C-fos启动子在人胶质瘤细胞株及正常组织的转录活性研究  

Transcriptional activity of C-fos promoter in human glioma cell lines

作　　者：王浩[1] 潘建青[1] 胡继良 冯诣 宋伟健[1] 罗杰[1] 刘欣民[1] 魏强国[1] 洪全球[1] 翟宝进[1] 

机构地区：[1]深圳市南山区人民医院神经外科,518052 [2]深圳市人民医院神经外科,518020

出　　处：《中华神经医学杂志》2015年第7期669-673,共5页Chinese Journal of Neuromedicine

摘　　要：目的观察C-fos、TERT、Survivin、E2F1、Cox2及CMV启动子在人胶质瘤细胞株U87、U373、U251中的转录活性以及C-fos、CMV启动子在正常组织中的转录活性。方法设计引物并应用PCR法从人胶质瘤基因组中克隆C-fos、TERT、Survivin、E2F1、Cox2启动子。将上述5种启动子以及CMV启动子序列插入到pGL4-Basic报告载体中。将构建的pGL4-Cfos、pGL4．TERT、pGL4-Survivin、pGL4．E2F1、pGL4．Cox2、pGL4-CMV重组质粒和内参质粒pRL—TK瞬时共转染胶质瘤细胞U87、U373、U251，双荧光素酶实验检测各启动子的转录活性；C57BL／6J小鼠10只，分为C-fos组、CMV组，每组5只，分别由尾静脉注射600μL含40μg pGL4-Cfos-luc、pGL4．CMV-luc质粒的生理盐水，24h后检测小鼠心、肝、脾、肺组织中荧光素酶的活性。结果各启动子基因序列经测序确认正确；TERT、Survivin、Cox2、E2F1、C-los启动子的转录活性（相对于CMV启动子1在U87细胞中依次为0．03％、0.33％、0．38％、0．24％、3．73％；在U373细胞中依次为0．95％、65．34％、0．32％、9．07％、70．83％，在U251细胞中依次为0．41％、15．57％、0．15％、0．51％、17．30％；CMV组小鼠心、肝、脾、肺组织荧光素酶活性依次为（93．40±75．06）RLU／mg蛋白、（12．33±6．49）RLU／mg蛋白、（22．60±7．50）RLU／mg蛋白、（950．47±538．74）RLU／mg蛋白；C—los组小鼠心、肝、脾、肺组织荧光素酶活性依次为（2．13±1．92）RLU／mg蛋白、（1．11±0．95）RLU／mg蛋白、（2．07±1．51）RLU／mg蛋白、（29．24±25．89）RLU／mg蛋白，与CMV组比较差异均有统计学意义（P〈0．05）。结论C-fos启动子在人胶质瘤细胞株中的转录活性高于TERT、Survivin、E2F1、Cox2，在正常组织中的转录活性低于CMV启动子。Objective To compare the transcriptional activities of C-fos, TERT, Survivin, E2F1, Cox2 and CMV promoters in human glioma lines U87, U373 and U251, and compare the transcriptional activities of C-los and CMV promoter in major tissues. Methods Genomic DNA of U87 cells was used as the template, human C-los, TERT, Survivin, E2F1 and Cox2 promoter segments were amplified by PCR and cloned into pGL-Basic vectors. The recombinant vectors pGL4-Cfos, pGL4-TERT, pGIA-Survivin, pGL4-E2F1, pGL4-Cox2 and pGL4-CMV were co-transfected respectively with pRL-TK into U87, U373 and U251 cells. The transcriptional activities were analyzed by Dual-Luciferase method. By tail vein injection pGL4-Cfos or pGL4-CMV vectors in C57BL/6J mice, the tissue luciferase activities in the heart, liver, spleen, lung tissues were analyzed. Results Five promoter fragments were successfully cloned and their recombinant vectors were successfully constructed by restriction enzyme digestion and sequencing. The transcriptional activities of TERT, Survivin, Cox2, E2F1 and C-los promoters （as compared with those of CMV promoter） were 0.03%, 0.33%, 0.38%, 0.24% and 3.73% in U87 cells, 0.95%, 65.34%, 0.32%, 9.07% and 70.83% in U373 cells, and 0.41%, 15.57%, 0.15%, 0.51% and 17.30% in U251 cells. The luciferase activities in heart, liver, spleen, lung tissues of pGL4-CMV vectors mice were （93.40±75.06） RLU/mg protein, （12.33±6.49） RLU/mg protein, （22.60±7.50） RLU/mg protein and （950.47±538.74） RLU/mg protein, those in pGL4-Cfos mice were （2.13±1.92） RLU/mg protein, （1.11±0.95） RLU/mg protein, （2.07±1.51） RLU/mg protein and （29.24±25.89） RLU/mg protein; the differences between them were significant （P〈0.05）. Conclusion The C-fos promoter has higher transcriptional activity in glioma cell lines than TERT, Survivin, E2F1 and Cox2 promoters; but it has much low transcriptional activity in normal tissues than that in CMV promoter.

关 键 词：神经胶质瘤 转录活性 靶向治疗 C-fos启动子 

分 类 号：R739.41[医药卫生—肿瘤]