本文關(guān)鍵詞： 微針 陰道粘膜 免疫應(yīng)答 佐劑系統(tǒng) 脂質(zhì)體　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：為發(fā)展有效的粘膜疫苗,本研究采用兩種類型的多功能脂質(zhì)體,一種為粒徑為200nm的甘露糖化脂質(zhì)A-脂質(zhì)體(MLLs)和另一種粒徑為50nm的隱形脂質(zhì)A-脂質(zhì)體(SLLs),兩者均裝載有模型抗原(OVA)及碳酸氫銨,并且組合在一起構(gòu)建包含pro-SLLs/MLLs微針陣列(proSMMA)。研究表明,proSMMA再水化后迅速溶解,釋放初始MLLs和SLLs。進(jìn)一步的研究證明在陰道粘膜接種proSMMA后,再次釋放的MLLs主要被陰道粘膜抗原提呈細(xì)胞所攝取,而SLLs則直接轉(zhuǎn)運(yùn)至引流淋巴結(jié),被其中的抗原提呈細(xì)胞所攝取。此外,SLLs/MLLs能夠促進(jìn)抗原產(chǎn)生溶酶體逃逸,從而有望通過APCs(抗原提呈細(xì)胞)表面的MHC-I途徑將抗原交叉提呈至淋巴細(xì)胞。產(chǎn)生溶酶體逃逸現(xiàn)象是由于溶酶體酸化脂質(zhì)體使其釋放NH4HCO3而生成CO2和NH4+/NH3再通過氣體膨脹使得溶酶體破裂。體內(nèi)追蹤實(shí)驗(yàn)表明,SLLs將運(yùn)載疫苗傳遞至淋巴結(jié)APCs,而MLLs將運(yùn)載疫苗傳遞至接種粘膜APCs。因此,proSMMA實(shí)際上是疫苗佐劑-雙遞送系統(tǒng),有望能夠誘導(dǎo)體液和細(xì)胞免疫應(yīng)答以預(yù)防通過性傳播的病原體。本次實(shí)驗(yàn)以卵清白蛋白(Ovalbumin,OVA)為模型抗原,將OVA和碳酸氫銨共同裝封在多功能脂質(zhì)體中構(gòu)建微針疫苗佐劑-雙遞送系統(tǒng)。結(jié)合微針給藥機(jī)制,開展其作為經(jīng)粘膜途徑接種的疫苗遞送系統(tǒng)。研究脂質(zhì)體微針的制備及表征和多功能脂質(zhì)體促進(jìn)APCs對(duì)抗原的攝取,以及研究多功能脂質(zhì)體對(duì)APCs功能影響及其部分機(jī)制等。通過分析脂質(zhì)體微針經(jīng)粘膜免疫應(yīng)答特點(diǎn),初步闡明其免疫應(yīng)答效果。本研究主要分為兩個(gè)部分,下面將對(duì)此兩部分內(nèi)容進(jìn)行簡(jiǎn)述。第一部分:不同類型多功能脂質(zhì)體微針陣列的構(gòu)建及其表征。脂質(zhì)體的制備方法分很多種,我們采用薄膜分散法成功制備了兩種多功能脂質(zhì)體SLLs和MLLs,并且同微針基質(zhì)相關(guān)物質(zhì)混合通過微針模具成功制備了微針陣列。在后續(xù)實(shí)驗(yàn)中為追蹤脂質(zhì)體在體內(nèi)及細(xì)胞內(nèi)的趨向,還制備了裝載熒光染料鈣黃綠素和磺酰羅丹明B(SRB)的脂質(zhì)體。隨后通過對(duì)脂質(zhì)體粒經(jīng),zeta電位,結(jié)合效率等方面進(jìn)行檢測(cè),從而完成對(duì)脂質(zhì)體及proSMMA的表征。第二部分:不同類型多功能脂質(zhì)體微針陣列的部分免疫機(jī)制考察,其中探討了微針經(jīng)粘膜接種的主要機(jī)制,包括通過共聚焦顯微鏡觀察脂質(zhì)體被APCs攝取后的細(xì)胞內(nèi)定位,通過流式細(xì)胞術(shù)考察脂質(zhì)體刺激APCs活化成熟以及抗原提呈,再將所制備的proSMMA通過陰道粘膜途徑接種后分析倆種脂質(zhì)體的體內(nèi)趨向。
[Abstract]:For the development of effective mucosal vaccine, this research adopts the multifunctional liposome two types, a particle size of mannosylated lipid A- 200nm liposomes (MLLs) and stealth liposome lipid A- a diameter of 50nm (SLLs), both of which are loaded with antigen (OVA) and model ammonium hydrogen carbonate, and together construct containing the pro-SLLs/MLLs micro needle array (proSMMA). The results show that proSMMA after rehydration of rapid dissolution, release of initial MLLs and SLLs. further demonstrated in the vaginal mucosa after inoculation with proSMMA, the MLLs is mainly released again vaginal mucous membrane antigen presenting cell uptake, and SLLs transfer directly to the draining lymph nodes, which are antigen-presenting cell uptake. Furthermore, SLLs/MLLs can promote the antigen of lysosomal escape, which is expected to pass APCs (antigen presenting cell surface antigen) MHC-I pathway cross presentation to lymphocytes. Have lysosomal escape phenomenon is due to the release of lysosomal acidification liposome NH4HCO3 and formation of CO2 and NH4+/NH3 by gas expansion. The lysosomal rupture in vivo tracking experiments show that SLLs will launch vaccine delivery to the lymph nodes of APCs, and MLLs will launch vaccine delivery to the mucosal vaccination APCs. so that proSMMA is actually a vaccine adjuvant - delivery systems. Is expected to be able to induce humoral and cellular immune responses to prevent sexually transmitted pathogens. The experiment with egg albumin (Ovalbumin, OVA) as a model antigen, OVA and ammonium bicarbonate common sealed micro needle vaccine adjuvant - build delivery system in multifunctional liposome. Combined with micro needle mechanism. To carry out the mucosal vaccination ways as vaccine delivery system. And the characterization of the micro needle of liposome preparation and multifunctional liposome to promote the uptake of antigen APCs, and Research on multi function Liposome on APCs function and its influence mechanism. Through the analysis of the liposome micro needle through the mucosal immune response characteristics, clarify its immune response. This research is mainly divided into two parts, the following will be two parts briefly. The first part: different types of multifunctional liposome construct micro needle array and characterization. Liposome preparation methods are many points, we use two kinds of thin film multifunctional liposome SLLs and MLLs dispersion were successfully prepared, and related with the micro needle matrix material mixed by micro needle mould were successfully prepared. The micro needle array to track the trend of liposomes in vivo and in subsequent cells in the experiment, also prepared a loaded fluorescent dye calcein and sulfonyl Luo Danming B (SRB) liposome. Then based on the liposome particle, zeta potential, combined with the efficiency and other aspects of detection, thus completing the liposome and proSMMA The second part: the research of characterization. The immune mechanism of different types of multifunctional liposome micro needle array, which explores the main mechanism of the micro needle by mucosal vaccination, including through confocal microscopy by APCs liposome after ingestion of the cellular localization, through liposome stimulated APCs activation and maturation of antigen-presenting flow cells were then analysis on the trend of two kinds of liposomes in vivo prepared proSMMA inoculated by the vaginal mucosa. After the way

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R945

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