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γδT细胞对血液肿瘤细胞杀伤作用的研究

发布时间:2018-05-16 02:28

  本文选题:γδT细胞 + 血液肿瘤 ; 参考:《中国人民解放军军事医学科学院》2017年硕士论文


【摘要】:血液肿瘤是血液系统一系列恶性疾病的总称,包括急性白血病、慢性白血病、多发性骨髓瘤、骨髓增生异常综合征、淋巴瘤和恶性组织细胞病等。在我国,血液肿瘤发病率较高。虽然联合化疗方案不断改进,支持治疗不断改善,血液肿瘤的初治CR不断提高,生存率也有明显提高。即使初始治疗达到完全缓解,但血液肿瘤复发率依然很高,尤其急性白血病长期生存率低。同时还有部分病例达不到CR,其长期生存率更低。复发、难治的病例至今仍治疗效果有限,缺乏理想的治疗手段。传统的肿瘤治疗方法有手术切除、放疗、化疗和靶向治疗,其中手术切除、放疗、化疗均是作用于肿瘤组织,只能局部清除肿瘤细胞,靶向治疗虽然能清除散在的肿瘤细胞,但存在肿瘤抗原变异和丢失、耐药等问题,均无法清除全身的肿瘤细胞,肿瘤的复发也就无法避免。肿瘤的免疫治疗作用于人体的免疫系统,通过提高人体免疫系统的清除肿瘤能力来达到治疗肿瘤的目的。在2013年,《Science》杂志将肿瘤免疫治疗评为年度十大科技突破之首,为血液肿瘤的治疗带来曙光。过继性免疫细胞治疗,是将采自患者的或健康供者的PBMC,体外扩增活化后,将获得的免疫细胞回输给患者,利用活化的免疫细胞抗肿瘤活性来清除患者体内的肿瘤细胞。目前,过继性免疫细胞治疗应用的比较多的人体的免疫细胞有T淋巴细胞、NK细胞和DC细胞。T淋巴细胞被认为是机体唯一能特异性识别杀伤肿瘤细胞的免疫细胞。根据表达不同的TCR,T淋巴细胞可分为αβT和γδT细胞。γδT细胞是表达TCRγδ的T细胞亚群,γδT细胞只占外周T淋巴细胞1%-5%,但在组织中却可高达20-50%。γδT细胞属于人体的固有免疫细胞,在体内起着抗肿瘤、抗病毒感染、抗细菌感染和免疫监视等作用,能促进未成熟B细胞分泌Ig A,Ig M,Ig G,调节体液免疫,能活化未成熟DC细胞。γδT细胞,能以MHC非限制性的方式识别多种肿瘤相关抗原,可通过以下方式杀伤肿瘤细胞:通过凋亡诱导蛋白配体途径Fas-Fas L和TRAILR诱导肿瘤细胞凋亡;分泌大量的细胞因子,作用于肿瘤细胞及其微环境,直接或协同其他免疫细胞杀伤肿瘤细胞;通过ADCC和穿孔素-颗粒酶B杀伤肿瘤细胞;通过某些膜表面受体如FcγR,经ADCC发挥细胞毒作用;作为APC,发挥提呈抗原作用。已有不少临床试验应用γδT细胞于肿瘤的治疗,已在结直肠癌、肾癌、非小细胞肺癌、乳腺癌和胰腺癌等肿瘤治疗上取得进展,取得良好疗效和安全性。将γδT细胞应用于血液肿瘤的治疗有三个优势:活化的γδT细胞对肿瘤细胞强大的杀伤活性;γδT细胞能够分泌大量细胞因子和提呈抗原协同其他免疫细胞杀伤肿瘤细胞;血液肿瘤患者常存在免疫低下或缺陷,因此常引发感染,特别是机会感染,γδT细胞属于固有免疫细胞,应用于肿瘤的治疗时能在清除肿瘤细胞的同时,抗病毒和细菌感染。目的:本研究的目的就是建立γδT细胞培养体系,探讨γδT细胞对多种血液肿瘤细胞的杀伤活性,并初步探讨影响γδT细胞杀伤的一些机制,为γδT细胞应用于血液肿瘤的治疗提供实验依据。内容:本文的研究内容主要分二个部分。首先是优化Zol联合IL-2的培养方案,建立γδT细胞培养体系,比较两种无血清T细胞培养基Op Tmizer和X-VIVO15培养基体外培养γδT细胞的效果和添加血清对无血清Op Tmizer和X-VIVO 15培养基体外培养γδT细胞的影响;然后是采用流式细胞术检测γδT细胞对Jurkat、THP-1、HL-60、K562、Raji、U-937和RPMI-8226细胞的杀伤活性,检测γδT细胞分泌IFN-γ和TNF-α的水平,检测γδT细胞表达CD107a的水平,检测延长与靶细胞共孵育时间对γδT细胞对靶细胞的杀伤活性的影响,检测Zol和美伐他汀预处理对γδT细胞杀伤肿瘤细胞的影响,并比较γδT与NK和CIK细胞对K562细胞的杀伤作用。方法:1、优化Zol联合IL-2的培养方案,采用RPMI-1640培养基,建立γδT细胞培养体系,在此基础上,比较两种无血清T细胞培养基Op Tmizer和X-VIVO15培养基体外培养γδT细胞的效果,并比较添加血清对无血清培养基扩增γδT细胞的影响。2、体外培养Jurkat、THP-1、HL-60、K562、Raji、U-937和RPMI-8226细胞,倒置显微镜下观察处于对数生长期,分别与γδT细胞共孵育,效靶比为10:1和20:1,共孵育4 h,用流式抗体标记后上流式细胞仪检测,比较γδT细胞对Jurkat、THP-1、HL-60、K562、Raji、U-937和RPMI-8226细胞的杀伤活性和不同的效靶比对γδT细胞杀伤的影响。将γδT细胞与K562细胞按10:1的效靶比共孵育48 h,分别于0、4、16、24、36和48 h用流式细胞仪检测γδT细胞分泌IFN-γ和TNF-α的水平。将HL-60和K562细胞分别与γδT细胞按10:1的效靶比共孵育4 h和20 h后,用流式细胞仪检测延长共孵育时间对γδT细胞杀伤K562细胞的影响。用不同浓度的Zol预处理K562细胞,分别与γδT细胞按10:1的效靶比共孵育4 h后,用流式细胞仪检测不同浓度的Zol对γδT细胞杀伤K562细胞的影响。用5μmol/L的美伐他汀预处理K562细胞,后与γδT细胞按10:1的效靶比共孵育4 h后,用流式细胞仪检测美伐他汀对γδT细胞杀伤K562细胞的影响。签署知情同意书后,抽取4位淋巴瘤患者的外周血,分离得到PBMC,采用不同的培养体系体外扩增γδT与NK和CIK细胞,后分别与K562细胞按5:1、10:1和20:1的效靶比共孵育4 h后,用流式细胞仪检测,比较γδT与NK和CIK细胞对K562细胞的杀伤作用。结果:1、采用Zol联合IL-2的培养方案可以在体外扩增外周血γδT细胞;Op Tmizer培养基比X-VIVO 15培养基体外培养γδT细胞的效果好(P0.05),适当加入血清能提高扩增效率(P0.05)。2、发现γδT细胞对Jurkat、THP-1、HL-60、K562、U-937和RPMI-8226细胞均有明显的杀伤活性(P0.05),对Raji细胞的杀伤作用较弱;随着与K562细胞共孵育时间的延长,γδT细胞分泌IFN-γ和TNF-α的水平基本呈现出时间依赖性增加,TNF-α自8h后逐渐增加;与K562细胞和HL-60细胞共孵育后,γδT细胞CD107a的表达水平均出现显著上调(P0.01)。延长γδT细胞与K562和HL-60细胞共孵育时间,γδT细胞对K562和HL-60细胞的杀伤活性也明显增强(P0.01)。Zol能提高γδT细胞对K562细胞的杀伤活性,而美伐他汀减弱了γδT细胞对K562细胞的杀伤活性;4位B细胞淋巴瘤患者均成功在体外扩增出γδT、NK和CIK细胞。效靶比为5:1时,γδT、NK和CIK细胞对K562细胞的杀伤活性无统计学差异(P0.05),效靶比为10:1和20:1时,γδT细胞对K562细胞的杀伤活性与NK细胞无统计学差异(P0.05),但明显强于较CIK细胞(P0.01)。结论:Zol联合IL-2的培养体系体外成功扩增健康人外周血γδT细胞;Op Tmizer培养基比X-VIVO 15培养基体外培养γδT细胞的效果好,适当加入胎牛血清(FBS)能提高扩增效率;γδT细胞可显著杀伤多种血液肿瘤细胞;γδT细胞对K562细胞的杀伤活性与NK细胞无统计学差异,明显强于CIK细胞,;在48 h内随着与k562细胞共孵育时间的延长,γδT细胞分泌IFN-γ的水平呈时间依赖性增加,而TNF-α自8 h后逐渐增加;与K562和HL-60细胞共孵育后,γδT细胞的CD107a分子的表达有水平明显上调增加;延长γδT细胞与K562和HL-60细胞共孵育时间,γδT细胞对K562和HL-60细胞的杀伤活性也明显增强(P0.01)。Zol能提高γδT细胞对K562细胞的杀伤活性,而美伐他汀减弱了γδT细胞对K562细胞的杀伤活性。总之,Zol联合IL-2体外培养的γδT细胞对多种血液肿瘤细胞均具有较高的杀伤活性。与靶细胞共孵育后,γδT细胞杀伤靶细胞的效率提高。提高效靶比和和延长与靶细胞共孵育时间,都能提高γδT细胞对靶细胞的杀伤活性。Zol能提高γδT细胞对K562细胞的杀伤活性,而美伐他汀减弱了γδT细胞对K562细胞的杀伤活性。为血液肿瘤的细胞免疫治疗提供实验依据。
[Abstract]:Blood tumor is the general name of a series of malignant diseases of the blood system, including acute leukemia, chronic leukemia, multiple myeloma, myelodysplastic syndrome, lymphoma and malignant histiocytic disease. In China, the incidence of blood tumors is high. Although combined chemotherapy regimens continue to improve, support treatment continues to improve, the initial blood tumor In the treatment of CR, the survival rate also improved. Even if the initial treatment reached complete remission, the recurrence rate of blood tumor was still very high, especially in the acute leukemia, the long-term survival rate was low. At the same time, some cases were less than CR, and the long-term survival rate was lower. The recurrence, refractory cases still had limited treatment effect and lack of ideal treatment. Traditional methods of tumor treatment include surgical resection, radiotherapy, chemotherapy and targeted therapy. Surgical excision, radiotherapy and chemotherapy are all responsible for tumor tissue, only local tumor cells can be removed. Targeting therapy can remove scattered tumor cells, but there are some problems, such as tumor antigen variation and loss, drug resistance, etc. In the 2013, magazine named the tumor immunotherapy as the first of the ten major scientific and technological breakthroughs of the year and brought the dawn of the treatment of blood tumor. The immune cell therapy is a PBMC that will be taken from a patient or a healthy donor. After in vitro expansion and activation, the acquired immune cells are returned to the patient, and the tumor cells in the patient are removed by the active immune cell antitumor activity. At present, the immune cells of adoptive immunotherapy should use more T lymphocytes in the human body. NK cells and DC cell.T lymphocytes are considered to be the only immune cells that can specifically identify tumor cells. According to the expression of different TCR, T lymphocytes can be divided into alpha beta T and gamma delta T cells. Delta T cells belong to the natural immune cells of the human body. They play the role of anti-tumor, antiviral infection, anti bacterial infection and immune surveillance in the body. It can promote the secretion of Ig A, Ig M, Ig G, modulate the humoral immunity and activate immature DC cells. The gamma delta T cells can identify various tumor related antigens in a MHC and non restrictive way, which can be passed through the MHC and non restrictive way. The following methods kill tumor cells: inducing apoptosis by Fas-Fas L and TRAILR through apoptosis inducing protein ligand pathway; secreting a large number of cytokines, acting on tumor cells and their microenvironment, killing tumor cells directly or in conjunction with other immune cells; killing tumor cells through ADCC and perforin granzyme B; through some membrane tables Surface receptors, such as Fc gamma R, play cytotoxic effects by ADCC; as APC, play an antigenic role. Many clinical trials have applied gamma delta T cells in the treatment of tumors, and have made progress in the treatment of colorectal cancer, kidney cancer, non small cell lung cancer, breast cancer and pancreatic cancer. Good efficacy and safety have been achieved. [delta] T cells are applied to blood. The treatment of tumor has three advantages: activated gamma delta T cells have a strong killing activity against tumor cells; gamma delta T cells can secrete a large number of cytokines and antigen in synergy with other immune cells to kill tumor cells; the patients with blood tumors often have immunodeficiency or defects, and therefore often cause infection, especially opportunistic infection, and gamma delta T cells belong to the tumor cells. The aim of this study is to establish a gamma delta T cell culture system, to explore the killing activity of gamma delta T cells to a variety of blood tumor cells, and to explore some mechanisms for the killing of gamma delta T cells, which should be used for the gamma delta T cells. The experimental basis for the treatment of blood tumor is provided. The content of this study is mainly divided into two parts. First, it is to optimize the culture scheme of Zol combined with IL-2, to establish a T cell culture system, to compare the effect of two kinds of serum free T cell culture medium Op Tmizer and X-VIVO15 culture medium in vitro culture of gamma delta T cells and to add serum to serum-free Op T. The effects of mizer and X-VIVO 15 culture medium on the culture of gamma delta T cells in vitro, and then using flow cytometry to detect the killing activity of gamma delta T cells on Jurkat, THP-1, HL-60, K562, Raji, U-937 and RPMI-8226 cells. The effect of time on the killing activity of gamma delta T cells on target cells was used to detect the effects of Zol and simvastatin on the killing of tumor cells by gamma delta T cells, and to compare the killing effects of gamma delta T and NK and CIK cells on K562 cells. Methods: 1, the culture scheme of Zol combined with IL-2 was optimized, and RPMI-1640 culture medium was used to establish the culture system of delta T cells. On the basis of the comparison of the effects of two serum-free T cell culture medium Op Tmizer and X-VIVO15 medium in vitro culture of gamma delta T cells, the effects of serum on the amplification of.2, Jurkat, THP-1, HL-60, K562, Raji, K562, and cells were observed in the logarithmic growth period under the inverted microscope. The effect target ratio was 10:1 and 20:1, and the target ratio was 4 h. The effect of gamma delta T cells on Jurkat, THP-1, HL-60, K562, Raji, U-937 and RPMI-8226 cells was compared with the effect of different target ratio on the killing of gamma delta cells. The level of IFN- gamma and TNF- alpha secreted by gamma delta T cells was detected by flow cytometry at 48 h, respectively. The effects of HL-60 and K562 cells on 4 h and 20 h were incubated with HL-60 and K562 cells according to the target ratio of 10:1, respectively. The effects of prolonged incubation time on the killing cells of gamma delta cells were detected by flow cytometry. After 4 h cells were incubated with gamma delta T cells according to the target ratio of 10:1 to 4 h, the effect of Zol on the killing of K562 cells by gamma delta T cells was detected by flow cytometry. The K562 cells were pretreated with the statins of 5 mu mol/L, and after 4 h were incubated with the 10:1 target ratio of the gamma delta T cells, the statins were detected by flow cytometer. After the informed consent book was signed, the peripheral blood of 4 patients with lymphoma was extracted and PBMC was isolated. The T and NK and CIK cells were amplified in vitro by different culture systems, and then the K562 cells were incubated with the target ratio of 5:1,10:1 and 20:1 for 4 h, respectively, and were detected by flow cytometry and compared with those of NK and CIK cells. Results: 1, the peripheral blood gamma delta T cells can be amplified in vitro by Zol combined with IL-2, and the effect of Op Tmizer culture based on X-VIVO 15 medium is good (P0.05), and the proper addition of serum can improve the amplification efficiency (P0.05).2. The cells had obvious killing activity (P0.05), and the killing effect on Raji cells was weak. With the prolongation of incubation time with K562 cells, the level of IFN- gamma and TNF- alpha secreted by delta T cells showed a time dependent increase, and TNF- alpha gradually increased since 8h; after incubation with K562 and HL-60 cells, the expression level of CD107a Delta T cell CD107a The incubation time of gamma delta T cells with K562 and HL-60 cells was prolonged, and the killing activity of K562 and HL-60 cells was significantly increased by the gamma delta T cells (P0.01).Zol could increase the killing activity of the delta T cells to K562 cells, while simvastatin weakened the killing activity of gamma delta T cells to the cells; 4 patients with lymphoma cell lymphoma were all Gamma delta T, NK and CIK cells were successfully amplified in vitro. When the target ratio was 5:1, there was no significant difference in the killing activity of T, NK and CIK cells to K562 cells (P0.05). The culture system successfully amplified healthy human peripheral blood gamma delta T cells in vitro; Op Tmizer culture was better than X-VIVO 15 culture medium in vitro culture of gamma delta T cells, and proper addition of fetal bovine serum (FBS) could improve the amplification efficiency; gamma delta T cells could kill a variety of blood tumor cells significantly, and the killing activity of gamma delta T cells to K562 cells was not statistically significant to NK cells. The difference was obviously stronger than that of CIK cells, and the level of IFN- gamma secreted by T cells increased in time with the prolongation of the incubation time with K562 cells in 48 h, while TNF- alpha increased gradually after 8 h, and the expression of CD107a molecules in the gamma delta T cells increased significantly after CO incubation with K562 and HL-60 cells; 60 cell incubation time, the killing activity of gamma delta T cells to K562 and HL-60 cells also increased significantly (P0.01).Zol can increase the killing activity of delta T cells to K562 cells, while simvastatin weakened the killing activity of delta T cells to K562 cells. In a word, the Zol combined with IL-2 in vitro gamma Delta T cells are higher for a variety of blood tumor cells. After incubating with target cells, the efficiency of gamma delta T cells to kill target cells increased. Improving target ratio and prolonging the incubation time with target cells could increase the killing activity of gamma delta T cells against target cells and increase the killing activity of.Zol Delta T cells to K562 cells, while simvastatin weakened the killing of K562 cells by gamma delta T cells. It provides experimental evidence for cellular immunotherapy of hematological malignancies.

【学位授予单位】:中国人民解放军军事医学科学院
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R733


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