新型靶向性自组装膀胱内部药物传输体系的构建和作用研究

Intravesical therapy is the most important strategy to reduce the high recurrence rate of bladder cancer, and its low efficiency at present mainly is due to the lack of targeted drug delivery systems, therefore, instilled drugs can’t efficiently target and break through the blood-urine barrier to get inside the cancer cells. To develop a novel targeted intravesical drug delivery system, our study will firstly construct and express a fusion protein FimH-HGFI in Pichia pastoris by flexible molecular-designing, and this protein vector has two merits: Fim H that is a type 1 fimbrial adhesin of uropathogenic E.coli can specially recognize its receptor uroplakin Ia in the plasma membrane of bladder caner cells, HGFI that is a hydrophobin protein can self assemble into a drug delivery vector. Then we will use high-resolution confocal microscope, transmission electron microscopy, flow cytometry and quartz crystal microbalance techniques to study the properties of this protein vector to illuminate its target function, self-assembling function, principle of drug load and release and cellular delivery mechanism. Finally, we will construct the mouse model of in situ bladder cancer and automatic installation system of bladder to prove the specificity and efficacy of this fusion protein drug deliver vector. Our study will solve the fundamental problem of the inefficiency of present strategy to break through the blood-urine barrier, and provide a new idea and system for in situ treatment of bladder cancer and other related diseases of the bladder.

膀胱药物灌输术是降低膀胱癌高复发率的主要疗法，其效率不高的主要原因是目前尚缺乏靶向药物运输体系，灌输药物不能高效靶向突破血-尿屏障进入到癌细胞内部。为开发一种新型靶向性自组装膀胱内部药物传输体系，本研究首先利用致病性大肠杆菌的菌毛顶部蛋白Fim H能特异性识别膀胱上皮癌细胞膜表面斑块蛋白Ia和疏水蛋白HGFI能自组装运载药物的特性，通过分子柔性设计, 在毕赤酵母中构建表达靶向性自组装融合蛋白药物传输载体FimH-HGFI；随后将利用高分辨激光共聚焦显微镜、透射电镜、流式细胞仪和石英晶体微天平等手段明确融合蛋白载体的靶向功能、自组装功能、载药和释药规律和细胞内运输机制；最后将通过构建小鼠原位膀胱癌模型和膀胱药物自动灌输系统来验证融合蛋白药物运输体系的特异性和有效性。本研究将会解决当前药物突破血-尿屏障效率低下的重要难题，为原位治疗膀胱癌及其它膀胱相关疾病提供新的思路和体系。

膀胱药物灌输术是降低膀胱癌高复发率的主要疗法，其效率不高的主要原因是目前尚缺乏靶向药物运输体系，灌输药物不能高效靶向突破血-尿屏障进入到癌细胞内部。本项目开发了一种新型靶向性自组装膀胱内部药物传输体系。基本原理是创新性利用致病性大肠杆菌的菌毛顶部蛋白FimH能特异性识别膀胱上皮癌细胞膜表面斑块蛋白Ia和疏水蛋白HGFI能自组装运载药物的特性，利用基因工程手段构建表达靶向性自组装融合蛋白药物传输载体FimH-HFBI。本项目在执行过程中，成功地开发了可表达融合蛋白的大肠杆菌表达系统。并利用建立的细胞实验和动物模型证明了构建表达的双功能融合蛋白载体针对膀胱癌细胞具有良好的靶向性特点，同时也具有可携带抗肿瘤药物的能力，是一种新型的靶向性药物传输载体。本研究成果解决当前药物突破血-尿屏障效率低下的重要难题，为原位治疗膀胱癌及其它膀胱相关疾病提供新的思路和体系。同时本课题还拓展了研究内容，开发了一种可特异性检测多种肿瘤细胞的多功能蛋白探针，证明了本项目研究成果的可拓展性。