摘要
外界电场的作用，可以产生细胞电通透(cell electropermeabilization)，有
利于细胞外的大分子物质进入细胞内，还可用于将药物或DNA转入活体组
织细胞中。本研究着重探讨细胞电通透在肿瘤治疗中的作用的两大方面一
抗肿瘤的电脉冲化疗和用于基因治疗的DNA电转染。电脉冲化疗
(electrochemotherapy，ECT)是抗肿瘤化学药物与电通透相结合的产物。
国外研究进入了多中心临床试验阶段，治疗浅表肿瘤获得了85．3％临床
缓解率的较好疗效。国内在本研究之前未见同类报告。需要特殊说明的是，
北京辛育龄等人所从事的应用直流电依靠电解等作用治疗肿瘤的“电化
疗”研究，其英文提法也为electrochemotherapy，但与本文所提到的
electrochemotherapy原理和方法有本质上的不同，所以我们将本文提到的
electrochemotherapy称为电脉冲化疗，以示与“电化疗”相区别。自1999
年起我们自行研制电脉冲化学治疗仪，并进行了人甲状腺癌和前列腺癌等
肿瘤的体外、体内的实验研究。DNA电转染(DNA electrotransfer)是DNA与
电通透相结合的产物。衰减波电脉冲进行的体外基因电转染早已在国外、
国内得到应用，被认为转染率低下且不能用于体内基因转染。本文所指的
DNA电转染是受到电脉冲化疗的启示后，应用方波电脉冲所进行的DNA真
核细胞转染技术。国外研究起始于5年以前，处于动物实验研究阶段，证
实方波电脉冲的DNA电转染技术具有较好的DNA转染效果，国内研究刚刚
起步。自2001年起我们将电脉冲化学治疗仪转型为多功能数控电通透仪，
开始了DNA体外、体内电转染的研究。
本文首先简要综述细胞电通透的治疗学应用及前景，然后用5个实
验，以肿瘤细胞系PC一3M为对象，分别进行了电脉冲化疗的体外、体内实
验和DNA电转染的体外、体内实验。探讨电通透的技术与抗肿瘤化学药物
的结合和与抗肿瘤DNA的结合，在肿瘤治疗中的作用及其临床应用前景。
实验一：电脉冲化疗对肿瘤细胞系PC-3M的体外作用。选用平阳霉
素作为抗癌药物与电脉冲相结合，以PC-3M为对象测定电脉冲化疗对
PC-3M细胞系的细胞毒作用。HE染色法观察上述实验后形态学改变。结
果表明，单用平阳霉素，低浓度无效，高浓度有效，无细胞毒作用的最高
浓度为O.1 u g／ml；单用电脉冲，无细胞毒作用的最大场强为1250V／cm；
0.1 u g／ml的平阳霉素与1250V／cm场强的电脉冲相结合，细胞生存分数
为52．6％，组织形态学改变明显。说明自行研制的电脉冲化学治疗仪适合
于电脉冲化疗使用。国人自主研制开发的“老药”平阳霉素可显著被电脉
冲增效，适用于电脉冲化疗。电脉冲化疗对前列腺癌细胞的生长有较强的
抑制作用。
实验二：电脉冲化疗对肿瘤细胞系裸鼠PC-3M皮下移植瘤的体内作
用。对裸鼠皮下移植瘤分4组，分别为对照组，单纯注射平阳霉素组，单
纯给予电脉冲组和平阳霉素+电脉冲组。采用的电通透参数为电场强度
l 300V／cm，脉冲时值100 u s，次数8次，频率1Hz。结果表明，平阳霉
素+电脉冲组取得了37．5％完全缓解率、62．5％部分缓解率的良好的治疗
效果，而其它各组肿瘤未受抑制。电脉冲化疗无明显局部及全身副反应，
是一种安全可靠的治疗手段。在一定参数下，平阳霉素可被电场显著增效，
起到较好的抗肿瘤效果。
实验三：方波脉冲基因电转染对肿瘤细胞系PC一3M的转染条件和效果
的确定。选用pEGFP—C1作为外源基因与方波电脉冲相结合，以PC一3M为
导入对象，探讨方波脉冲基因电转染对PC一3M转染的电脉冲电场强度，脉
冲时值，脉冲次数和反应体系大小。在电场强度600V／cm、脉冲时值20ms、
次数1次、200 u 1反应体系的条件下，基因转染效率可达35%，细胞生
存分数55％。说明上述条件是人前列腺癌细胞系转染适合条件；自行研
制的多功能数控点通透仪可用于DNA电转染。
实验四：细胞电通透技术应用到DNA肌肉转染的基因转染率和达到
高效电转染的参数条件的确定。以EGFP质粒作为报告基因，进行体内肌
肉组织电转染实验，确定适合转染的电场强度、脉冲时值和质粒剂量，通
过荧光显微镜确定转染率。质粒15 u g注射到小鼠的胫前肌，在注入后
30s给与如下参数电脉冲200V／cm，20ms，1Hz，8次。此时转染率最高，
可达73．2±7．2％。DNA注射后不会在细胞外立即降解，l小时内给予电脉
冲不影响转染率；15 u g为最适当的DNA剂量：EGFP蛋白的表达，7天达
到高峰。与单纯EGFP质粒肌肉注射相比，DNA肌肉电转染可极大提高
DNA转染率。DNA肌肉电转染在基因治疗学的研究中有应用价值。
实验五：观察体内DNA电转染对裸鼠人前列腺癌细胞系PC-3M。皮下
移植瘤的体内作用。将裸鼠皮下移植瘤分为5组：对照组，PCDNA一3质粒
肌肉内电转染组(相当于单纯给予电脉冲)，TLMP一3质粒肌肉注射组，
TIMP一3质粒肌肉内电转染组，TIMP一3质粒肿瘤内电转染组。肌肉电转染
采用的参数为电场强度200V／cm，脉冲时值20ms，8次，1Hz；肿瘤内电
转染采用的参数为电场强度600V／cm，脉冲时值20ms，1次。TIMP一3质
粒肌肉内电转染组和TIMP一3质粒肿瘤内电转染组肿瘤生长受到抑制，与
Therapeutic effect of cell electropermeabilization
on cancer cell line PC-3M
Cell electropermeabilization is a technique used in biochemical and pharmacological studies for the in vitro introduction of nonpermeant molecules into living cells. But electric pulses can be used as well in vivo for the delivery of drugs or DNA into cells of tissues. This study plays emphasis on therapeutic effects of cell electropermeabilization in vivo, in particular of the antitumour electrochemotherapy (i.e., the combination of a cytotoxic nonpermeant drug with permeabilizing electric pulses delivered to the tumours) and of in vivo DNA electrotransfer for gene therapy. Over the last decade a new cancer treatment modality, electrochemotherapy, has emerged. The cytotoxicity of bleomycin can be augmented several 100-fold by cell electropermeabilization. Drug delivery by cell electropermeabilization has been in experimental use for cancer treatment since 1991. The rates of complete response (CR) after once-only treatments were between 9 and 100% depending on the technique used. It is quite different from Dr. Xin's ECT theory, which is based on chemical changes caused by electric current. DNA electrotransfer for gene therapy is an emerging field in which results are rapidly accumulating. DNA electrotransfer mechanisms, as well as the potentialities of DNA electrotransfer to become an efficient non-viral approach for gene therapy are concerned. The electropermeabilization apparatus we made which have gained patent, can be applied both in electrochemotherapy and DNA electrotransfer. Five years past since our groups took part in the research in cell electropermeabilization' application. Now have got funds from government to support the research.
The application and therapeutic prospects were reviewed in this paper.
And five experiments were undertaken to access the efficacy of electrochemotherapy and DNA electro transfer, Which were two major parts of cell electropermeabilization. The therapeutic prospects of cell electropermeabilization were concerned and foundation works were done for future clinical trials.
Experiment 1 investigates the efficacy of electrochemotherapy with pingyangmycin (bleomycin A5)for the treatment of cancer cell line PC-3M in vitro. The cancer cells were treated by pingyangmycin alone, electric pulse alone and electrochemotherapy, i.e. electric pulses were delivered after pingyangmycin was given. Cytotoxicity was examined with MTT assay and histological examination was performed. The maximum nontoxic dose for pingyangmycin was 0.1 u g/ml; The maximum voltage not showing cytotoxicity was 1250V/cm; The 50% inhibitory concentration was 36 times reduced in electrochemotherapy than pingyangmycin alone. The apparatus we designed is fit for the research of electrochemotherapy. The cytotoxicity of pingyangmycin can be enhanced by electric impulses. Pingyangmycin maybe a novel drug for electrochemotherapy.
Experiment 2 investigates the anti-tumour effectiveness of electrochemotherapy on human cancer cell line PC-3M in vivo. The dose of intralesional pinyangmycin was based on tumor volume. This was followed by 8 100-microsec pulses of electricity at an amplitude of 1300V/cm. The antitumor effects of single electrochemotherapy treatments were investigated using tumors on nude mice. Animals were followed based on periodic tumor volume determinations. Histologic analysis was used to confirm response data. Animals were randomly assigned to one of four different treatment groups. These groups received no treatment, drug only, electric pulses only, or drug combined with electric pulses. A single electrochemotherapy treatment
protocol for small tumors resulted in a 100% response rate and a 37.5% complete response rate. In contrast, tumors in the groups that received no treatment, electric pulses only, and drug only progressed. Our data support the role of pingyangmycin as the drug of choice for electrochernotherapy. Electrochemotherapy with bleomycin may have promise for treatment o
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