同时下调巨噬细胞和树突状细胞CD40表达水平诱导移植免疫耐受的纳米药物递送系统研究

发布时间：2018-02-26 18:38

本文关键词： 纳米颗粒移植免疫 CD40 RNA干扰免疫调节　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景与目的:近年来,纳米技术发展迅速,纳米颗粒在医学、生物学中的应用越来越广泛,在多种疾病的诊断与治疗中发挥了非常重要的作用。纳米药物可以通过将药物分子吸附在颗粒表面或者包裹在其中,实现高效携带递送药物分子,包括多肽、蛋白质、核酸、小分子药物等。CD40是肿瘤坏死因子超家族成员之一,主要表达于抗原提呈细胞。CD40/CD40L通路是T淋巴细胞活化中一条重要的共刺激途径。组织器官移植是器官衰竭终末期病人最主要的治疗方式。但是因移植免疫排斥所导致的移植组织、器官失败与副作用始终困扰着患者。目前,慢性移植免疫排斥是诱导长期移植免疫耐受需要解决的最主要的难题。RNA干扰是一种生物体内通过RNA降解靶基因mRNA,特异性沉默特定基因表达的生物学现象。然而siRNA药物具有在体内生理环境中不稳定和难以主动跨越细胞膜等障碍,因此siRNA药物的体内治疗应用需要一种稳定的体内给药系统。本研究目的是开发一种新型siRNA体内给药系统,在体外与体内水平降低树突状细胞与巨噬细胞CD40的表达水平,延长同种异型皮肤移植物的存活时间。方法:本研究首先通过双乳化法制备获得包载siRNA的PEG5k-PLGA10k纳米颗粒,应用粒度仪对纳米颗粒的粒径与表面电势进行表征,应用冷冻透射电镜观察纳米颗粒的形貌。将PLGA/Cy5-siRNA NPs、PLGA/FAM-siRNA NPs与DC1.2、RAW264.7细胞共同培养,随后应用流式细胞术与激光共聚焦显微镜进行检测。将PLGA/siCD40 NPs与DC1.2、RAW264.7细胞共同培养,应用q PCR与流式细胞术检测CD40的表达。C57BL/6小鼠通过尾静脉注射PLGA/Cy5-siRNA NPs,16 h后处死,分离获得外周血单个核细胞、脾细胞、骨髓细胞,应用流式细胞术检测PLGA/Cy5-siRNA NPs在小鼠体内的巨噬细胞和DC中分布情况。C57BL/6小鼠在尾静脉注射PLGA/siCD40 NPs后处死,分离骨髓细胞体外诱导获得骨髓来源树突状细胞,加入LPS刺激,应用流式细胞术检测LPS刺激活化骨髓来源树突状细胞的相关细胞标记。应用Balb/c向C57BL/6小鼠同种异型皮肤移植模型验证PLGA/siCD40 NPs的生物学效应。结果:1.双乳化法成功制备获得的PLGA/si CD40 NPs粒径为103 nm左右,表面电位为27.5 m V左右,在水溶液中呈较规整球形结构。2.PEG5k-PLGA10k纳米颗粒能够在体外向DC1.2、RAW264.7细胞质内递送siRNA。3.PLGA/si CD40 NPs能够降低DC1.2、RAW264.7细胞CD40的表达。4.PLGA/Cy5-siRNA NPs可以在体内向树突状细胞和巨噬细胞递送siRNA。5.PLGA/si CD40 NPs能够抑制LPS刺激活化骨髓诱导树突状细胞。6.PLGA/si CD40 NPs能够延长小鼠同种异型皮肤移植物的存活时间。结论:1.通过双乳化法成功制备获得了PEG5k-PLGA10k纳米颗粒。2.PEG5k-PLGA10k纳米颗粒可以在体外和体内水平将siRNA递送至细胞内。3.PEG5k-PLGA10k纳米颗粒可以在体外和体内水平将CD40 siRNA递送至细胞内,显著降低CD40的表达。4.PLGA/si CD40 NPs可以显著延长Balb/c小鼠皮肤移植物在C57BL/6小鼠同种异型皮肤移植模型中的存活时间。
[Abstract]:Background and purpose: in recent years, the rapid development of nanotechnology, nano particles in medicine, biology is applied more and more widely, play a very important role in the diagnosis and treatment of various diseases. Nano drug by drug molecules adsorbed on the particle surface or in one package, to achieve efficient delivery of carrying drug molecules, including peptide, protein, nucleic acid, small molecule drugs such as.CD40 tumor necrosis factor superfamily, is mainly expressed on antigen-presenting cells.CD40/CD40L T cell activation pathway is an important pathway. Organ transplantation for patients with end-stage organ failure is the main treatment. But due to graft rejection the transplanted tissue, organ failure and side effects has plagued the patients. At present, the immune rejection is induced by long-term chronic allograft tolerance need to be solved The main problem of.RNA interference is a kind of organism by RNA degradation of target gene mRNA, biological phenomenon of specific silencing of specific gene expression. However, with the in vivo physiological environment is not stable and it is difficult to take the initiative across the cell membrane barriers such as siRNA drugs, so siRNA drug body treatment application needs a stable in vivo system. The purpose of this study is to develop a new type of siRNA in drug delivery system, decreased the expression of dendritic cells and macrophages with CD40 in vitro and in vivo, prolong skin allograft survival time of graft. Methods: This study firstly by double emulsion prepared PEG5k-PLGA10k nanoparticles encapsulated siRNA, application of particle size analyzer the particle size and surface potential were characterized by morphology observation of nanoparticles by freezing TEM. PLGA/Cy5-siRNA NPs, PLGA/FAM-siRNA NPs and DC 1.2, co cultured RAW264.7 cells were detected by flow cytometry and confocal laser microscopy. The application of PLGA/siCD40 NPs and then DC1.2, co cultured RAW264.7 cells, using Q PCR and flow cytometry to detect the expression of CD40 in.C57BL/6 mice by intravenous injection of PLGA/ Cy5-siRNA NPs, 16 h were isolated from peripheral blood. Mononuclear cells, spleen cells and bone marrow cells of.C57BL/6 mice were distributed in NPs after intravenous injection of PLGA/siCD40 in mice and macrophages in DC using flow cytometry to detect PLGA/Cy5-siRNA NPs from bone marrow cells in vitro, induced by dendritic cells derived from bone marrow cells, stimulated with LPS, detection of LPS related cell activation marker of bone marrow stimulation dendritic cells by flow cytometry. The application of Balb/c to the biological effect of C57BL/6 mice model of allogeneic skin transplantation to verify the PLGA/siCD40 NPs. Results: the preparation of 1. double emulsion method was PLGA/si CD40 NPs particle size was about 103 nm, the surface potential is 27.5 m V, in aqueous solution had a regular spherical structure of.2.PEG5k-PLGA10k nanoparticles to DC1.2 in vitro, RAW264.7 siRNA.3.PLGA/si CD40 NPs in the cytoplasm of delivery can reduce DC1.2, RAW264.7 cells and the expression of CD40.4.PLGA/Cy5-siRNA NPs siRNA.5.PLGA/si CD40 NPs in vivo delivery to dendritic cells and macrophages can inhibit LPS stimulated activation of bone marrow dendritic cells induced by.6.PLGA/si CD40 NPs could prolong skin allograft survival time of grafts. Conclusion: 1. by a double emulsion method is successfully prepared by PEG5k-PLGA10k.2.PEG5k-PLGA10k nanoparticles in in vitro and in vivo delivery of siRNA to the intracellular.3.PEG5k-PLGA10k nano particles in vitro and in vivo level will be CD 40 siRNA delivered to cells significantly reduced the expression of CD40..4.PLGA/si CD40 NPs significantly prolonged the survival time of skin graft of Balb/c mice in allogeneic skin transplantation model of C57BL/6 mice.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392.4

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