微/纳秒复合脉冲电场协同诱导细胞融合的基础研究

Cell electrofusion is the most effective and promising approach in cell fusion which is the core technology of biological preparation. Currently, electrofusion is mainly induced by microsecond pulse electric field (μsPEF). Because of the sensitivity of the μsPEF to cell size, it is difficult to achieve effective electrofusion between the cells with disparate size under μsPEF, and this affects the benefit of the biological preparation seriously. However, the size of the cell has an insignificant influence on the electroporation of nanosecond pulsed electric field (nsPEF). Considering of these, an innovative idea that “electrofusion synergistically induced by micro/nano second composite PEF” is proposed, and researches of this study will focus on: the effect of the inner and outer membrane of the cell caused by combined effect of μsPEF and nsPEF, the mechanism of the cell electrofusion synergistically induced by μsPEF and nsPEF and the fundamental issues about optimization for the window parameters of the micro/nano second composite pulsed electric field. We will develop the prototype producing the micro/nano second composite PEF for electrofusion, at the same time, we will carry out the theoretical and experimental studies on the cell electrofusion induced by the micro/nano second composite PEF. The study, which has a great significance for promoting the development of electrofusion and is expected to achieve innovative theoretical achievements in the mechanism of the combined effect and the synergistic induction for electrofusion under the micro/nano second composite PEF, aims to provide an innovative and efficient physical means for cell electrofusion.

细胞融合是生物制备的核心技术，而电融合是最有效、最具前景的一种细胞融合技术。现有的电融合主要是基于微秒脉冲电场(μsPEF)的电融合技术。由于μsPEF对细胞尺寸敏感，难以实现尺寸悬殊细胞的有效融合，严重影响了生物制备的效益；而纳秒脉冲电场(nsPEF)则具有对尺寸不敏感的互补特性。为此，本项目提出“微/纳秒复合脉冲电场(μs/nsPEF)协同诱导细胞电融合”的创新思路，重点围绕(μs/nsPEF)对细胞内外膜的联合作用机理、μs/nsPEF诱导细胞融合的协同机制，以及μs/nsPEF窗口参数的优化等基础问题，结合μs/nsPEF电融合样机的开发和研制，同时展开μs/nsPEF协同诱导细胞电融合的理论和实验研究。本项目旨在为细胞电融合提供一种高效的新型物理手段，有望在微/纳秒复合脉冲电场的联合作用机制和协同诱导细胞电融合机理方面取得创新性理论成果，对于推进细胞电融合技术的发展具有重要意义。

细胞融合是生物制备的核心技术，而电融合是最有效、最具前景的一种细胞融合技术。现有的电融合主要是基于微秒脉冲电场(μsPEF)的电融合技术。由于μsPEF对细胞尺寸敏感，难以实现尺寸悬殊细胞的有效融合，严重影响了生物制备的效益；而纳秒脉冲电场(nsPEF)则具有对尺寸不敏感的互补特性。本项目围绕所提出的“微/纳秒复合脉冲电场协同诱导细胞电融合”，揭示了微/纳秒复合脉冲电场协同诱导细胞融合的机理及规律，研制出微/纳秒复合脉冲发生器，搭建细胞融合实验平台，开展细胞染色实验和杂交瘤培养实验，验证了微/纳秒复合脉冲电场对细胞融合效率提升的有效性。基于所建立的细胞融合有限元仿真模型，分别从孔密度、孔半径和跨膜电压三个维度进行了仿真分析，均证实了微/纳秒复合脉冲电场相较于微秒脉冲电场和纳秒脉冲电场所具有的优势；基于全固态开关技术，率先研制出一种电压幅值调节范围宽(0-7kV)、脉宽调节范围大(300 ns-100 μs)、智能化程度高、人机交互友好的微纳秒复合脉冲细胞融合样机；以小鼠骨髓瘤细胞和淋巴细胞为实验对象，先后通过细胞染色实验和杂交瘤培养实验，直接证实同剂量的微/纳秒复合脉冲电场相较于传统的微秒脉冲电场提高融合效率1.5-2倍；此外，还从脉冲电场极性的角度来解决细胞常规电融合技术细胞死亡率过高这一瓶颈问题，开创性地提出采用双极性脉冲电场以提高细胞融合效率的构想，并分别通过理论仿真和细胞实验予以证实。依托所提出的创新思路研制出实验样机，申请发明专利4项，已授权3项，为下一步开发具有完全自主知识产权的电融合装置奠定了基础。