论文摘要
1型糖尿病（Type1 diabetes mellitus,T1DM）又称为胰岛素依赖性糖尿病,是一种与机体免疫细胞平衡紊乱有关的自身免疫病。当机体因遗传变异、病毒感染等因素诱发机体免疫系统紊乱,自身反应性T细胞异常扩增,胰岛组织中出现大量淋巴单核细胞的浸润,引起分泌胰岛素的β细胞破坏,血糖持续性增高,最终导致显性糖尿病。因此,自身反应性T细胞在1型糖尿病发生、发展过程中起着重要的作用。目前仍无有效的1型糖尿病根治方法。非肥胖型糖尿病小鼠品系（non-obese diabetic,NOD）具有很多类似人类自身免疫性糖尿病的特征,能自发1型糖尿病,是当前研究1型糖尿病的主要动物模型。该品系小鼠有独特H-2g7单倍体（Kd、I-Ag7、I-Enull、Db）,通常于4～5周龄开始出现胰岛炎,进行性破坏胰岛β细胞。许多研究发现insulin B15-23肽能够刺激NOD小鼠的特异性CD8+T细胞的克隆增殖并伴有IFN-γ的产生。本课题试图制备包裹细胞毒药物阿霉素的、能特异性识别细胞毒T细胞的H-2Kd- insulin B15-23肽-阿霉素脂质体,用于1型糖尿病小鼠特异性T细胞的清除,期望为人类1型糖尿病等自身免疫性疾病的治疗提供了一个新的研究思路。一、H-2Kd和β2m蛋白的表达和纯化采用本实验室所构建的原核表达H-2Kd和β2m的两个基因工程菌株,诱导表达的H-2Kd和β2m蛋白,采用洗涤包涵体的方法初步纯化H-2Kd和β2m蛋白。经一系列纯化条件摸索后,最后使用Profinia亲和层析柱纯化大量的H-2Kd和β2m蛋白。二、H-2Kd- Insulin B15-23肽复合物的复性折叠将纯化获得的H-2Kd、β2m蛋白与insulin B15-23肽以一定的比例加入折叠缓冲液中进行稀释复性,形成H-2Kd-insulin B15-23肽复合物单体。作为进一步与载药脂质体相连的靶向弹头,为制备H-2Kd-insulin B15-23肽-阿霉素脂质体奠定基础。同时,以生物素化的H-2Kd- insulin B15-23肽复合物单体制备成四聚体,用于检验H-2Kd-insulin B15-23肽-阿霉素脂质体对特异性细胞毒T细胞的靶向性清除作用。三、阿霉素脂质体的制备及初步鉴定阿霉素具有强细胞毒性,将其包裹在脂质体内,可减少其毒副作用。以镍金属螯合磷脂（DOGS-NTA-Ni）为主要材质,采用薄膜分散法,制备阿霉素脂质体。采用建立阿霉素的标准曲线的方法来测定阿霉素的包封率,包封率为46%。使用透射电子显微镜和激光动态光散射仪分析阿霉素脂质体的理化性质,从仪器的分析结果可以看出阿霉素脂质体粒径大小均一、较为稳定。四、H- 2Kd-Insulin B15-23肽-阿霉素脂质体的制备将H-2Kd-insulin B15-23肽复合物单体和DOPC/DOGS-NTA-Ni阿霉素脂质体按浓度比2∶1等体积混合,4℃条件下孵育过夜,期望把羧基末端含有6-His-tag的H-2Kd-insulin B15-23肽复合物单体展示在阿霉素脂质体的表面。进一步采用SephadexG-100凝胶柱除去没有络合的游离蛋白。收集各组分,用Bradford法测定,发现出现两个蛋白吸收峰,对蛋白的收集组使用电镜等方法进行分析,证明H-2Kd -insulin B15-23肽复合物单体已经与脂质体相连接。H-2Kd-insulin B15-23肽-阿霉素脂质体的药理作用还有待进一步研究。
论文目录
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