单细胞实时荧光成像监测稳态声空化介导细胞内吞递送基因的实验研究

The delivery of naked gene is not efficient enough because it is hard to transfer over membranes and easy to be degraded in cells. Cellular endocytosis wraps up genes and directs them with endosomes. It is a safe, efficient way to mediate gene delivery. We found in our previous study that ultrasound combining with microbubbles could enhance the expression and gathering of clathrin and increase the endosomes inside the cell membrane. Inertial cavitation produced ‘sonoporation’ can hardly explain the phenomena. Stable cavitation enhances membrane permeability by pushing and plugging. Therefore, we hypothesize that stable cavitation stimulates cellular endocytosis and can mediate gene delivery. This study will develop the research as follows. Firstly, the mechanism in stimulating cellular endocytosis will be explored. The equipment producing stable acoustic cavitation will be set up. The sing-cell real-time fluorescence imaging of endosomes will be carried out using ORF gene clone technique to mark specific endocytosis protein with fluorescence. The endosomes in cells will be observed by transmission electron microscopy. Secondly, the mechanism will be explored in enhanced gene delivery mediated by cellular endocytosis stimulated by stable acoustic cavitation. The mechanism will be studied by co-location of naked gene and endocytosis pathway under confocal fluorescence microscope using marking with fluorescence in live cells. The effect of endocytosis will be verified by specific endocytosis inhibited tests. This study will contribute to a new gene delivery system.

裸基因不易穿越细胞内膜性结构、易被降解，致递送效率低。细胞内吞可通过囊泡包裹裸基因并定向转运，是安全、高效的基因递送方式。前期研究发现，超声辐照微泡介导基因转染时，可增强网格蛋白介导的内吞活动，使细胞内侧面内吞泡数目明显增多。致“声孔效应”的瞬态空化难以解释这种现象。稳态空化通过推、拉细胞膜增加细胞膜通透性。因此，我们假设稳态声空化刺激细胞内吞，并可介导基因递送。本课题拟通过建立稳态声空化系统，采用ORF（开放阅读框）基因克隆荧光标记内吞特异性蛋白进行单细胞实时荧光成像，实时动态直接观察细胞内空载内吞泡的形成和转归；通过活细胞荧光标记技术，共聚焦显微镜下活细胞内目的基因与内吞通路荧光共定位的方法，确定内吞泡包裹目的基因，监测载基因内吞泡的走向，并通过基因转染效率测定和内吞抑制实验，观察稳态声空化介导基因递送的生物学效应及对细胞内吞的依赖性，为新型基因递送系统的研发奠定实验基础。

超声联合微泡促进细胞摄取的效果确实，但超声联合微泡促进细胞摄取的机制还不明确。基于前期研究，我们认为超声联合微泡有可能刺激了细胞的内吞活动，这与已经证实的“声孔效应”不一致。本研究中，我们假设刺激细胞内吞的声空化不是致“声孔”的瞬态空化，而是相对柔和的稳态空化，即稳态空化刺激了细胞内吞，进而促进细胞摄取。研究开展过程中，我们成功建立了分别用于群体细胞研究和单细胞实时观察的稳态声空化体系，并以实时荧光成像的方式显示，稳态空化条件下细胞内吞活动增强。由于腺相关病毒为载体的基因递送依赖于细胞内吞，我们研究超声辐照微泡对腺相关病毒为载体的基因转染的促进作用，结果发现，超声辐照微泡不仅促进以腺相关病毒为载体的基因转染，而且在基因转染过程中，通过透射电镜观察和荧光共聚焦显微镜观察细胞内吞行为，发现超声辐照微泡确实增加细胞内吞行为的活跃程度。以上两方面的研究结果证明稳态空化刺激细胞内吞进而促进细胞摄取的假设成立。本研究稳态声空化体系的建立有助于此空化条件下细胞各种行为的进一步研究，稳态声空化增强细胞内吞活动的研究结果有助于形成安全、有效的促进细胞对基因和药物摄取的新的给药方式。