【摘要】：我們新合成了多種殼聚糖的陽(yáng)離子衍生物,并用其作為載體包裹重組非顆粒形式的乙肝HBcAg蛋白作為候選疫苗。為了評(píng)價(jià)其作為疫苗的可行性,以及誘導(dǎo)通過(guò)“內(nèi)吞體至胞漿”途徑形成交叉遞呈的潛力,我們利用一系列試驗(yàn)對(duì)候選疫苗進(jìn)行篩選,這些方法包括透射電鏡、體外包裹及釋放試驗(yàn)以及兩個(gè)可以反映對(duì)內(nèi)吞體的破壞能力的試驗(yàn):體外的溶血試驗(yàn)和與RAW264.7共孵育后進(jìn)行共聚焦顯微鏡觀察。我們利用選取的殼聚糖衍生物(它們包括Ntrimethylaminoethylmethacrylate chloride—Methylmethacrylate(TM),Chitosan—Methylmethacrylate (CM),Chitosan—Ntrimethylaminoethylmethacrylate chloride—Methylmethacrylate(CTM) and Chitosan—N-dimethylaminoethylmethacrylate hydrochloride—Methylmethacrylate(CDM))包裹重組的乙肝HBcAg蛋白對(duì)小鼠進(jìn)行免疫并且評(píng)價(jià)其免疫效果。與被接種純蛋白溶液的組相比,這些接受候選疫苗免疫的小鼠被誘導(dǎo)出很強(qiáng)的特異性體液以及細(xì)胞免疫。在這些候選疫苗之中,CTM包裹的蛋白疫苗的免疫原性是最強(qiáng)的。從體內(nèi)的免疫接種效果以及體外的一系列試驗(yàn)來(lái)看,通過(guò)納米粒的包裹,候選疫苗可以通過(guò)“內(nèi)吞體到胞漿”的途徑來(lái)引起交叉遞呈,同時(shí)也說(shuō)明了此疫苗投放系統(tǒng)是大有潛力的。 誘導(dǎo)針對(duì)乙肝病毒抗原有效的細(xì)胞以及體液免疫的疫苗策略,是治療慢性乙肝的關(guān)鍵。傳統(tǒng)的核酸疫苗具有能有誘導(dǎo)體液免疫和細(xì)胞免疫的能力,然而它的效率不是很理想。為了提高相應(yīng)核酸疫苗的效果,我們也研究了幾種新合成的殼聚糖的陽(yáng)離子衍生物作為納米顆粒疫苗佐劑的應(yīng)用情況。這些殼聚糖衍生物納米粒在生理?xiàng)l件下顯示更好的可溶性,增加了吸附親水性分子的能力。這些納米顆粒在體外可以保護(hù)DNA分子,延緩脫氧核糖核酸酶Ⅰ的分解作用。溶血試驗(yàn)和激光共聚焦顯示這些納米具有膜破壞活性因此具有潛在的加強(qiáng)基因轉(zhuǎn)染的能力。我們用這些納米粒去包裹pVAX—HBc的DNA疫苗,然后免疫C57BL/6小鼠,隔一周注射,共接
[Abstract]:We have synthesized several cationic derivatives of chitosan and used them as carriers to encapsulate non-granular hepatitis B HBcAg protein as candidate vaccine. To evaluate its feasibility as a vaccine and the potential for inducing cross-presentation through the endocytosomal to cytoplasmic pathway, we used a series of tests to screen candidate vaccines, including transmission electron microscopy. In vitro encapsulation and release tests and two tests reflecting the damage to endocytosis: hemolysis test in vitro and confocal microscopy after co-incubation with RAW264.7. The selected chitosan derivatives (including Ntrimethylaminoethylmethacrylate chloride-Methylmethacrylate (TM) Chitosan-Methylmethacrylate (CM) Chitosan-Ntrimethylaminoethylmethacrylate chloride-Methylmethacrylate (CTM) and Chitosan-N-dimethylaminoethylmethacrylate hydrochloride-Methylmethacrylate (CDM) were used to immunize mice and evaluate their immunological effects. Compared with those inoculated with pure protein solution, the mice immunized with the candidate vaccine were able to induce highly specific humoral and cellular immunity. Of these candidate vaccines, the CTM encased protein vaccine is the strongest immunogenicity. From the results of immunization in vivo and a series of experiments in vitro, through the encapsulation of nanoparticles, the candidate vaccine can cause cross-presentation through the "endocytosomal to cytoplasmic" pathway. It also shows that the vaccine delivery system has great potential. The key to the treatment of chronic hepatitis B is to induce effective cellular and humoral immunity against hepatitis B antigen. The traditional nucleic acid vaccine has the ability to induce humoral and cellular immunity, but its efficiency is not ideal. In order to improve the effect of nucleic acid vaccine, we also studied the application of several newly synthesized cationic derivatives of chitosan as adjuvant of nano-particle vaccine. These chitosan nanoparticles exhibited better solubility under physiological conditions and increased their ability to adsorb hydrophilic molecules. These nanoparticles can protect DNA molecules and delay the decomposition of deoxyribonuclease 鈪，

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