【作者】 洪帆；

【导师】 张明智；

【作者基本信息】 郑州大学 ， 肿瘤学， 2010， 硕士

【摘要】 背景和目的恶性淋巴瘤(Malignant Lymphoma, ML)是原发于淋巴结或淋巴结外组织或器官的一种恶性肿瘤,来源于淋巴细胞及其前体细胞。在我国恶性淋巴瘤虽相对少见,但近年来新发病例逐年上升,每年至少超过25000例,已成为严重威胁人类健康的一类疾病。恶性淋巴瘤的治疗以化疗为主,同时配合放疗和生物治疗等综合治疗。尽管一部分恶性淋巴瘤经过化疗等综合治疗后可以达到长期持续缓解,但总的来说恶性淋巴瘤因为其侵袭性、原发和继发耐药等原因,治疗效果往往不尽人意。因此,恶性淋巴瘤的攻克需要发展新的治疗途径与手段。目前研究较多的是肿瘤的免疫治疗,其中CpG ODN是肿瘤免疫治疗研究的热点。现已有多篇文献报道,CpG ODN对多种实体瘤的免疫治疗效果显著,但有关淋巴造血系统的恶性肿瘤研究较少。过去几十年里大量的证据显示,病原体中的核酸不仅能作为生命的基本物质而存在,而且还可以激活宿主的免疫系统。细菌中未甲基化5’CpG3’二核苷酸(CpGoligodeoxynucleotide, CpG ODN)对各种免疫细胞亚群有强大的免疫激活作用。它们能直接激活巨噬细胞、单核细胞和树突状细胞等抗原递呈细胞,释放多种细胞因子,如白介素(IL)-12和IL-6等,再由这些细胞因子间接激活自然杀伤(NK)细胞及T细胞,发挥抗病毒、抗细菌感染及抗肿瘤作用。本研究运用人工合成的含有CpG基序的寡聚脱氧核苷酸刺激人外周血单个核细胞(Peripheral blood mononuclear cell,PBMC),观察其对PBMC的活化刺激作用,以及由此介导的对淋巴瘤hut-78细胞的杀伤作用,旨在为淋巴瘤的免疫治疗提供一种新途径。方法淋巴细胞分离液分离健康人外周抗凝血,体外细胞培养技术常规培养PBMC；将实验分为四组：①阴性对照组：PBS+PBMC;②阳性对照组：LPS+PBMC；③实验组：CpG ODN+PBMC；④联合组：CpG ODN+LPS+PBMC.ELISA法检测CpG ODN活化PBMC后分泌IL-12.INF-γ的含量,流式细胞仪测活化PBMC表面标记CD80、CD86分子的表达；MTT法检测CpG ODN活化PBMC对hut-78细胞杀伤作用,倒置显微镜下观察细胞形态学变化。结果1 CpG ODN活化后PBMC培养液上清中IL-12和INF-γ的表达水平CpG ODN活化后PBMC培养液上清中IL-12和INF-γ的表达水平显著升高。经LPS组、CpG ODN组及LPS+ CpG ODN组活化后的PBMC产生IL-12的含量分别60.54±1.82 pg.mL-1、115.50±19.34 pg.mL-1、120.75±13.71 pg.mL-1；产生INF-γ的含量分别为158.74±18.46 pg.mL-1、262.75±30.21 pg.mL-1、265.25±29.26 pg.mL-1。实验组结果与空白对照组差异有统计学意义(P<0.01)。2 CpG ODN诱导PBMC表面标志的表达与对照组相比,PBMC在经LPS组、CpG ODN组刺激后细胞表面标志协同刺激分子CD80、CD86的表达水平均明显增高(P<0.01)；CpG ODN组刺激的PBMC表达协同刺激分子CD80、CD86明显大于阳性对照LPS组(P<0.05)。3 CpG ODN活化的PBMC对HUT-78细胞杀伤作用CpG ODN活化后的PBMC对HUT-78细胞杀伤活力显著提高。经LPS组.CpG ODN组及CpG ODN+ LPS组活化后的人外周血单个核细胞对HUT-78细胞杀伤活力分别为41.86±2.93%、63.65±7.98%、67.05±3.56%。其实验组0D值与空白对照组差异有统计学意义(P<0.05)。4形态学观察结果通过观察发现HUT-78细胞与经CpG ODN刺激后的PBMC共同培养48小时后,形态学改变较明显：有些细胞边界不清,相互融合,部分细胞可见核浓缩。结论1经CpG ODN2006活化PBMC分泌细胞因子IL-12和INF-γ水平显著提高。2 CpG ODN刺激PBMC表面表达的协同刺激分子CD80、CD86水平明显增高3 CpG ODN显著增强人外周血单个核细胞对HUT-78细胞杀伤活力。更多还原

【Abstract】 Background and ObjectiveMalignant lymphoma is a malignant proliferative disease generates from the human immune system cells and their precursor cells. Although malignant lymphoma in China is relatively rare, yet new cases have been increasing in recent years, at least more than 25,000 cases a year, which often occurs in young and seriously endangers the health and lives of human beings. Currently, combined chemotherapy is the common modality of the treatment of malignant lymphoma. It plays an important role in improving the curative rate and remarkably prolonging the disease-free interval of sufferers, but it has inevitably adverse reactions, such as bone marrow suppression and the decreasing of immune system, which make it difficult for the patients to complete the full course of treatment. Moreover, Primary or secondary drug resistance during treatment has become one of the tough problems of clinical therapy of the malignant tumor. Therefore, it is time to seek new ways and means of treatment for malignant lymphoma. More current researches are focus on immunotherapy of cancer and CpG ODN. A number of articles have been published that treatment with CpG ODN on solid tumors was significantly. However, the study of malignant tumors of lymphoid system is less.DNAs in bacteria and virus, which contain numethylated CPG motifs activate host defense mechanisms leading to innate and acquired immune responses. Synthetic oligodeoxynucleotides(ODNs) containing unmethylated CPG motifs Minic the immunostimulatory qualities of bacterial DNA and elicit a coordinated set of T helper 1 (Thl)-like immune responses, including direct activation of B cells, plasmacytoid dendritic cells (PDCs), macrophages, antigen present cell (APC) and indirect activation of T cells, natural Killer (NK) cells. CpG ODN-induced activation of innate immunity protects against lethal challenge with a wide variety of pathogens. However, we can only find some reports on antitumor activity of CPG ODN in solid tumors.So, in this study, we will investigate the antitumor effects of PBMC activated by Synthetic oligodeoxynucleotides (ODN) containing unmethylated CPG dinucleotides (CPG ODN) in vitro and explore the mechanisms of anti-tumor action of CPG ODN, which offers a new way for the treatment of the lymphoma.MethodsSeparating the circumference anti-hemoglutination of healthy persons with Lymphocyte separation medium, human peripheral blood mononuclear cells (PBMC) were cultured with technology of cell culture in vitro. We established four groups in the experiment:control group:PBMC+PBS; Positive control group:PBMC+LPS; ODN2006 group:PBMC+ODN2006; Combination group:PBMC+ODN2006+LPS. The secretions of INF-γand IL-12 from PBMC determined using ELISA method. After treated with these groups for 48 hours, Co-stimulatory molecules of CD80, CD86 on the surface of PBMC were analyzed by flow cytometry. PBMC activated by CPG ODN was co-cultured with hut-78 cells for 48 hours, and then MTT method was used to detect PBMC-mediated antitumor activity. We examined cell morphology with the inverted microscope.Results1. The effect of CpG ODN on INF-γand IL-12 of PBMCThe secretions of INF-γand IL-12 from PBMC treated with CpG ODN were increased significantly. The levers of IL-12 from PBMC of control group, Positive control group, CpG ODN group and Combination group was 15.50±10.12, 60.54±1.82 pg-mL-1,115.50±19.34 pg·mL-1,120.75±13.71 pg·mL-1,respectively; The levers of INF-γfrom PBMC of these four groups was 85.50±10.01, 158.74±18.46 pg·ml-1,262.75±30.21 pg·mL-1,265.25±29.26 pg·mL-1 separately.2. The effect of PBMC treated with CpG ODN on Co-stimulatory molecules of CD80, CD86 of PBMCPBMC treated with CPG ODN and Combination group exhibited more expression of Co-stimulatory molecules of CD80 and CD 86 than control group and Positive control group.3. The effects of PBMC Stimulated by CPG ODN on hut-78 cellsThe growth of hut-78 cells could be inhibited partially after being treated by PBMC activated by CPG ODN for 48h. The Cytotoxicity of control group, Positive control group, CPG ODN group, and Combination group was 24.93±7.81%, 41.86±2.93%,63.65±7.98%,67.05±3.56%.4. The influence of PBMC stimulated by CPG ODN on cell morphology of hut-78 cells co-cultured PBMCAfter co-cultured with PBMC stimulated by CpG ODN, morphological changes of hut-78 cells were more obvious:some cell boundaries unclear, and some cells shows nuclear enrichment.Conclusions1. CpG ODN could increase the secretions of INF-γand IL-12 of PBMC2. CpG ODN could significantly exhibited more Co-stimulatory molecules of CD80 and CD 86.3. CpG ODN could increase the cytotoxicity of PBMC on HUT-78 cells in vitro.更多还原