基于硅玻基底材料的细胞电融合芯片的仿真及实验研究
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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。
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.
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。
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.
引文
[1]罗立新.细胞融合技术与应用[M].北京:化学工业出版社, 2003.
[2]陈光荣,肖克宇,翁波.细胞融合技术及其在生物医药中的应用[J].动物医学进展. 2004(01): 34-37.
[3]胡宁.基于SOI的细胞电融合原理性芯片的研制与细胞排队及融合研究[D].重庆:重庆大学生物医学工程专业, 2007.
[4]李志勇.细胞工程[M].北京:科学出版社, 2003: 127.
[5]李志勇.细胞工程[M].北京:科学出版社, 2003: 86-92.
[6]霍乃蕊,孟利梅.细胞融合技术的应用研究进展[J].动物医学进展. 2005(03): 184-186.
[7] Okada Y, Bikens J. The introduction of cell fusion with non-activity Xitai virus[J]. Nature, 1958, (1): 103-110.
[8] Harris H, Watts JW. Hybrid cells derived from mouse and man: artificial heterokaryons of mammalian cells from different species[J]. Nature, 1965, 205: 640-646.
[9]赵志强.一种基于MEMS和细胞电场效应的细胞融合方法的研究[D].重庆:重庆大学生物医学工程专业, 2006: 18-19.
[10] Senda M. Plant Cell Physoil [J]. Plant Cell Rep, 1979, 20(1): 441-443.
[11]汪睦和,谢廷栋.细胞电穿孔电融合电刺激原理技术及其应用[M].天津科学技术出版社, 2000.
[12] Zimmermann U, Pilwat G. The relevance of electric field induced changes in the membrane structure to basic membrane research and clinical therapeutics and diagnosis[C]. Abstract IV-19-(H) of the 6th International Biophysics Congress, Kyoto, Japan, 1978: 140.
[13]王克明.细胞融合技术及其进展[J].浙江科技学院学报. 2004, 16, 2: 114.
[14] Zimmermann U, Büchner KH, Arnold WM. Electrofusion of cells: Recent developments and relevance for evolution [M]. In Allen M J, Usherwood P N R (eds.) Charge and Field Effects in Biosystems, Abacus Press, 1984: 293-318.
[15] Zimmermann U, Vienken J, Pilwat G, et al. Electrofusion of cells: principles and potential for the future[M]. In Cell Fusion (CIBA Foundation Symposium 103), Pitman, London, 1984: 67-73.
[16] Weaver JC. Understanding conditions for which biological effects of nonionizing electromagnetic fields can be expected[J]. Bioelectrochemistry, 2002, 56(1): 207-209.
[17] Zimmermann V, Scheurich P. The cell fusion of method with electro-mechanical fusion[J]. Planta, 1981, 151: 26-32.
[18]沈羡云.神舟四号上的细胞融合[J].中国航天, 2003, 9: 10-12.
[19] ZimmermannU, Schnettler R, Hannig K. Biotechnology in space: Potentials and perspectives [J]. Biotechnology, 1988, 6: 637-672.
[20]曹毅.基于微电极阵列的高通量细胞电融合方法研究[D].重庆:重庆大学生物医学工程专业, 2009.
[21]郑慧琼,王六发,陈爱地,等.烟草细胞的空间电融合[J].科学通报, 2003, 13: 1438-1441.
[22] Wiegand R, Weber G, Zimmermann K, et al. Laser-induced fusion of mammalian-cells and plantprotoplasts[J]. J. Cell Sci., 1987, 88: 145-149.
[23] Steubing RW, Cheng S, Wright WH, et al. Laserinduced cell-fusion in combination with optical tweezers-the laser cell-fusion.trap[J]. Cytometry, 1991, 12: 505-510.
[24] Wiegand R, Weber G, Zimmermann K, et al. Laser-induced fusion of mammalian cells and plant protoplasts[J]. J. Cell Sci., 1987, 88: 145-149.
[25]张闻迪,赵百,王秋,等.激光诱导泥鳅卵细胞融合——一种新的细胞融合方法[J].生物工程学报, 1988, 4: 298-303.
[26]张闻迪,赵白,姚纪花,等.超远缘鱼类-斑马鱼和泥鳅-受精卵的激光融合[J].青岛海洋大学学报(自然科学版), 1992, 3: 11-17.
[27] Masuda S, Washizu M, Nanba T. Novel method of cell fusion in field constriction area in fluid integrated circuit[J]. IEEE Transac. Indus. Appl., 1989, 4: 732-737.
[28] Lee SW, Shim DS, Kim YK, et al. Micromachined cell fusion device[C]. The 18th Annual International Conference of the IEEE Engineering in Medicine and Biology, Amsterdam, Holland, 1996: 258-259.
[29] Wang J, Lu C. Microfluidic cell fusion under continuous direct current voltage[J]. Appl. Phys. Lett., 2006, 89: 234102.
[30] Zhao ZQ, Zheng XL, Yang J, et al. Dielectrophoretic Manipulation of Cells by Using Interdigitated Microelectrodes[C]. The 1st IEEE International Conference on Nano/Micro Engineered and Molecular Systems, Zhuhai, China, 2006: 1021-1024.
[31]胡宁,杨军,侯文生,等.基于SOI基底的高通量细胞电融合芯片[J].高等学校化学学报, 2009, 30: 42-45.
[32]曹毅,杨军,胡宁,等.一种细胞电融合芯片的研制[J].仪器仪表学报, 2009, 30(2): 298-302.
[33]胡宁,杨军,郑小林,等.基于聚酰亚胺基底的细胞电融合芯片的研制[J].分析化学, 2009, 37(28): 1247-1250.
[34] Skelley AM, Kirak O, Suh H, et al. Micro?uidic control of cell pairing and fusion [J]. Nature Methods, 2009, 6(2): 147-152.
[35]邓亲恺.现代医学仪器设计原理[M].北京:科学出版社, 2004: 17-21.
[36] Weaver JC. Electroporation of cells and tissues[J]. Plasma Sci., 2000, 28(1): 24-33.
[37]苏明皓.细胞膜在脉冲电场下的穿透机理及研究[D].天津:天津理工大学光学工程专业, 2005: 4-6.
[38]曹毅,杨军,阴正勤,等.高效细胞电融合芯片中的电场分析[J].分析化学, 2008, 36(5): 593-598.
[39]马慧,王刚. COMSOL Multiphysics基本操作指南和常见问题解答[M].北京.人民交通出版社, 2009,9: 9-12.
[40] William B, Zimmerman J. COMSOL Multiphysics有限元分法多物理场建模与分析[M].北京.人民交通出版社, 2007. 9: 75-76.
[41]徐敬波,赵玉龙,蒋庄德,等.基于SOI的集成硅微传感器芯片的制作[J].半导体学报, 2007, 28(2): 302-307.
[42]姜京伟,程阔.有限元网格划分剖析[J].山西交通科技, 2007, 5: 71-73.
[43]王明强,朱永梅,刘文欣.有限元网格划分方法应用研究[J].机械设计与制造, 2004, 1: 22-24.
[44] Zimmermann U, Pilwat G, Becker. F. et al. Effects of external electrical fields on cell membranes[J]. Bioelectrochemistry. 1976, 3: 53-58.
[45] Jacob, Forster. Microbiological implications of electric-field effects[J]. Microbiology. 1981,21. (3): 225-233.
[2]陈光荣,肖克宇,翁波.细胞融合技术及其在生物医药中的应用[J].动物医学进展. 2004(01): 34-37.
[3]胡宁.基于SOI的细胞电融合原理性芯片的研制与细胞排队及融合研究[D].重庆:重庆大学生物医学工程专业, 2007.
[4]李志勇.细胞工程[M].北京:科学出版社, 2003: 127.
[5]李志勇.细胞工程[M].北京:科学出版社, 2003: 86-92.
[6]霍乃蕊,孟利梅.细胞融合技术的应用研究进展[J].动物医学进展. 2005(03): 184-186.
[7] Okada Y, Bikens J. The introduction of cell fusion with non-activity Xitai virus[J]. Nature, 1958, (1): 103-110.
[8] Harris H, Watts JW. Hybrid cells derived from mouse and man: artificial heterokaryons of mammalian cells from different species[J]. Nature, 1965, 205: 640-646.
[9]赵志强.一种基于MEMS和细胞电场效应的细胞融合方法的研究[D].重庆:重庆大学生物医学工程专业, 2006: 18-19.
[10] Senda M. Plant Cell Physoil [J]. Plant Cell Rep, 1979, 20(1): 441-443.
[11]汪睦和,谢廷栋.细胞电穿孔电融合电刺激原理技术及其应用[M].天津科学技术出版社, 2000.
[12] Zimmermann U, Pilwat G. The relevance of electric field induced changes in the membrane structure to basic membrane research and clinical therapeutics and diagnosis[C]. Abstract IV-19-(H) of the 6th International Biophysics Congress, Kyoto, Japan, 1978: 140.
[13]王克明.细胞融合技术及其进展[J].浙江科技学院学报. 2004, 16, 2: 114.
[14] Zimmermann U, Büchner KH, Arnold WM. Electrofusion of cells: Recent developments and relevance for evolution [M]. In Allen M J, Usherwood P N R (eds.) Charge and Field Effects in Biosystems, Abacus Press, 1984: 293-318.
[15] Zimmermann U, Vienken J, Pilwat G, et al. Electrofusion of cells: principles and potential for the future[M]. In Cell Fusion (CIBA Foundation Symposium 103), Pitman, London, 1984: 67-73.
[16] Weaver JC. Understanding conditions for which biological effects of nonionizing electromagnetic fields can be expected[J]. Bioelectrochemistry, 2002, 56(1): 207-209.
[17] Zimmermann V, Scheurich P. The cell fusion of method with electro-mechanical fusion[J]. Planta, 1981, 151: 26-32.
[18]沈羡云.神舟四号上的细胞融合[J].中国航天, 2003, 9: 10-12.
[19] ZimmermannU, Schnettler R, Hannig K. Biotechnology in space: Potentials and perspectives [J]. Biotechnology, 1988, 6: 637-672.
[20]曹毅.基于微电极阵列的高通量细胞电融合方法研究[D].重庆:重庆大学生物医学工程专业, 2009.
[21]郑慧琼,王六发,陈爱地,等.烟草细胞的空间电融合[J].科学通报, 2003, 13: 1438-1441.
[22] Wiegand R, Weber G, Zimmermann K, et al. Laser-induced fusion of mammalian-cells and plantprotoplasts[J]. J. Cell Sci., 1987, 88: 145-149.
[23] Steubing RW, Cheng S, Wright WH, et al. Laserinduced cell-fusion in combination with optical tweezers-the laser cell-fusion.trap[J]. Cytometry, 1991, 12: 505-510.
[24] Wiegand R, Weber G, Zimmermann K, et al. Laser-induced fusion of mammalian cells and plant protoplasts[J]. J. Cell Sci., 1987, 88: 145-149.
[25]张闻迪,赵百,王秋,等.激光诱导泥鳅卵细胞融合——一种新的细胞融合方法[J].生物工程学报, 1988, 4: 298-303.
[26]张闻迪,赵白,姚纪花,等.超远缘鱼类-斑马鱼和泥鳅-受精卵的激光融合[J].青岛海洋大学学报(自然科学版), 1992, 3: 11-17.
[27] Masuda S, Washizu M, Nanba T. Novel method of cell fusion in field constriction area in fluid integrated circuit[J]. IEEE Transac. Indus. Appl., 1989, 4: 732-737.
[28] Lee SW, Shim DS, Kim YK, et al. Micromachined cell fusion device[C]. The 18th Annual International Conference of the IEEE Engineering in Medicine and Biology, Amsterdam, Holland, 1996: 258-259.
[29] Wang J, Lu C. Microfluidic cell fusion under continuous direct current voltage[J]. Appl. Phys. Lett., 2006, 89: 234102.
[30] Zhao ZQ, Zheng XL, Yang J, et al. Dielectrophoretic Manipulation of Cells by Using Interdigitated Microelectrodes[C]. The 1st IEEE International Conference on Nano/Micro Engineered and Molecular Systems, Zhuhai, China, 2006: 1021-1024.
[31]胡宁,杨军,侯文生,等.基于SOI基底的高通量细胞电融合芯片[J].高等学校化学学报, 2009, 30: 42-45.
[32]曹毅,杨军,胡宁,等.一种细胞电融合芯片的研制[J].仪器仪表学报, 2009, 30(2): 298-302.
[33]胡宁,杨军,郑小林,等.基于聚酰亚胺基底的细胞电融合芯片的研制[J].分析化学, 2009, 37(28): 1247-1250.
[34] Skelley AM, Kirak O, Suh H, et al. Micro?uidic control of cell pairing and fusion [J]. Nature Methods, 2009, 6(2): 147-152.
[35]邓亲恺.现代医学仪器设计原理[M].北京:科学出版社, 2004: 17-21.
[36] Weaver JC. Electroporation of cells and tissues[J]. Plasma Sci., 2000, 28(1): 24-33.
[37]苏明皓.细胞膜在脉冲电场下的穿透机理及研究[D].天津:天津理工大学光学工程专业, 2005: 4-6.
[38]曹毅,杨军,阴正勤,等.高效细胞电融合芯片中的电场分析[J].分析化学, 2008, 36(5): 593-598.
[39]马慧,王刚. COMSOL Multiphysics基本操作指南和常见问题解答[M].北京.人民交通出版社, 2009,9: 9-12.
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[41]徐敬波,赵玉龙,蒋庄德,等.基于SOI的集成硅微传感器芯片的制作[J].半导体学报, 2007, 28(2): 302-307.
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