發(fā)布時(shí)間：2018-02-26 18:38

  本文關(guān)鍵詞： 納米顆粒 移植免疫 CD40 RNA干擾 免疫調(diào)節(jié)　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景與目的:近年來,納米技術(shù)發(fā)展迅速,納米顆粒在醫(yī)學(xué)、生物學(xué)中的應(yīng)用越來越廣泛,在多種疾病的診斷與治療中發(fā)揮了非常重要的作用。納米藥物可以通過將藥物分子吸附在顆粒表面或者包裹在其中,實(shí)現(xiàn)高效攜帶遞送藥物分子,包括多肽、蛋白質(zhì)、核酸、小分子藥物等。CD40是腫瘤壞死因子超家族成員之一,主要表達(dá)于抗原提呈細(xì)胞。CD40/CD40L通路是T淋巴細(xì)胞活化中一條重要的共刺激途徑。組織器官移植是器官衰竭終末期病人最主要的治療方式。但是因移植免疫排斥所導(dǎo)致的移植組織、器官失敗與副作用始終困擾著患者。目前,慢性移植免疫排斥是誘導(dǎo)長(zhǎng)期移植免疫耐受需要解決的最主要的難題。RNA干擾是一種生物體內(nèi)通過RNA降解靶基因mRNA,特異性沉默特定基因表達(dá)的生物學(xué)現(xiàn)象。然而siRNA藥物具有在體內(nèi)生理環(huán)境中不穩(wěn)定和難以主動(dòng)跨越細(xì)胞膜等障礙,因此siRNA藥物的體內(nèi)治療應(yīng)用需要一種穩(wěn)定的體內(nèi)給藥系統(tǒng)。本研究目的是開發(fā)一種新型siRNA體內(nèi)給藥系統(tǒng),在體外與體內(nèi)水平降低樹突狀細(xì)胞與巨噬細(xì)胞CD40的表達(dá)水平,延長(zhǎng)同種異型皮膚移植物的存活時(shí)間。方法:本研究首先通過雙乳化法制備獲得包載siRNA的PEG5k-PLGA10k納米顆粒,應(yīng)用粒度儀對(duì)納米顆粒的粒徑與表面電勢(shì)進(jìn)行表征,應(yīng)用冷凍透射電鏡觀察納米顆粒的形貌。將PLGA/Cy5-siRNA NPs、PLGA/FAM-siRNA NPs與DC1.2、RAW264.7細(xì)胞共同培養(yǎng),隨后應(yīng)用流式細(xì)胞術(shù)與激光共聚焦顯微鏡進(jìn)行檢測(cè)。將PLGA/siCD40 NPs與DC1.2、RAW264.7細(xì)胞共同培養(yǎng),應(yīng)用q PCR與流式細(xì)胞術(shù)檢測(cè)CD40的表達(dá)。C57BL/6小鼠通過尾靜脈注射PLGA/Cy5-siRNA NPs,16 h后處死,分離獲得外周血單個(gè)核細(xì)胞、脾細(xì)胞、骨髓細(xì)胞,應(yīng)用流式細(xì)胞術(shù)檢測(cè)PLGA/Cy5-siRNA NPs在小鼠體內(nèi)的巨噬細(xì)胞和DC中分布情況。C57BL/6小鼠在尾靜脈注射PLGA/siCD40 NPs后處死,分離骨髓細(xì)胞體外誘導(dǎo)獲得骨髓來源樹突狀細(xì)胞,加入LPS刺激,應(yīng)用流式細(xì)胞術(shù)檢測(cè)LPS刺激活化骨髓來源樹突狀細(xì)胞的相關(guān)細(xì)胞標(biāo)記。應(yīng)用Balb/c向C57BL/6小鼠同種異型皮膚移植模型驗(yàn)證PLGA/siCD40 NPs的生物學(xué)效應(yīng)。結(jié)果:1.雙乳化法成功制備獲得的PLGA/si CD40 NPs粒徑為103 nm左右,表面電位為27.5 m V左右,在水溶液中呈較規(guī)整球形結(jié)構(gòu)。2.PEG5k-PLGA10k納米顆粒能夠在體外向DC1.2、RAW264.7細(xì)胞質(zhì)內(nèi)遞送siRNA。3.PLGA/si CD40 NPs能夠降低DC1.2、RAW264.7細(xì)胞CD40的表達(dá)。4.PLGA/Cy5-siRNA NPs可以在體內(nèi)向樹突狀細(xì)胞和巨噬細(xì)胞遞送siRNA。5.PLGA/si CD40 NPs能夠抑制LPS刺激活化骨髓誘導(dǎo)樹突狀細(xì)胞。6.PLGA/si CD40 NPs能夠延長(zhǎng)小鼠同種異型皮膚移植物的存活時(shí)間。結(jié)論:1.通過雙乳化法成功制備獲得了PEG5k-PLGA10k納米顆粒。2.PEG5k-PLGA10k納米顆�？梢栽隗w外和體內(nèi)水平將siRNA遞送至細(xì)胞內(nèi)。3.PEG5k-PLGA10k納米顆�？梢栽隗w外和體內(nèi)水平將CD40 siRNA遞送至細(xì)胞內(nèi),顯著降低CD40的表達(dá)。4.PLGA/si CD40 NPs可以顯著延長(zhǎng)Balb/c小鼠皮膚移植物在C57BL/6小鼠同種異型皮膚移植模型中的存活時(shí)間。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392.4

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