乏氧条件启动HRE调控HSV-TK／GCV效应链对人肺腺癌A549细胞杀伤作用的观察  

Hypoxic condition switching on the killing effect of HRE regulated HSV-TK/GCV system on human lung adenocarcinoma A549 cells

作　　者：司英健[1] 光丽霞[1] 袁发焕[2] 张克斌[1] 

机构地区：[1]第三军医大学新桥医院实验技术中心,重庆400037 [2]第三军医大学新桥医院肾内科,重庆400037

出　　处：《中华肿瘤防治杂志》2008年第4期248-251,共4页Chinese Journal of Cancer Prevention and Treatment

摘　　要：目的:观察由缺氧反应元件(HRE)调控的单纯疱疹病毒胸苷激酶/丙氧鸟苷(HSV-TK/GCV)系统对乏氧环境中的人肺腺癌细胞系A549细胞的杀伤作用。方法:将HRE调控HSV-TK和增强型绿色荧光蛋白(EGFP)双基因共表达的真核载体pHRE-TK通过脂质体导入A549细胞,分别在乏氧(O2浓度为3%)和常氧(O2浓度为21%)环境中培养,荧光显微镜观察重组载体转染A549细胞后报告基因EGFP表达变化,并进行荧光光密度(FOD)分析;再予以GCV处理5d后,用MTT法检测GCV对培养细胞的杀伤效果。设立分别仅含有HRE、HSV-TK以及两者均无的3个荧光表达载体(pHRE、pTK、pEGFP)为对照组。结果:在乏氧环境中,由HRE调控的重组载体pHRE-TK和pHRE转染A549细胞后,其FOD值高于无HRE调控的载体pTK和pEGFP转染组P<0.01;而在常氧环境中各组细胞之间FOD值差异无统计学意义,P>0.05。携带HSV-TK基因的重组载体pHRE-TK和pTK转染A549细胞后,细胞对GCV的敏感性均高于无HSV-TK基因的pHRE组细胞,P<0.01;敏感性随GCV浓度增加而增大。在乏氧环境中培养的pHRE-TK组细胞对GCV的敏感性高于pTK组(P<0.01),亦高于在常氧环境中培养的pHRE-TK组细胞,P<0.01;而在常氧环境中,pHRE-TK组与pTK组细胞对GCV的敏感性差异无统计学意义,P>0.05。结论:在乏氧的A549细胞内,HRE使报告基因EG-FP表达增强;HRE能增强HSV-TK/GCV系统对乏氧环境中的A549细胞的杀伤效率。OBJECTIVE： To observe the killing effect of hypoxia response element （HRE） regulated herpes simplex virus-thymidine kinase/gancyclovir（HSV-TK/GCV） system on human lung adenocarcinoma A549 cells under hypoxia condition. METHODS： A549 cells were transfected by recombinant fluorescent expression vectors pHRE-TK, pHRE, pTK and pEGFP, respectively, which contained HRE and HSV-TK gene, HRE gene, HSV-TK gene, and single EGFP gene, respectively. Then, the cells of these four groups were exposed to normoxia （21% oxygen） or hypoxia （3% oxygen） condition. The expression of enhanced green fluorescent protein （EGFP） was monitored by fluorescent microscopy. The sensitivity of A549 cells to the anti-tumour drug gancyclovir （GCV） was determined with the method of tetrazolium （MTT） after treated with GCV for 5 days. RESULTS： The EGFP fluorescence optical density （FOD） value of pHRE and pHRE-TK group cells exposed to hypoxia was significantly higher than that of pTK and pEGFP group cells, P〈0.01 ; There was no difference among the cells of the four groups when exposed to normoxia, P〉0.05. The pTK and pHRE TK group cells exposed to hypoxia was more sensitive to GCV than pHRE group cells, and the higher the GCV concentration, the more the difference. The pHRE-TK and pTK group cells showed almost the same sensitivity to GCV under the normoxia condition, P〈0.05, but the pHRE-TK group cells under hypoxia condition was much more sensitive to GCV than pTK group cells under the same condition and pHRE-TK group cells un der the normoxia condition, P〈 0. 01. CONCLUSIONS： HRE could up-regulate expression of EGFP in A549 cells under hypoxia condition. HRE could enhance the killing effect of HSV-TK/GCV system on A549 cells under hypoxia condition.

关 键 词：肺肿瘤 腺癌 缺氧 反应条件 单纯疱疹病毒属 胸苷激酶 更昔洛韦 

分 类 号：R734.2[医药卫生—肿瘤]