细胞膜在脉冲电场下的穿透机理及研究
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摘要
在过去的20年,电穿孔技术的机理和应用受到了很大的关注。电场对细胞的作用能被看成是:将细胞看成被电介质(细胞膜)包围的导体(细胞质),当把电场加到细胞上,这造成电量在细胞膜上的聚集,最后造成膜上产生电压。如果膜上的电压超出临界值,表面膜的结构会产生变化,导致孔洞的产生,这个过程就是电穿孔。
随着体外电穿孔实验被应用于转染细胞,现在已经形成了许多应用分类。从本质上说,任何大小的分子都可以被导入细胞的细胞质中或被传送透过细胞膜。在活的有机体内的电穿孔应用包括将抗癌药物传输至实体瘤,抗癌药物本身是有力的但相对于膜却又是不可渗透的。电化学疗法(ECT)现在已经进行到了临床试验阶段。有机体内的试验表明了ECT可比被动渗透传输更多的抗癌药物至细胞内部。由一定的药物加上电脉冲治疗可以组成新型抗癌方法。
本文首先介绍了细胞电穿孔的机理、发展过程及其应用。
然后结合对细胞膜电穿孔的认识,介绍了课题组研制的基因导入仪的原理及其研制过程。重点介绍了该仪器的系统组成包括高压脉冲产生电路、闸流管触发电路、信号处理电路、组合电容等,随后介绍仪器的性能指标、外观及输出波形并对实验中的温度测量问题提出解决方案。
接着,对细胞膜电穿孔的理论模型进行了分析。通过对细胞电穿孔的数学及物理模型进行系统研究,为以后的实验提供理论依据。同时分析了各电场参数与电穿孔效率的关系,电场各参数包括脉冲幅度、脉冲宽度、脉冲个数、脉冲波形等。
最后,我们研究并报道了电脉冲对治疗肿瘤的作用,在实验装置、实验方法等方面进行了有益的改进,并取得了良好的实验结果,研究结果得到同行专家关注与认可。同时,为进一步的动物实验研究以及临床肿瘤治疗的研究提供了可供参考的方法和数据。我们发现:通过动物实验,在电脉冲和抗癌药物的共同作用下,接种在动物身上的肿瘤能够得到有效的抑制或治愈,可见治疗效果是很明显的。
The fundamental effects of an electric field on a cell can be described by considering the cell to be a conductive body (the cytoplasm) surrounded by a dielectric layer (the cell membrane).When a short electric pulse is applied to this cell, the resulting current causes accumulation of electrical charges at the cell membrane and consequently a voltage across the membrane. If the membrane voltage exceeds a critical value, structural changes in the surface membrane occur with transmembrane pore formation, a process known as electroporation. The applications and mechanistic understanding have received growing attention over the past two decades.
Several classes of applications have been identified, with in vitro electroporation widely used to transfect suspended or anchored cells in laboratory apparatus by introducing DNA. Essentially, any size molecule can be introduced into the cell cytoplasm or transported across a membrane. In vivo applications include the local delivery of potent but relatively membrane impermeable anticancer drugs into solid tumors. Electrochemotherapy (ECT) has now reached clinical trials. Motivating in vivo experiments showed that orders of magnitude more anticancer drugs are delivered into cytoplasm than by passive permeation. New anticancer treatment, which consisted of established drugs with added electric pulse, would in principle be attractive.
Firstly, the thesis introduces the research on the mechanism and development of electroporation and its applications.
Secondly, it introduces the gene pulser transfection apparatus which was designed by our group. We make a point of the principle and specifications of this apparatus. It includes the high-voltage generator, thyristor trigger circuit, single processing circuit and capacitance of combination. Because of the temperature influence, we must examine the temperature in the experiment. We propose optical fiber sensor in the apparatus.
Thirdly, theoretic model of the electroporation is analyzed. With the research on the mathematic and physical model, the research is referenced by the experiment. And the relationship between the effect of electroporation and the electric parameter is introduced.
At last, we reported a new approach for the treatment of sarcoma, Electroporation Therapy (EPT). Based on the experiment of Sichuan University, we improved the method of the electroporation and the experiment got good results. At the same time, the medical expert recognized the result. From the experiment, we discovered the approach could obtain remarkable effect.
随着体外电穿孔实验被应用于转染细胞,现在已经形成了许多应用分类。从本质上说,任何大小的分子都可以被导入细胞的细胞质中或被传送透过细胞膜。在活的有机体内的电穿孔应用包括将抗癌药物传输至实体瘤,抗癌药物本身是有力的但相对于膜却又是不可渗透的。电化学疗法(ECT)现在已经进行到了临床试验阶段。有机体内的试验表明了ECT可比被动渗透传输更多的抗癌药物至细胞内部。由一定的药物加上电脉冲治疗可以组成新型抗癌方法。
本文首先介绍了细胞电穿孔的机理、发展过程及其应用。
然后结合对细胞膜电穿孔的认识,介绍了课题组研制的基因导入仪的原理及其研制过程。重点介绍了该仪器的系统组成包括高压脉冲产生电路、闸流管触发电路、信号处理电路、组合电容等,随后介绍仪器的性能指标、外观及输出波形并对实验中的温度测量问题提出解决方案。
接着,对细胞膜电穿孔的理论模型进行了分析。通过对细胞电穿孔的数学及物理模型进行系统研究,为以后的实验提供理论依据。同时分析了各电场参数与电穿孔效率的关系,电场各参数包括脉冲幅度、脉冲宽度、脉冲个数、脉冲波形等。
最后,我们研究并报道了电脉冲对治疗肿瘤的作用,在实验装置、实验方法等方面进行了有益的改进,并取得了良好的实验结果,研究结果得到同行专家关注与认可。同时,为进一步的动物实验研究以及临床肿瘤治疗的研究提供了可供参考的方法和数据。我们发现:通过动物实验,在电脉冲和抗癌药物的共同作用下,接种在动物身上的肿瘤能够得到有效的抑制或治愈,可见治疗效果是很明显的。
The fundamental effects of an electric field on a cell can be described by considering the cell to be a conductive body (the cytoplasm) surrounded by a dielectric layer (the cell membrane).When a short electric pulse is applied to this cell, the resulting current causes accumulation of electrical charges at the cell membrane and consequently a voltage across the membrane. If the membrane voltage exceeds a critical value, structural changes in the surface membrane occur with transmembrane pore formation, a process known as electroporation. The applications and mechanistic understanding have received growing attention over the past two decades.
Several classes of applications have been identified, with in vitro electroporation widely used to transfect suspended or anchored cells in laboratory apparatus by introducing DNA. Essentially, any size molecule can be introduced into the cell cytoplasm or transported across a membrane. In vivo applications include the local delivery of potent but relatively membrane impermeable anticancer drugs into solid tumors. Electrochemotherapy (ECT) has now reached clinical trials. Motivating in vivo experiments showed that orders of magnitude more anticancer drugs are delivered into cytoplasm than by passive permeation. New anticancer treatment, which consisted of established drugs with added electric pulse, would in principle be attractive.
Firstly, the thesis introduces the research on the mechanism and development of electroporation and its applications.
Secondly, it introduces the gene pulser transfection apparatus which was designed by our group. We make a point of the principle and specifications of this apparatus. It includes the high-voltage generator, thyristor trigger circuit, single processing circuit and capacitance of combination. Because of the temperature influence, we must examine the temperature in the experiment. We propose optical fiber sensor in the apparatus.
Thirdly, theoretic model of the electroporation is analyzed. With the research on the mathematic and physical model, the research is referenced by the experiment. And the relationship between the effect of electroporation and the electric parameter is introduced.
At last, we reported a new approach for the treatment of sarcoma, Electroporation Therapy (EPT). Based on the experiment of Sichuan University, we improved the method of the electroporation and the experiment got good results. At the same time, the medical expert recognized the result. From the experiment, we discovered the approach could obtain remarkable effect.
引文
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[3] 黄卡玛,李颖等.近年来弱电磁场(波)生物效应机理研究进展[J].中国医学物理学报, 2000,17(1):36-40
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[6] 王保义,刘长军,郭庆功等.电穿孔—解释生物体非热效应的关键机理[J].四川大学学报(自然科学版)1997,34(3):310
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[9] S.J.Singer and G.L.Nicolson, The fluid mosaic model of the structure of cell membranes, Science,1972,175:720-731
[10] 熊兰,孙才新等.细胞膜电穿孔的机理及应用前景的初步探讨[J].重庆大学学报.2000,23(4):76-79
[11] J.C.Weaver, Electroporation of cells and tissues, IEEE Trans. Plasma Sci., 2000 Vol.28.No1:24-33
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