【作者】 刘媛媛；

【导师】 张连生；

【作者基本信息】 兰州大学 ， 内科学， 2010， 硕士

【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(P<0.05),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(P<0.05)。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。更多还原

【Abstract】 Objective:CD4+CD25+ Treg cells which have the nature of immune suppression and immune incompetence are about 5%-10% of peripheral CD4+ T cells in healthy people, and participate in process of immune suppression. The numbers of Treg cells are higher in peripheral blood and tumor tissues of a variety of cancer patients and are responsible for inducing and maintaining peripheral tolerance. Treg cells have two subsets, natural Treg and adaptive Treg. Adaptive Treg cells can be differentiated from CD4+ T by the change of expression on the costimulatory molecules of APC or the change of the cytokines. Treg have been suggested to play an important immunopathological role in human cancer by lowering the intrinsic T-cell immunity towards tumor-associated antigen, resulting in tumor immune evasion. Some reports have indicated that Treg depletion can be useful in tumor biotherapy. Treg depletion would eliminate immune-suppression mediated by Treg leading to enhance T-cell activity. Another study has confirmed that immature DC can induced CD4+T cells to adaptive Treg. The current strategies of cancer immunotherapy focus on breaking immune tolerance and enhance the host T cell response. Pseudomonas aeruginosa-mannose sensitive hamemagglutination vaccine (PA-MSHA vaccine) is prepared from Pseudomonas aeruginosa which contains mannose-sensitive hemagglutinin pili. It has been widely used for anti-inflammatory treatment, even for anti-tumor treatment as immune modulator. According to reports, PA-MSHA vaccine could improve clinical efficiency as adjuvant therapy for a variety of cancer such as leukemia, malignant lymphoma, lung cancer, breast cancer, gastric cancer, liver cancer and so on. This vaccine can increase the antigen presenting function of dendritic cells (DCs) or Th1/Th2 proportion, and regulate unbalance between cellular immune and humoral immune to achieve the purpose of effective treatment of tumors. However, the specific mechanism is still not very clear. Acute myeloid leukemia (AML) is a malignant hematopoietic disorder characterized by proliferation of immature myeloid precursors with considerable impairment of the immune system. Compared with healthy controls, AML patients had a higher proportion of CD4+CD25+ T cells in peripheral blood.Professor Sun reported PA-MSHA vaccine can improve immune function in patients with acute leukemia. The level of IL-2, the activity of NK cells, the ratio of CD4+/CD8+ in PA-MSHA vaccine treatment group are significantly higher than those in control group.But whether PA-MSHA vaccine can take effect on anti-tumor through reducing the number and function of the Treg cells or not, has not been reported. This study will detect the effect of the dendritic cells derived from acute myeloid leukemia and induced by PA-MSHA on CD4+CD25+ regulatory T cells, and is to investigate the immune regulatory fuctions of PA-MSHA to acute myeloid leukemia.Methods:The buffy coat separated from peripheral blood cells of untreated newly diagnosed patients with acute myeloid leukemia by leukapheresis was diluted 1:1 with phosphate buffered saline(PBS). Lymphocytes were isolated using Ficoll lymphocyte separating medium, and the cells were incubated in RPMI1640 complete mediums for 3h. Then after the suspended cells were removed, recombinant human granulocyte-macrophage colony-stimulating factor(rhGM—CSF)and recombinant human interleukin-4(rhlL-4) were added into medium and the culture were incubated for 7 days. Then the cells were devided into three groups. As the control group, the first group was not given any treatment. The second group was added with PA-MSHA. Tumor necrosis factor-a(TNF-a) added in the third group, which continued to incubat 24 hours. The morphological feature was observed with inverted microscope everyday. The phenotypes of cells were detected by flow cytometry, and the proliferation effect of three groups of AML-DCs on Allogeneic T lymphocytes was investigated by a mixed lymphocyte reaction system which was analyzed by MTT assay. In the 8th day, the CD4+ T cells separated from peripheral blood cells of healthy adult by magnetic cell sorting (MACS) system were respectively incubated with the DCs from the three groups for 7 days.Then the supernatant concentration of IL-10, TGF-p was detected by ELISA kit. The cells of all the groups were harvested respectively and further tested for the expression of CD4+CD25+ T cells by flow cytometry. The RT-PCR method was used to examine Foxp3mRNA expression.Results:The morphology of dendritic cells in PA-MSHA group and TNF-a group were irregular with slender synapses on their surface, and the expressions of CDla,CD80, CD83, CD86and HLA-DR on mature dendritic cells in PA-MSHA group and TNF-a group were significantly higher on day 8 than the control group, and proliferation indexes of the two groups was also higher than the control group as the more numbers of stimulating cells. In addition, the levels of IL-10 and TGF-β, the expressions of CD4 and CD25 on Treg, and the expression of Foxp3mRNA in PA-MSHA group and TNF-a group are all higher than the control group.Conclusion:PA-MSHA can further promote the maturation of AML-DC, and thus inhibit the differentiation from CD4+ T cells to Treg. So PA-MSHA can enhance anti-tumor activity for acute myeloid leukemia patients.更多还原