【作者】 夏斌；

【导师】 郑小林；

【作者基本信息】 重庆大学 ， 生物医学工程， 2010， 硕士

【摘要】 细胞融合技术也称为细胞杂交技术,是指在生物、化学、物理等外力作用下,两个或多个细胞或者原生质体紧密接触,从而发生细胞膜、细胞质和细胞核的融合并形成杂种细胞的技术。细胞融合后可获得来自亲本细胞的遗传物质,从而产生新的生物或遗传特性,可能培养成为新的物种、品系或成为新的细胞工程产品。细胞融合技术的发展先后经历了生物诱导、化学诱导和物理诱导等几个发展阶段。其中细胞电融合技术由于与传统的细胞融合技术相比,具有操作简便、效率高、对细胞不产生毒害、适于仪器应用和规范操作等优点而具有很大的发展潜力。但是,传统的细胞电融合系统依然存在一些问题,如不易观察、设备成本高等,束缚了该技术的广泛使用。为了解决这些问题,产生了一种基于芯片的细胞电融合技术。我们的课题组也开展了该类技术的研究,并已研制出了几种细胞电融合芯片,实现了在低压驱动下的细胞电融合。根据前期研究结果,为了进一步改善芯片结构,达到更好的融合效果,课题组又提出了一种基于硅玻基底材料的细胞电融合芯片,这种芯片不仅可以进一步降低驱动电压,提高融合效率,还可以在倒置荧光显微镜下使用,从而为观察荧光标记的细胞融合提供了条件。本论文首先采用有限元分析的方法分别对这种基于硅玻基底材料的细胞电融合芯片和课题组前期研制的基于SOI材料芯片的内部电场分布情况进行了仿真,并对仿真结果进行了对比。仿真结果表明,在芯片内部产生相同场强的情况下,采用基于硅玻基底材料的细胞电融合芯片可以大大的降低外部驱动电压的幅度,并且相同尺寸的微通道与电极结构设计使得芯片内部电场分布更加均匀,可以有效的提高融合效率。这为芯片的加工和封装提供了有力的理论支持。然后利用封装好的芯片搭建了细胞融合实验平台,采用HEK-293细胞和NIH-3T3细胞分别进行了同种细胞排队、融合实验研究。实验结果与仿真结果吻合,使用10V以内的驱动电压就可以使细胞发生排队和融合,这大大的小于前期芯片所需的数十伏特,并且采用均一化的电极结构,使细胞融合效率也有较大的提高。另外,论文还利用不同荧光标记的方法对HEK-293细胞和NIH3T3细胞的异源细胞排队、融合进行了探索,在荧光显微镜下直接观察到了分别标有绿色荧光和蓝色荧光的细胞之间发生了融合。对无菌条件下融合的细胞进行培养鉴定,获得了成活的杂合体细胞,更加有力的证明了该技术的优越性,也为后期进一步的体细胞与干细胞融合实验研究奠定了基础。更多还原

【Abstract】 Cell fusion technology is also called cell hybridization, in which two or more cells (protoplasts) are contacted one by one and then their membrane, cytoplasm and nuclear are fused by using some biological, chemical or physical methods. The hybrid may obtains genetic material from two or more parent cells. Thus, it achieves new biological or genetic characteristics and may be used to develop new species, strain or cell engineering products. The development of cell fusion technology has gone through three stages: biological, chemical and physical inducement, in these cell electrofusion was developed as a new physical mediated way for cell fusion. Compared to traditional cell fusion methods, cell electrofusion technology has many advantages such as easy operation, high efficiency, harmless to cells, suitable for instrumentation applications, and so on. However, there are still some problems in the traditional cell electrofusion method including difficult to observing and high voltage required, which hindered its wider use. In order to solve these problems, a new cell electrofusion technology based on chip has been set up. In our group, related study has also been carried out and several kinds of cell electrofusion chips has been developed, which could be used for cell fusion under low-voltage driven. According to the previous studies, a new chip based on silicon-glass substrates is developed in our group in order to further improve the chip architecture and achieve better fusion effect. This chip can not only reduce the drive voltage and improve fusion efficiency, but also can be used under an inverted fluorescence microscope to observe the fusion of fluorescence labeled cells.In this thesis, first the simulation of the electric field within both silicon-glass based chip and SOI based chip is be done. The simulation results show that using the silicon-glass based chip can greatly reduce driven voltage, and the same size of the micro-channel and electrodes structure design makes the electric field distribute more uniform, which can effectively improve the cell fusion efficiency. The simulation results provide a strong theoretical support to chip fabrication and packaging. Then the packaged chip is used to do some alignment and fusion experiments on HEK-293 and NIH-3T3 cells. Experimental and simulation results are matched each other, using the driven voltage under 10V could induce cell alignment and fusion, which is much lower than using previous chips, and the uniform electrodes structure also improves the fusion efficiency. Otherwise, the experiment on heterologous cell alignment and fusion of HEK-293 and NIH-3T3 is done. Cell-fusion process of different kinds of fluorescent cells is directly observed under the fluorescence microscope, and survival heterozygous cells are obtained, which validates the advantage of this new technology and lay foundation for the further fusion research of somatic cells and stem cells.更多还原