增强型胸苷激酶基因表达载体的构建及其杀伤性的研究进展  

Construction of enhanced thymidine kinase gene expression vector and its experimental study on killing efficiency

作　　者：宋秋灵[1] 

机构地区：[1]山东省菏泽市立医院耳鼻喉科,山东菏泽274000

出　　处：《中国医学工程》2017年第10期14-18,共5页China Medical Engineering

摘　　要：目的探讨人端粒酶催化亚单位(h TERT)启动子及巨细胞病毒原核(CMV)增强子联合调控胸苷激酶基因增强型表达载体的改良构建及其在杀伤鼻咽癌细胞中的效应与安全性评价。方法对链接h TERT启动子及CMV增强子、胸苷激酶基因进行酶切(模板为p GL3空载体),以构建增强型表达载体;选取h TERT启动子单调控胸苷激酶基因的表达载体作为对照组。另选用人鼻咽癌细胞(端粒酶阳性,实验组)、人乳腺癌细胞(对照组)及正常人脐静脉内皮细胞(端粒酶阴性)为标本,分别对其采用荧光显微镜观察荧光蛋白表达、聚合酶链式反应(PCR)检测胸苷激酶基因m RNA的表达、Telochaser法检测细胞端粒酶活性,同时分析鼻咽癌细胞增殖及抑制受增强型载体的影响作用。结果 h TERT启动子及CMV增强子对胸苷激酶基因的增强型表达载体进行联合调控,可以成功改良构建;通过荧光显微镜下观察显示人鼻咽癌细胞和人乳腺癌细胞均有荧光表达,但后者较前者在表达数量及强度方面有所加强,且对照组的人乳腺癌细胞亦有荧光表达存在,但ECV-304细胞无荧光表达。在PCR定量检测方面,结果显示增强型载体组(实验组)的胸苷激酶基因m RNA的表达量显著强于对照组的单启动载体(P<0.05)。在细胞端粒酶活性方面,ECV-304细胞为阴性,肿瘤细胞端粒酶活性均为阳性。四甲基偶氮唑蓝(MMT)检测结果显示,增强型载体组可明显抑制鼻咽癌细胞的体外增殖,其细胞存活率较对照组显著降低(P<0.05)。动物体内实验结果显示改良重建的增强型载体对裸鼠鼻咽癌移植瘤生长具有明显抑制作用(抑瘤率为56.5%),显著高于单启动子载体组(抑瘤率为43.3%)(P<0.05);实验组与其他对照组抑瘤率比较差异均有统计学意义(P<0.05)。实验组裸鼠肝肾病理检查显示,肝、肾组织结构正常,均无明显损害。结论以h TERT启动子及CMV增强子对胸苷激酶基因的�【Objective】To investigate the improved construction of enhanced expression vector of h TERT promoter combined with cytomegalovirus（CMV） enhancer for regulating thymidine kinase gene, and the effect and safety evaluation of killing nasopharyngeal carcinoma cells.【Methods】Link h TERT promoter, CMV enhancer and thymidine kinase gene were digested（template for p GL3 empty vector） to construct enhanced expression vector; the expression vector of thymidine kinase gene controlled by h TERT promoter was selected as the control group. Human nasopharyngeal carcinoma cells（telomerase positive, research group）, human breast cancer cells（control group） and normal human umbilical vein endothelial cells（telomerase negative） were selected as samples, fluorescence microscopy was used to observe the expression of fluorescent protein, the expression of thymidine kinase gene m RNA was detected by polymerase chain reaction（PCR）, and telomerase activity was detected by Telochaser assay, at the same time, the effect of enhanced vector on the proliferation and inhibition of nasopharyngeal carcinoma cells was analyzed.【Results】h TERT promoter and CMV enhancer for regulating the expression vector of thymidine kinase gene could be successfully constructed; human nasopharyngeal carcinoma cells and breast cancer cells had fluorescence expression under fluorescence microscopy, and the latter was strengthened in expression number and expression intensity, there was fluorescence expression in human breast cancer cells in the control group, but no fluorescence expression in ECV-304 cells. In the quantitative detection of PCR, the results showed that the expression of thymidine kinase gene m RNA in the enhanced vector group was significantly stronger than that in the control group, and the difference was statistically significant（P〈0.05）. In the telomerase activity of cells, ECV-304 cells were negative, and tumor cells were positive; the results of MMT detection showed that the proliferation of nasopharyn

关 键 词：鼻咽癌 胸苷激酶基因 端粒酶 巨细胞病毒原核 

分 类 号：R739[医药卫生—肿瘤]