PD-L1/ PD-1 signaling pathway has become an important strategy for tumor immunotherapy. Objective: To study the effect of the application of shPD-1 protein on the proliferation of T-lymphocytes, the secretion of cytokines and the ability to stimulate CTL. The effects of PD-L1/ PD-1 on the immunostimulating ability of DC in patients with liver cancer were also discussed. Methods: After the peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of the tumor patients by Ficoll density gradient centrifugation, the mononuclear cells (Mo) were obtained by the adhesion method, and the recombinant human granulocyte/ macrophage colony stimulating factor (rhGM-CSF) and the interleukin-4 (rh) were used. IL-4, human-tumor necrosis factor (rhTNF-1) in vitro induced culture of DCs; observation of the administration or absence of shPD-1 protein pairs The effect of DC was detected by flow cytometry. The proliferation ability of DC-stimulated autologous T-lymphocytes was detected by flow cytometry. The level of IL-12p70 in the supernatant was determined by ELISA. Results: After the DC surface PD-L1 was closed, the DC-stimulated T-cell proliferation and the secretion of IL-12p70 increased significantly, and the ability of the CTL to kill the CTL significantly increased. Conclusion: The soluble human PD-1 protein (shPD-1) can significantly improve the ability of the DC-activated T cells after the DC surface PD-L1 molecule is closed to promote D.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392;R735.7

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