【作者】 杨孔；

【导师】 郭聪；

【作者基本信息】 四川大学 ， 动物学， 2002， 硕士

【摘要】 细胞膜是电场作用的关键靶位点，它由液晶态和晶态两种状态的磷脂双分子层构成，具有一定的流动性和通透性。细胞膜对于一些小的分子是自由通过的，对大分子物质等则具有选择性，形成通透性屏障。细胞膜受到高强度脉冲电场处理时，其通透性障碍受到抑制，电穿孔就是形成短暂的通透性结构或膜上形成微孔。这种微孔能维持20多毫秒，最后因膜的流动性而闭合。不同的生物材料用相同的电场处理，穿孔的几率不同；同样，相同的生物材料用不同的电场处理，穿孔的几率也不同。本实验用天津理工学院生厂的基因脉冲导入仪处理Hela细胞，在不同的电场参数(电压，电容，脉冲次数)处理下，电穿孔的几率不同，从实验中找到了穿孔的最佳条件，在扫描电镜下观察到了电穿孔，电穿孔的深度与电压有关。Hela细胞受到电场处理时，表面的微绒毛受到不同程度的损伤。 利用电穿孔把抗肿瘤药物(环磷酰胺)导人Hela细胞，经台盼蓝染色和细胞流式仪测定，细胞的存活率大大降低，凋亡和死亡率大大增加，在小剂量时就能达到很大的胞质毒性。运用丫啶染色可以看到典型的凋亡细胞，核固缩、断裂。还发现，当药物达到一定的浓度时，电化学处理细胞的存活率与药物的浓度无关，即低剂量组与高剂量组的细胞存活率相等；在相同的药物浓度范围内，化疗组的细胞存活率明显与浓度有关；只用电场处理的细胞存活率与对照组相比发现，电场使细胞的存活率降低。 将电穿孔与环磷酰胺结合用于治疗KM小鼠S180肉瘤细胞异种移植肿瘤，电化学组的治愈率和有反应的比例都比只用电场和只用环磷酰胺(化疗)及对照组高。用电场处理肿瘤后，肿瘤部位不同程度的发生水肿，但随后水肿消失，表面结痂，肿瘤体积缩小直至消失。剥离未完全消退的电化学处理肿瘤与对照组肿瘤，比较发现，前者的表面几乎没有血管分布，对照组的血管非常丰富。更多还原

【Abstract】 Cell membrane is the crucial target of electromagnetic pulse field (EMPF). In is composed of bilayer phosphate and protein including liquid crystal and solid crystal,and has the nature of fluidity and permeability. The permeability barrier is withheld when the cell membrane is exposed to a short high voltage EMPF. The electroporation is the transient pore or permeable structure on the cell membrane that will be last for 20 milliseconds and will be closed at the end because of the fluidity. The electroporation ratio of the different kinds of cell exposed to the same EMPF is different,so do the same cell exposed to the different EMPF. Furthermore,the viability of cell and the kind of buffers also affect the ratio of poration. In this paper,Hela cell handled by EMPF with various voltages,times,capacity,is contrast put in with unhandled cell,the handled cell is found to have been electropored. The frequency,pores number,depth of poration are relative to the EMPF parameters. At the same time,the microvilli are damaged. Furthermore,the pore likes of volcanic vent and ejecta have been seen,then the best parameters of EMPF to Hela cell have been found. All above provide primary evidence for the electrochemotherapy of Hela cell.CTX is delivered into Hela cell by electroporation. By cell flow sorter and the stains of typannan,the life ratio is reduced very much,the apoptosis ratio and the death ratio are increase severely,and the cytotoxicity at the low pose is so tense as the high pose. By the stain of yading,the quintessential apoptosis cell with nuclear condensity and the flaw have been founded. Furthermore,the life ratio has nothing todo with the density of ami tumor drug when the density reaches somedegree.As a result of EMPF influence,the life ratio of cell handled only byEMPF is less low than the control cell’ s.The subcutaneous xenograft of slSO tumor cell on the KM rats has been used for experiments evaluating the response of tumors to various therapies. The cure ratio and the partial response ratio of the rats theraped by ECT is more high than the rats theraped by drugs only or EMPF only. The control rats have no response. Results show severe necrosis and edema in nearly all the rats treated by the drug and the pulse. The group with only drug treatment did not show any significant edema and the tumor volume of the control group was found to increase more much,but the tumor volume regressed with drug in combination with electric field.Phlyctenes were observed at the sites of electrode contact with the skin,superficial leukonecrosis of some nodules was visible within the following 3 days,and scabs were observed a few days later,and the tumor volume decrease until regression. Separating the tumor from the body of rats. The tumor of rats treated by the drug and the pulse compared to the tumor of rats treated only with the drug or the pulse has little of vasculature,and the control has quite a few of vasculatures. Also,histopathological studies indicated that all the tumor cell was necrosis and the tissue from the tumor site showed complete absence of tumor cells when the tumor volume was complete regression . No tumor reappeared within 20 days.更多还原