摘要
目的观察E1B-55分子缺失的腺病毒(E1B-55molecule deleted adenovirus,Ad-delE1B55)在联合各种化疗药物后针对9种人食管癌细胞系所产生的协同作用。方法采用细胞的增殖检测法(MTT)法检测Ad-delE1B55和(或)4种化疗药物在9种食管癌细胞中的细胞毒性,并根据所得数据计算50%抑制浓度(IC50);使用流式细胞学方法检测表达绿色荧光蛋白基因的腺病毒(Ad-GFP)对各种细胞的感染率;细胞经过处理后,通过流式细胞学方法检测细胞的凋亡和细胞周期;用蛋白印迹western blot法检测腺病毒在细胞内产生的蛋白,以检验病毒复制与细胞毒性的关系;通过使用裸鼠的动物实验,验证体外实验的联合效应。结果聚合酶链反应(PCR)结果显示,Ad-delE1B55在所感染的细胞中能够复制,针对E1B55的PCR结果证明了其缺失。MTT结果说明Ad-delE1B55在9种食管癌细胞中均产生细胞毒性,但敏感性并不相同;通过细胞毒性结果计算了Ad-delE1B55对9种食管癌细胞株的IC50。通过Ad-GFP感染细胞,计算出病毒感染率,将其与Ad-delE1B55对细胞的IC50作对比,发现两者之间无明显相关性;将9种食管癌细胞株的p53基因型与Ad-delE1B55对细胞的IC50作对比,发现两者之间也无明显相关性。Western blot结果显示Ad-delE1B55感染细胞后48小时,其复制水平达到峰值。在讨论化疗药物与Ad-delE1B55联合给药时的顺序时,不论是western blot结果还是细胞周期结果均显示同时给药效果最好。所以,Ad-delE1B55分别与5-氟尿嘧啶(5-FU),丝裂霉素(MMC),足叶乙甙(VP-16)或顺铂(CDDP)4种化疗药物以同时给药形式联合应用于食管癌细胞株,MTT结果显示腺病毒与5-FU、MMC、VP-16联合能够产生抗肿瘤的协同作用,但与CDDP联合应用不能产生协同作用;动物实验中联合应用Ad-delE1B55与5-FU产生的抗肿瘤作用均好于单独应用任意一种,与体外实验一致,证明了抗肿瘤的协同作用。结论在食管癌细胞中腺病毒�
Objective To observe the possible combinatory antitumor effects of EIB-55 molecules deleted adenovirus （Ad-delE1B55） and chemotherapeutic agents in nine human esophageal carcinoma cells. Methods Cytotoxicity of Ad delEiB55 and/or four kinds of chemotherapeutic agents in nine kinds of esophageal cancer cells were tested with MTT assay. The ICS0 was calculated according to the MTT data; the infection rate of Ad GFP in tumor cells was tested by flow cytometry;tumor cells with or without treatment were used for examining cell apoptosis and cell cycles by flow cytometry;relationship of viral replication with cytotoxicity to cells was detected with western blot assay; the in vivo synergy of ad-delE1B55 with chemotherapy was confirmed in nude mice inoculated with tumor ceils. Results PCR result showed that Ad-delE1B55 proliferated in infected cells and EIB55 was deleted. MTT results indicated that Ad-delE1B55 produced cytotoxic effects on all the carcinoma cells,but the cytotoxicity was variable. We calculated IC50 with the cytotoxic effects and compared them with p53 status and the infectivity. The results showed that the cytotoxieity was not directly linked with the p53 status of the tumors or with the infectivity to respective tumors. Western blot results showed that Ad-delE1B55 replication reached the peak 48 hours after infection. Both western blot result and cell cycle result showed administration of 5-FU could minimally inhibit the viral replication and a simultaneous treatment with the Ad and 5-FU achieved better cytotoxicity than sequential treatments. MTT results showed a simultaneous combinatory treatment with Ad-delE1B55 and an anticancer agent, 5-FU, MMC, or VP-16, produced greater cytotoxic effects than that with monotherapy treatment. CDDP, however, did not achieve the combinatory effects in most of the cells tested. We also confirmed the antitumor effects by the combination of Ad- delE1B55 with 5-FU in vivo. Coneluslon Ad-delEIB55 produced combinatory antitumor effects with 5 FU,MMC and VP-16 but not
出处
《临床荟萃》
CAS
2014年第4期417-423,共7页
Clinical Focus
基金
河北省医学科学研究重点课题计划资助(20120125
20120128)
关键词
食管肿瘤
腺病毒科
抗肿瘤联合化疗方案
esophageal neoplasms
adenoviridae
antineoplastic combined chemotherapy protocols
