可溶性人PD-1蛋白對肝癌患者樹突狀細胞功能的影響
發(fā)布時間：2019-05-22 06:59
【摘要】： 原發(fā)性肝癌(Hepatocellular carcinoma,HCC)是一種嚴重威脅人類健康和生命安全的惡性腫瘤,為全球常見腫瘤的第5位,其死亡率居惡性腫瘤的第3位[1],我國肝癌死亡率在城市僅次于肺癌,在農(nóng)村則次于胃癌,其發(fā)病率逐年增高,每年全球HCC死亡率高達50多萬人,而新發(fā)病人數(shù)則達到60多萬人,其中約50%發(fā)生于我國[2]。肝癌惡性程度高、預后差、病死率高,多數(shù)患者在經(jīng)確診時,已屬于中晚期[3],由于患者的肝功差,且多伴有轉移,僅10%-20%的患者可行姑息性手術切除,切術后5年生存率僅為25%-50%。因此,新的能夠有效抑制肝癌細胞生長的方法將是目前研究的關鍵。隨著對腫瘤免疫學的深入研究,證明了機體對腫瘤存在特異性免疫反應,這為腫瘤治療指明了新方向,使免疫療法成為腫瘤治療的重要手段之一,其中以DC瘤苗為基礎的生物免疫治療成為近年來人們研究的熱點,目前部分DC瘤苗已進入Ⅰ、Ⅱ期臨床試驗[4]。樹突狀細胞(DC)作為目前已知的專職且功能最為強大的抗原遞呈細胞(APC),參與抗原的捕捉、加工、處理和遞呈,是淋巴細胞活化、增生、發(fā)揮效應的始動環(huán)節(jié),主要參與細胞免疫和T細胞依賴的體液免疫,在抗腫瘤免疫應答中起關鍵作用[5]。體外和動物實驗表明,以適當形式的腫瘤抗原負載DC制備瘤苗,可激活能夠識別、殺傷腫瘤細胞的特異性T細胞,并可產(chǎn)生免疫記憶效應。大量的臨床試驗研究也表明DC瘤苗抗腫瘤免疫治療具有良好的應用前景[6]。在人體的抗腫瘤免疫機制中,T細胞介導的細胞免疫為主要方式。初始T細胞的活化需要兩個信號,即由T細胞抗原受體(TCR)與DC上的抗原肽-MHC分子復合物結合提供的抗原特異性信號,又需要DC上的B7共刺激分子與T細胞上的CD28分子結合提供的共刺激信號[7]。只有雙信號共同刺激才能有效激活T細胞反應,若缺乏第二信號刺激,將使T細胞處于克隆無能或無應答狀態(tài)[8],因此B7/CD28共刺激信號對T細胞的活化起著至關重要的作用。近年來,PD-L1/PD-1作為B7/CD28協(xié)同刺激分子超家族的一個重要成員,其作用已被越來越多人重視[9]。PD-L1作為B7家族的一個新成員,主要表達于造血細胞,如靜息的T細胞、B細胞、單核細胞、樹突狀細胞[10],其受體PD-1主要表達在活化的T細胞、B細胞、髓系細胞和胸腺細胞[11]。DC上的PD-L1與其受體結合后,可產(chǎn)生抑制信號,并誘導抗原特異性T細胞凋亡、無反應性和衰竭,對機體的免疫應答起負性調(diào)控作用[12]�；A研究表明,腫瘤微環(huán)境中存在的VEGF、IL-10、TGF-β等細胞因子可誘導樹突狀細胞PD-L1表達上調(diào),使得腫瘤患者的DC成熟和功能障礙,激活T淋巴細胞能力降低,不能產(chǎn)生有效的抗腫瘤免疫反應,使腫瘤細胞發(fā)生免疫逃逸[13]。研究發(fā)現(xiàn),PD-L1是參與腫瘤免疫逃逸過程的一個重要分子,因此封閉PD-L1/PD-1信號傳導通路已成為腫瘤免疫治療的一個重要策略。 目的:本實驗觀察了應用shPD-1蛋白封閉PD-L1/PD-1信號傳導通路,對肝癌患者DC刺激自體T淋巴細胞增殖、分泌細胞因子和刺激CTL殺傷能力的影響,進而探討通過阻斷負性調(diào)節(jié)信號PD-L1/PD-1來增強肝癌患者DC的免疫刺激能力,為DC瘤苗在臨床中的應用提供新的思路。 方法:以Ficoll密度梯度離心法從腫瘤患者外周血中分離出外周血單個核細胞( PBMC)后,再以粘附法獲得單核細胞(Mo),應用重組人粒細胞/巨噬細胞集落刺激因子( rhGM-CSF)、白細胞介素-4( rhIL-4)、α-腫瘤壞死因子(rhTNF-α)體外誘導培養(yǎng)DCs;觀察給予或不給予shPD-1蛋白對DC的影響。應用流式細胞術檢測DC免疫表型,MTT法檢測DC刺激自體T淋巴細胞的增殖能力, ELISA法測定上清液中IL -12p70的水平,MTT法檢測效應淋巴細胞對靶細胞的殺傷活性。 結果:封閉DC表面PD-L1分子后,DC刺激T細胞增殖,分泌IL -12p70能力顯著提高,促進CTL殺傷反應的能力顯著增強(P0.05)。 結論:可溶性人PD-1蛋白(shPD-1)封閉DC表面PD-L1分子后能顯著提高DC活化T細胞的能力,促進DC誘導的抗腫瘤免疫反應。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392;R735.7
本文編號：2482765
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392;R735.7
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