| Cell electrofusion has gradually developed into the most effective and promising cell fusion technology because of its obvious advantages such as simple operation,non-toxic to cells and easy real-time observation.However,both scientific research and practical engineering applications show that there are still a series of difficult problems to be solved.Firstly,the existing cell electrofusion methods are mainly based on the electrofusion technology of cells with different sizes and radii induced by microsecond pulsed electric field or nanosecond pulsed electric field.Because microsecond pulsed electric field is sensitive to cell size,it is difficult to realize the effective fusion of cells with large size,which seriously affects the benefit of biological preparation;secondly,under the action of nanosecond pulsed electric field,the pore size is only nano scale,and the ability to maintain the pore is weak.The interaction process between membrane fusion and genetic material between cells requires that the pores on the cell membrane continue to "open" for a period of time.The pores are easy to close or disappear,which is not conducive to the effective progress of cell fusion;In order to solve the above difficult problems,from the perspective of high-voltage engineering and new technology of electrical theory,this paper proposes a new pulse form of inducing cell electrofusion-Nano / microsecond composite pulse electric field to induce cell electrofusion,so as to achieve the purpose of accurately targeting the contact area of connected cells.Therefore,this paper deeply studied the joint action mechanism of nano / microsecond composite pulsed electric field on cell inner and outer membranes Key basic problems such as the synergistic mechanism and pulse parameters of cell fusion induced by nano / microsecond composite pulsed electric field and the influence law of cell intrinsic parameters on cell fusion.The main achievements are as follows:(1)The mechanism of cell electrofusion based on electroporation effect under pulsed electric field was revealed.Based on dynamic simulation,the dynamic change process of "formation" and "development" of micropores during cell electroporation and electrofusion is explained.Through finite element simulation,the effects of pulsed electric field parameters and cell intrinsic parameters on cell fusion efficiency were explored,and the temporal and spatial distributions of transmembrane potential,pore density and pore half diameter of cell inner and outer membranes under nanosecond pulsed electric field,microsecond pulsed electric field and nano / microsecond composite pulsed electric field were compared.It is found that when the nanosecond pulsed electric field acts on the fused cells alone,a large number of "micropores" will be generated in the cell contact area.However,due to the small pore radius,the pores on the cell membrane disappear rapidly after the action of nanosecond pulsed electric field,which is difficult to create basic conditions for the effective cell fusion.When microsecond pulsed electric field acts on cell fusion alone,the density of "micropores" in the contact area of the two cells is high and the pore radius is large,while a large number of pores will be generated in other parts.Because there are too many "micropores" and large pore diameter,it is very easy to cause cell death.When using nano / microsecond composite pulsed electric field to cooperate with cell fusion,it can "accurately target perforation" in the cell contact area,and there are no pores or few pores in the rest of the cell,so as to ensure the smooth progress of cell fusion.(2)Based on power electronics technology and solid-state switching technology,a nano / microsecond composite pulse generator is designed by combining the full bridge circuit with the classical Marx circuit.The switch structure group of anti series connection is designed to build a switching module to ensure the safe and stable operation and fast switching of bipolar power supplies used in parallel at different voltage levels.The normally open normally closed series switch is used as the key component of the anti misdirect on module of the circuit,which effectively reduces the influence of the false trigger signal of the control terminal on the main circuit.Finally,a nano / microsecond composite pulse generator with wide output voltage amplitude adjustment range,wide pulse width adjustment range and adjustable polarity is developed.(3)The effects of pulsed electric field intensity,pulse width and pulse polarity on cell electroporation and electrofusion were analyzed.Through molecular dynamics simulation and cytological experiments,it is found that the pulse with high electric field intensity is conducive to "perforation" on the cell membrane,while the pulse with large pulse width is conducive to "expanding perforation",and the generation of large-size pores is more conducive to cell fusion.In the study of pulse polarity,it is found that bipolar microsecond pulsed electric field can improve the cell fusion rate and ensure low cell mortality.It is concluded that under the action of equal dose pulsed electric field,compared with the traditional unipolar microsecond pulsed electric field,bipolar microsecond pulsed electric field can significantly improve the efficiency of cell fusion.(4)The mechanism of cell fusion induced by nano / microsecond composite pulsed electric field was clarified.The ability of electroporation and the distribution area of electroporation under different combinations of pulsed electric fields were studied by using molecular probes of different sizes.It was confirmed that the synergistic pore expansion effect of nano / microsecond composite pulsed electric field and the ability of targeted perforation in the cell contact area.Cell staining experiment and hybridoma culture experiment were carried out.The results showed that compared with the microsecond pulse electric field used in conventional cell electrofusion,the nano /microsecond composite pulse electric field with equal pulse dose could significantly improve the cell fusion rate and ensure less killing effect on cells.The efficiency of cell fusion can be further improved by changing the polarity of nano / microsecond composite pulsed electric field.In addition,the conclusion also shows that the effect of nanosecond/ microsecond composite pulsed electric field on cell fusion is the synergistic effect of nanosecond pulsed electric field and microsecond pulsed electric field in the composite pulsed electric field,rather than the result of a single effect.To sum up,this paper proposes a new technology of cell fusion induced by nano /microsecond composite pulsed electric field,and carries out in-depth theoretical and Experimental Research on cell electrofusion combined with the development and development of pulse generator.Based on molecular dynamics simulation,the coarsening and all atom model of cell membrane was constructed,and the dynamic development law of cell membrane electroporation was clarified;Based on the finite element simulation,the mechanism and law of cell fusion induced by nanosecond pulsed electric field,microsecond pulsed electric field and nano / microsecond composite pulsed electric field are revealed;The nano / microsecond composite pulse generator was developed,and the bioelectricity experimental platform of cell fusion was built.The effects of pulse electric field intensity,pulse width and pulse polarity on cell electroporation and electrofusion were studied;The cell staining experiment and hybridoma culture experiment confirmed the effectiveness of nano / microsecond composite pulsed electric field on the synergistic perforation and targeted perforation of cell membrane and the improvement of cell fusion efficiency.It provides necessary theoretical basis,hardware basis and technical support for promoting the application of nano / microsecond composite pulsed electric field in cell electrofusion technology. |
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