【作者】 王宁；

【导师】 单风平；

【作者基本信息】 中国医科大学 ， 免疫学， 2010， 硕士

【摘要】 前言1975年Hughes等从猪脑提取液中分离出具有内源性吗啡样活性物质蛋氨酸脑啡肽(Methionine Enkephalin, MENK)。蛋氨酸脑啡肽的早期研究主要集中在作为镇痛剂和神经递质方面,但随着研究的深入,人们发现其受体在免疫系统以及某些肿瘤组织和正常组织细胞均有分布。MENK通过与免疫细胞上受体结合发挥免疫调节作用。树突状细胞(Dendritic cell, DC)是目前所知的机体内功能最强大的抗原提呈细胞,参与机体多种自身免疫性疾病、免疫缺陷病、肿瘤及一些炎症反应的病理过程,在机体的抗感染免疫及肿瘤免疫应答过程中起着极其重要的作用,是基础与临床研究的热点。未成熟DC在机体的外周组织中捕获和加工抗原,然后开始向淋巴器官转移,并渐进式成熟,与此同时DC表面重要的功能分子,如：MHC-Ⅱ、CD40和CD80(CD86)的表达增加。由于DC在免疫调节中的重要作用,使人们越来越多地关注DC的研究与应用,特别是在抗肿瘤方面的应用。因此,研究DC已经成为一个热点领域。鉴于MENK在免疫系统中的显著调节作用,所以研究MENK对DC的作用对于阐明MENK对免疫系统作用机理是十分必的,国内外未见报道。本研究就MENK对小鼠髓系DC2.4细胞株成熟过程中表型及功能的变化的影响作用进行探讨。方法采用本教研室保存并传代的小鼠髓系DC2.4细胞株进行实验,复苏培养细胞,应用扫描电镜技术、酸性磷酸酶活性检测、流式细胞仪检测技术、酶联免疫吸附试验等检测DC表型和功能的各种指标。结果1、MENK处理的DC表面可见很长的突起；而未作处理的DC(即RPMI1640组)的突起很短甚至没有突起。2、蛋氨酸脑啡肽组和LPS组的树突状细胞的酸性磷酸酶活性均低于RPMI1640组(P<0.05)。3、MENK和LPS类似,能够上调树突状细胞表面MHC-Ⅱ、CD86、CD40分子的表达。4、ELISA结果显示DC在未做任何处理时(即RPMI 1640组)IL-12分泌的量少,MENK和LPS处理的DC能够分泌高水平的IL-12(P<0.05)。结论1、适宜浓度的蛋氨酸脑啡肽显著促进DC形态学的成熟。2、适宜浓度的蛋氨酸脑啡肽作用使DC细胞内的酸性磷酸酶活性下降,表明DC吞噬抗原能力下降,递呈能力增加,趋向成熟。3、适宜浓度的蛋氨酸脑啡肽显著增加DC的MHC-Ⅱ、CD40和CD86分子的表达,刺激DC发育成熟。4、适宜浓度的蛋氨酸脑啡肽显著增加培养上清中IL-12的含量,说明MENK促进DC成熟的同时,上调DC的功能。更多还原

【Abstract】 IntroductionIn 1975, Methionine-enkephalin(MENK) with endogenous morphine-like activity was found in extract from pig brain. The study found that met-enkephalin may regulate the endocrine system and immune response. Met-enkephalin earlier studies found on as paregoric and neurotransmitters, but with a detailed study, people found that receptors in the central nervous system, immune system and certain cell in tumor tissues and normal tissue were distributed. MENK with the opioid receptors, regulate the immune system function. Dendritic cells(DC) are professional antigen-presenting cells while play an important role in initiating T cell responses against microbial pathogens and tumors. DC participate in a variety of autoimmune disease, the body immune deficiency disease, cancer and some inflammatory pathological process. In the body’s anti-infection immunity and tumor immune response, DC play an extremely important role and is basic and clinical research focus. Immature DC capture and process exogenous antigens in peripheral tissues and then begin to migrate to lymphoid organ, at the same time, the DC surface expression of MHC-Ⅱ、CD40 and CD80(CD86) increased. The DC of important role in regulate the immune system function have led to increasing attention to the research and application of DC, especially anti-tumor function. Therefore, the study of DC has become a hot area. In view of the significant regulatory role in immune system, the function of MENK on DC is necessary to clarify the mechanism of the role of MENK on the immune system, the study has not been reported at domestic and abroad. Thus, the purpose of this study is to explore the mechanism of immunomodulatory effects of met-enkephalin on dendritic cells.MethodsWe used scanning probe microscope, activity assay for acid phosphatase, flow cytometry and ELIS A to study the effects on DC by MENK. Results1、MENK-treated DC displayed a more mature morphology with long protrusions; un-treated DC displayed short protrusions than the stimulated DC.2、The MENK and LPS group compared with control group, acid phosphatase activity decreased(P<0.05).3、The MENK similarly to LPS, induced up-regulation of MHC-Ⅱ、CD86 and CD40 expression.4、MENK-or LPS-stimulated DC secreted higher concentration of IL-12 than the control DC (P<0.05).Conclusion1、Suitable concentration of MENK significantly improve the morphology mature of DC.2、Suitable concentration of MENK decrease the activity of intracellular acid phosphatase, this show a low endocytic capacity, increased antigen presenting capacity and gradually matured DC.3、Suitable concentration of MENK induce up-regulation of MHC-Ⅱ、CD86 and CD40 expression of DC and stimulate DC maturation.4、Suitable concentration of MENK stimulate DC secreted higher concentration of IL-12, this indicates that MENK promote DC maturation, while up the fuction of DC.更多还原