【作者】 孙敬儒；

【导师】 郭聪；

【作者基本信息】 四川大学 ， 动物学， 2002， 硕士

【摘要】 电穿孔是指在电脉冲作用下，细胞膜脂双层上形成瞬时微孔，使细胞膜的通透性和膜电导瞬时增大，亲水分子、病毒颗粒、DNA、蛋白质以及染料颗粒等在正常情况下不能通过细胞膜的分子得以进出细胞的一个生物物理过程。 目前，许多科技工作者利用电穿孔的这一特性，将之与现代医学相结合，开展了癌症治疗方面的新探索。 基于电穿孔在癌症治疗方面的潜力，我们课题组利用基因脉冲导入仪产生的强电脉冲与一些抗癌药物相结合，进行了一系列实验，所取得的一系列结果为使用电穿孔治疗癌症的可行性提供了依据。 论文共分四部分。第一部分研究了强电脉冲可导致离体鸡血细胞产生电穿孔，获得了最直接的形态学证据，并对电穿孔产生的机理作了初步探讨。第二部分研究了电穿孔提高抗癌药物细胞毒性的实验，利用了MTT法、染色体畸变和微核等手段，结果证明在电穿孔作用下，抗癌药物的细胞毒性得到显著提高。第三部分初步研究了强电脉冲对HeLa细胞生长的影响，通过荧光染色、DNA Ladder、流式细胞术等方法，我们研究发现强电脉冲可诱导HeLa细胞发生凋亡。第四部分是动物肿瘤模型的建立，选用昆明小鼠、S-180肉瘤细胞为研究对象，选用环磷酰胺作抗癌药物，实验结果表明，电场+药物组的治疗效果最佳，与对照组相比，呈显著差异。根据这四部分的实验结果我们可以知道，强电脉冲作用于癌组织时，在癌细胞膜上形成电穿孔，使抗癌药物可以有效地进入细胞，发挥最大效用。同时电脉冲诱导了细胞凋亡，使细胞进入程序化细胞死亡。另外，癌组织在形成过程中，会产生大量的微血管以便从血液中摄取营养，供其高速增长的需要，而电场的作用可以抑制微血管的形成，也就减少了对癌细胞的营养供应，降低了其新陈代谢速度，也就抑制了癌组织的生长。 目前利用强电脉冲致细胞膜电穿孔特性的主要趋势是：根据电穿孔时细胞膜天然屏障功能的减弱或丧失，导致离子和溶水性分子极易透过膜的特性，向细胞内导入微粒，其应 四J；】大学硕士学位论文 川领域包括以卜L个方面：电转染、电插入、电融合、电导入、建立基冈库、电穿孔促进 钓物的运送、电穿孔促进皮肤的药物传递等。 随着时间的推移，对电穿孔技术的研究必将日益）’泛而深入，可以相信，强电脉冲结 合抗癌药物的研究和应用在我国将得到长足发展。更多还原

【Abstract】 Electroporation is believed to be the transient creation of micropores in lipid bilayer of cell membrane due to the effects of electric pulses. It is a biophysics process,which can increase the permeation and conductance of cell membrane. This increase allows many molecules,such as aqueous affinity molecules,virosomes,DNA,proteins and dye granules,etc,which can not pass through cell membrane in normal conditions,to go through cell membrane.Recently,many scientists have taken advantage of characteristics of electroporation,combined with morden medicine,to develop new anticancer methods.Because of the promising application of electroporation in curing cancer,our research program has used the high-intensity electric pulses produced by LN-101 Gene Pulser Transfection Apparatus,combined with several anticancer drugs in a series of experiments. The results showed curing cancer by electroporation was a feasible way.My thesis includes four sections. In the first section,that electroporation can be induced on chick’s erythrocytes by high-intensity electric pulses was investigated. The most direct proof of morphology was acquired. The mechanism of electroporation has been preliminarily studied. In the second section,that electroporation can enhance the cytotoxicity of some anticancer drugs was researched. The methods,such as MTT colorimetry,chromosome aberration and micronucleus have been used in this section. The results implied that electroporation can increase the cytotoxicity remarkably. In the third section,the effects of high-intensity electric pulses on the growth of HeLa cells were studied. The results showed that the high-intensity electric pulses can induce apoptosis in HeLa cells. The following experimental methods wereused in this section:fluorescence staining,DNA Ladder assay,flow cytometry,and so on. In the fourth section,an animal tumor model has been constructed. KM mouse and S-180 sarcoma cell have been chosen as researching targets and the cyclophosphamide was chosen as the anticancer drug. The results showed that the experimental group of electric field combined with drugs has the best effect on cancer,compared with the control group.From the above research,we know that electroporation can be induced in cancer cell membrane by the effects of high-intensity electric pulses,which can make anticancer drugs pass through cancer cell membrane and take effect to the largest degree.. At the same time,high-intensity electric pulses can induce apoptosis,making cancer cell go into the programmed cell death. In addition,electric field can inhibit the formation of vas capillaries,which were largely produced during the formation of cancer tissues to take nutrition from blood for those tissues. As a result,high-intensity electric pulses can decrease the supply of nutrition for cancer cells,and slow the speed of metabolism of them. In conclusion,electric field has inhibited the growth of cancer.At present,the tendency of exploting the electroporation lies in electrotransfection,electroinsertion,electrofusion,electrotransformation and constructing genebank,and so on.All of these applications base on the fact that the membrane permeability barrier can be temporarily suppressed when cells are subjected to transient high-intensity pulsed electric fields,so particles can be transferred into cells to take advantage of the traits of the free exchange of molecules between the cell interior and exterior.As time going on,the study on electroporation will be more and more expanding and profound. We believe the research and application of high-intensity electric pulses combined with anticancer drugs will have a full development.更多还原