人参皂苷Ro调控STAT5通路促进白血病细胞向DC定向分化作用与机制研究

发布时间：2018-04-22 14:11

本文选题：人参皂苷Ro + 白血病　；参考：《济南大学》2017年硕士论文

【摘要】：白血病是一种特殊的血液系统恶性肿瘤。白血病患者体内的白血病细胞增殖失去控制,从而在骨髓和其他造血组织中大量积累,抑制了正常造血功能。传统的骨髓干细胞移植及化疗存在供源受限、微小残留病灶复发等问题。肿瘤生物治疗是目前研究的热点,其中诱导白血病细胞向免疫细胞定向分化呈现良好的应用前景。树突状细胞(DC)是体内功能最强大的抗原提呈细胞(APC),是T淋巴细胞免疫应答的启动者,在识别肿瘤抗原、活化T细胞杀伤肿瘤细胞中发挥着关键作用。然而白血病患者体内DC数量明显减少并且存在功能缺陷,这是特异性的肿瘤抗原不能有效激发机体特异性抗肿瘤细胞免疫应答的主要因素之一。DC在免疫应答中发挥重要的作用,已经被用来作为治疗肿瘤及免疫相关疾病,但DC在体内含量少并且难分离纯化限制了DC在临床上的应用。因此,如何提高白血病患者DC数量及功能成为肿瘤领域研究的重点与难点。直接诱导白血病细胞分化为DC是潜在的有效方法,这种白血病来源的DC不仅具有了抗原递呈细胞的特性,同时携带有白血病抗原,是肿瘤免疫治疗的一个重要研究方向。目前体外诱导白血病源DC主要采用粒细胞巨噬细胞-集落刺激因子(GM-CSF)联合白介素4(IL-4)的方法,但存在诱导及以体内转输后效果不稳定及价格昂贵的缺点。中药治疗肿瘤具有疗效好、副作用小等优点,成为目前白血病治疗领域研究热点。人参皂甙Ro是人参的主要有效单体成分之一,研究显示其可以通过诱导肿瘤细胞凋亡、抑制肿瘤细胞浸润和转移、抑制血管形成等多个方面发挥抗肿瘤作用;但其是否具有诱导肿瘤细胞向DC分化的作用尚未见报道。JAK/STAT是调控DC分化及功能的关键信号通路,可启动下游靶基因的转录与表达,调控DC的发育分化与功能,是目前DC发育分化领域研究的热点。STAT5是STAT家族的重要成员,已证实STAT5通过活化DC特异性和转录因子Id2诱导DC的发育分化。但目前调节STAT信号通路调控白血病来源DC分化的关键机制尚不清楚,更缺乏药物靶向研究。本实验目的为探讨人参皂苷Ro联合细胞因子(GM-CSF与IL-4)促进白血病源树突状细胞(DC)分化的作用。筛选细胞因子诱导白血病源DC敏感细胞株;采用细胞因子联合人参皂苷Ro诱导筛选的敏感细胞株人单核白血病细胞(THP-1)向DC分化。培养体系中分别加入小(5μmol/L)、中(10μmol/L)、大(20μmol/L)剂量人参皂苷Ro,流式细胞术检测白血病来源DC表面标志CD1a、MHC-II、CD80、CD83、CD86及CCR7的表达;逆转录-聚合酶链式反应(RT-PCR)检测白血病来源DC MHC-II、CD83、CD86及CCR7 mRNA转录水平;酶联免疫吸附法(ELISA)检测培养上清TNFa、IL-6蛋白水平。检测结果显示,THP-1是细胞因子诱导DC分化的敏感白血病细胞株,STAT5调控Id2在THP-1向DC分化中具有重要作用。与单纯细胞因子诱导比较,人参皂苷Ro联合细胞因子作用后白血病来源DC表面标志CD1a、MHC-II、CD80、CD83、CD86及CCR7的比例显著升高(P0.05),MHC-II、CD83、CD86及CCR7的mRNA转录水平显著升高(P0.05),培养上清TNFa、IL-6蛋白水平显著升高(P0.05),提示人参皂苷Ro联合细胞因子具有明显促进白血病细胞向DC分化的作用;同时发现人参皂苷Ro明显升高白血病来源DC STAT5、Id2的mRNA水平,并且呈剂量依赖性(P0.05),提示靶向调控STAT5信号通路可能是人参皂苷Ro诱导白血病细胞向DC的关键机制。以上结果提示,THP-1为GM-CSF与IL-4细胞因子诱导白血病源DC的敏感细胞株;人参皂苷Ro具有明显促进白血病源DC诱导的作用,靶向调控STAT5信号通路可能是人参皂苷Ro诱导白血病细胞向DC的关键机制。
[Abstract]:Leukemia is a special malignant tumor of the blood system. The proliferation of leukemic cells in the leukemia patients is not controlled, thus accumulating in the bone marrow and other hematopoietic tissues and inhibiting the normal hematopoiesis. The traditional bone marrow stem cell transplantation and chemotherapy have the problem of limited source of donor and the recurrence of small residual focus. Therapy is a hot spot of current research, which induces a good prospect of leukemic cells to be directed to Immunocytic differentiation. Dendritic cells (DC) are the most powerful antigen presenting cells (APC) in the body. It is the promoter of the immune response of T lymphocytes. It plays a key role in identifying tumor antigenic and activating T cells to kill tumor cells. However, there is a significant decrease in the number of DC in the patients with leukemia and the existence of functional defects. This is one of the main factors that the specific tumor antigen can not effectively stimulate the immune response of the body..DC plays an important role in the immune response and has been used as a treatment for cancer and immune related diseases, but DC is contained in the body. Less and difficult separation and purification limit the clinical application of DC. Therefore, how to improve the number and function of DC in leukemia patients is the key and difficult point in the field of cancer research. Direct induction of leukemia cells to differentiate into DC is a potential effective method. The DC derived from this leukemia is not only characterized by antigen presenting cells, but also carried by the leukemia cells. Leukemic antigen is an important research direction in tumor immunotherapy. At present, the main method of inducing leukemia source DC in vitro is the method of granulocyte macrophage colony stimulating factor (GM-CSF) combined with interleukin 4 (IL-4), but there is a shortcoming of instability and high price after induction and transfer in vivo. The therapeutic effect of traditional Chinese medicine on tumor is effective. Good, small side effects and so on, it has become a hot spot in the field of leukemia treatment. Ginsenoside Ro is one of the main effective monomer components of ginseng. The study shows that it can induce tumor cell apoptosis, inhibit tumor cell infiltration and metastasis, inhibit vascular formation and other sides to play anti-tumor effect, but it can induce swelling. The role of tumor cell differentiation to DC has not yet been reported that.JAK/STAT is the key signaling pathway to regulate the differentiation and function of DC, which can activate the transcription and expression of the target genes of the downstream target, regulate the development and differentiation and function of the DC, which is a key member of the STAT family in the field of DC development and differentiation, and has confirmed that STAT5 is specific and turned through the activation of DC. The transcriptional factor Id2 induces the development and differentiation of DC. However, the key mechanism of regulating the differentiation of leukemia source DC by the STAT signaling pathway is still unclear, and it lacks the drug targeting study. The purpose of this experiment is to explore the role of ginsenoside Ro combined cytokine (GM-CSF and IL-4) in promoting the differentiation of leukemia derived dendritic cells (DC). DC sensitive cell line of the source of white blood disease; the human mononuclear leukemic cells (THP-1), which were induced by cytokines and ginsenoside Ro, were differentiated into DC. In the culture system, small (5 mu mol/L), medium (10 mu), and large (20 mu mol/L) dose ginsenoside Ro were used, and the flow cytometry was used to detect the DC surface marker CD1a, MHC-II, CD. 80, CD83, CD86 and CCR7 expression; reverse transcription polymerase chain reaction (RT-PCR) was used to detect the transcriptional level of DC MHC-II, CD83, CD86 and CCR7 mRNA, and enzyme linked immunosorbent assay (ELISA) was used to detect and cultivate the supernatant TNFa and protein level. THP-1 has an important role in the differentiation of DC. Compared with simple cytokine induction, ginsenoside Ro combined with cytokines, the proportion of leukemia source DC surface markers CD1a, MHC-II, CD80, CD83, CD86 and CCR7 increased significantly (P0.05). Significantly increased (P0.05), suggesting that ginsenoside Ro combined with cytokines can significantly promote the differentiation of leukemic cells to DC, and that ginsenoside Ro significantly increases the mRNA level of leukemia source DC STAT5, Id2, and is dose-dependent (P0.05), suggesting that the target regulation of STAT5 signaling pathway may be the Ro induction of Ginsenoside induced leukemia. The key mechanism of cell to DC. The results suggest that THP-1 is a sensitive cell line of leukemia source DC induced by GM-CSF and IL-4 cytokine, and ginsenoside Ro can obviously promote the DC induction of leukemia source, and the target regulation of STAT5 signaling pathway may be the key mechanism of ginsenoside Ro induced leukemic cell to DC.

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7

【参考文献】