脂质体介导的光基因高效转染的微流控方法研究

Blind-causing diseases such as retinal degeneration may bring serious influence on the quality of life, as well as enormous social and economic problems. However, there is no efficient treatment method. On the degenerated retina, optogenetic transfection method can enable cells express light-sensitive proteins and reconstruct the photoelectric conversion capabilities of retina. Thus, this method has enormous potential in the application of the treatment of retinal degeneration. However, existing transfection methods in the body have many problems such as a poor targeting, a low transfecion ratio, a bad transformation effect, and a considerable side effect, which hinder the wide application of these methods.. This project proposes a new strategy for the treatment of retinal degeneration. Precise and controllable transfection of embryonic stem cells outside of the body will be used to overcome the difficulty of the optogenetic transfecion in the body. Transfected stem cells can reconstruct the photoreception capability of retina. Microfluidic technology will be used as the main manipulation method, and giant lipid vesicles are used as optogentic carriers. Accurate pairing and fusion of vesicles and cells are used to realize high-efficiency optogenetic transfecion of embryonic stem cells. Controllable and optogenetic embryonic stem cells are analyzed, evaluated and screened, which will be transplanted to retina. Outside special photostimulation will be used to control the differentiation of stem cells and the expression of action potential, which can further repair and reconstruct the photoreception capability. Thus, the exploration of this new method has important significance in basic research and clinical application.

视网膜变性疾病等致盲性眼病严重影响人类生存质量，带来巨大社会和经济问题却缺乏有效治疗手段。在变性视网膜上通过光基因转染使细胞表达外源性光敏感蛋白，可重建其光电转换能力。该方法具有治疗视网膜变性疾病的巨大应用潜力。但是，现有体内细胞转染方法存在靶向性差、转染率低、转化效果差、副作用大等问题，严重阻碍其广泛应用。. 本课题提出一种新的视网膜变性疾病治疗策略，以体外精确可控的胚胎干细胞转染来克服体内光基因转染面临的难题，移植转染后干细胞可重建视网膜光感受能力。将微流控技术作为主要操作手段，巨型脂质体作为光基因载体，通过脂质体-细胞准确配对融合，实现光基因向胚胎干细胞准确高效导入；分析、评价并筛选导入可控光敏感基因的胚胎干细胞并用于视网膜移植；借助外界特定光刺激以控制干细胞的分化及分化细胞的动作电位表达，进而达到修复或重建光感受能力的目的。该方法的探索具有重要基础研究和临床应用意义。

视网膜变性疾病等致盲性眼病严重影响人类生存质量，带来巨大社会和经济问题却缺乏有效治疗手段。在变性视网膜上通过光基因转染使细胞表达外源性光敏感蛋白，可重建其光电转换能力。该方法具有治疗视网膜变性疾病的巨大应用潜力。但是，现有体内细胞转染方法存在靶向性差、转染率低、转化效果差、副作用大等问题，严重阻碍其广泛应用。.通过两年的研究工作，我们完成了光敏感基因ChR2质粒载体的构建。在脂质体包载芯片及包载方法研究基础上，实现了质粒载体的有效包载，并得到了大量尺寸均一的包载脂质体。研制出微流控融合芯片，通过高通量配对及融合操作实现了大量包载脂质体与干细胞的高效融合。利用培养细胞，测试分析了转染ChR2细胞的动作电位表达情况，证实了转染细胞具有光刺激下的动作电位，并研究了培养细胞的一些生物学特性。研究工作实现了预期目标，为后期基因向视网膜的导入研究奠定了坚实的基础。