PA-MSHA刺激的AML-DC对Treg作用的研究

发布时间：2018-08-13 17:31

【摘要】： 目的：CD4+CD25+调节性T细胞(Regulatory T cells,Treg)占正常人体外周CD4+T淋巴细胞的5%-10%,参与体内免疫抑制过程,具有免疫抑制和免疫无能性。在多种肿瘤患者外周血及肿瘤组织局部的数量增多,诱导维持外周免疫耐受。Treg可分为天然性Treg和获得性Treg。在一定的环境条件下由于抗原提呈细胞(antigen-presenting cell,APC)表面共刺激分子表达的改变或细胞因子种类的变化,使CD4+T细胞分化为获得性Treg。Treg通过降低对肿瘤相关抗原的初始T细胞免疫,导致肿瘤免疫逃逸,从而在肿瘤患者的免疫病理中发挥重要作用。有报道表明,减少Treg的数量在肿瘤生物治疗中至关重要。减少Treg的数量就可以减少Treg介导的免疫抑制作用,从而增强T细胞的效能。另有研究证实,未成熟树突状细胞可促使CD4+T细胞向获得性Treg分化,从而促进Treg的产生。目前肿瘤免疫治疗策略的研究重点集中在打破免疫耐受和提高宿主T细胞反应。铜绿假单胞菌注射液(Vaccine of Pseudomonas aeruginosa with Mannose Sensitive Hemagglutination pili,PA-MSHA)是从带甘露糖敏感血凝菌毛的铜绿假单胞菌中制备而来,因其产生免疫调节作用及低毒无副作用而倍受青睐,目前已广泛应用于抗感染和抗炎治疗,甚至作为免疫调节剂用于抗肿瘤治疗。据报道,PA-MSHA作为白血病、恶性淋巴瘤、肺癌、乳腺癌、胃癌、肝癌等恶性肿瘤的辅助治疗可提高临床有效率,其抗肿瘤的可能机制在于通过增加树突状细胞的抗原递呈能力,提高Th1/Th2的比例,调节细胞免疫和体液免疫的不平衡状态,达到有效的治疗肿瘤的目的,但其具体作用机制尚不十分明确。急性髓细胞白血病(Acutemyeloid leukemia,AML)是造血干细胞异常克隆增生导致的一种恶性肿瘤性疾病,伴有严重的免疫系统损伤。与正常人相比,AML患者外周血和骨髓血中均含有更高比例Treg。孙文平报道了PA-MSHA疫苗能明显提高急性白血病患者的免疫功能,增强其自身的免疫杀伤能力。PA-MSHA疫苗治疗组患者其IL-2水平、NK细胞活性、CD4+/CD8+比值均明显高于对照组。但是PA-MSHA疫苗是否可以通过降低AML患者体内的Treg细胞的数量和功能,从而发挥其抗肿瘤作用,还尚未有报道。本研究通过检测经铜绿假单胞菌注射液刺激的急性髓细胞白血病源性树突状细胞(dendritic cells derived from aute myeloid leukemia,AML-DCs)对Treg作用的影响,探讨PA-MSHA对AML的免疫调节作用。方法：用来源于初诊未治急性髓细胞白血病患者的经由外周血细胞单采术得到的富集白细胞层与PBS液1：1稀释,采用淋巴细胞分离液进行淋巴细胞分离,3h后弃悬浮细胞,加入重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF),重组人白细胞介素-4(rhIL-4)和RPMI1640完全培养基体外培养至第7天,分为三组：第一组为对照组,不给予任何处理；第二组加入PA-MSHA;第三组加入TNF-a。继续培养24小时。每天取三组DC在倒置显微镜下观察细胞形态。第8天用流式细胞仪检测其细胞表型,并行混合淋巴细胞反应检测AML-DCs对淋巴细胞的增殖作用。将第8天的三组AML-DCs分别与MACS法分离得到的正常人外周血CD4+T细胞共培养7天,然后ELISA法分别测量三组上清液IL-10、TGF-β水平,流式细胞仪检测CD4、CD25的表达,RT-PCR测量Foxp3mRNA水平。结果：PA-MSHA组和TNF-a组的DC呈现明显的树突状形态,这两组DC的CD1a、CD80、CD83、CD86和HLA-DR表达较对照组明显升高(P0.05),且这两组DC诱导的T细胞增殖指数随刺激细胞数量的增加而增加,并明显高于对照组。此外,PA-MSHA组和TNF-a组DC诱导Treg产生后所测的IL-10、TGF-β水平,CD4、CD25的表达及Foxp3 mRNA水平均较对照组明显降低(P0.05)。结论：PA注射液可以促进AML-DC的进一步成熟,并抑制初始T细胞向Treg方向分化,从而增强对急性髓细胞白血病细胞的抗肿瘤活性。
[Abstract]:Objective: CD4 + CD25 + regulatory T cells (Treg) account for 5% - 10% of the normal human peripheral CD4 + T lymphocytes, participate in the process of immunosuppression in vivo, with immunosuppression and immune anergy. CD4 + T cells differentiate into acquired Treg. Treg induces tumor immune escape by reducing the initial T cell immunity to tumor-associated antigens under certain environmental conditions due to changes in the expression of costimulatory molecules on the surface of antigen-presenting cells (APC) or changes in the types of cytokines. It has been reported that reducing the number of Treg is essential in tumor biotherapy. Reducing the number of Treg can reduce Treg-mediated immunosuppressive effects and thus enhance T cell potency. Current studies on tumor immunotherapy strategies have focused on breaking immune tolerance and enhancing host T cell responses. Vaccine of Pseudomonas aeruginosa with Mannose Sensitive Hemagglutination pili (PA-MSHA) is a mannose-sensitive Pseudomonas aeruginosa strain. PA-MSHA has been widely used in anti-infection and anti-inflammation therapy, even as an immunomodulator in anti-tumor therapy. It is reported that PA-MSHA as an adjuvant therapy for leukemia, malignant lymphoma, lung cancer, breast cancer, gastric cancer, liver cancer and other malignant tumors can be improved. The possible mechanism of its anti-tumor effect is to increase the antigen presenting ability of dendritic cells, increase the ratio of Th1/Th2, regulate the imbalance of cellular immunity and humoral immunity, so as to achieve the goal of effective treatment of tumor, but the specific mechanism is not very clear. AML is a malignant disease caused by abnormal clonal proliferation of hematopoietic stem cells, accompanied by severe immune system damage. Compared with normal people, the peripheral blood and bone marrow blood of AML patients contain a higher proportion of Treg. Sun Wenping reported that PA-MSHA vaccine can significantly improve the immune function of patients with acute leukemia and enhance their own immunity. The levels of IL-2, NK cell activity and CD4 +/CD8 + ratio in PA-MSHA vaccine group were significantly higher than those in control group. However, whether PA-MSHA vaccine can exert its anti-tumor effect by reducing the number and function of Treg cells in AML patients has not been reported. The effect of dendritic cells derived from aute myeloid leukemia (AML-DCs) on Treg was studied to investigate the immunomodulatory effect of PA-MSHA on AML.
Methods: The enriched leukocyte layer obtained from untreated patients with acute myeloid leukemia was diluted with PBS solution 1:1 by peripheral blood mono-collection. Lymphocyte isolation was carried out with lymphocyte isolation solution. The suspension cells were discarded after 3 hours and recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was added to recombinant human leukocyte fine. Interleukin-4 (rhIL-4) and RPMI 1640 were cultured in vitro for 7 days and divided into three groups: the first group was the control group without any treatment; the second group was added PA-MSHA; the third group was added TNF-a. Mixed lymphocyte reaction was used to detect the proliferation of AML-DCs. On the 8th day, three groups of AML-DCs were co-cultured with normal human peripheral blood CD4+T cells separated by MACS for 7 days, then the levels of IL-10 and TGF-beta in the supernatants of three groups were measured by ELISA, the expressions of CD4 and CD25 were detected by flow cytometry, and Foxp3 mRNA water was measured by RT-PCR. Flat.
Results: DCs in PA-MSHA group and TNF-a group showed obvious dendritic morphology. The expressions of CD1a, CD80, CD83, CD86 and HLA-DR in the two groups were significantly higher than those in the control group (P The levels of IL-10, TGF-beta, CD4, CD25 and Foxp3 mRNA were significantly lower than those in the control group (P 0.05).
CONCLUSION: PA injection can promote the maturation of AML-DC and inhibit the differentiation of initial T cells toward Treg, thus enhancing the antitumor activity of AML-DC cells.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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