摘要
目的:构建含人p21启动子的双荧光素酶报告载体,感染肿瘤细胞后用于检测p53激活的抗肿瘤药物筛选。方法:从人血白细胞DNA中克隆p21启动子,用于控制Firefly荧光素酶的表达;Renilla荧光素酶基因和EGFP基因与IRES相连,在CMV启动子控制下表达;将上述元件和基因克隆至载体pLL3.7中,应用酶切和测序反应验证后再转染至U251和Eca109肿瘤细胞建立细胞检测模型。结果:成功构建含人p21启动子、大小约13kb的pLL3.7-Dluc重组质粒,经LipofecttamineTM转染后用于细胞药物筛选;采用MTT方法对8种不同药物对U251和Eca109细胞处理后进行IC50检测,其中姜黄素和丹参酮ⅡA在U251肿瘤细胞中的IC50分别为20和8μg/mL,Eca109细胞的IC50分别为12和25μg/mL;药物作用转染后U251细胞48h后作荧光素酶检测,计算F/R值并SPSS 13.0分析,姜黄素和丹参酮ⅡA的P值分别为0.003和0.024,药物作用后荧光检测结果显著;该实验在Eca109细胞模型中姜黄素和丹参酮ⅡA取得了一致性的结果,P值分别为0.001和0.015。人参皂苷Rb1与阴性对照组相比差异有统计学意义,P=0.022。结论:通过p21途径检测双荧光素酶基因的表达状况,提示姜黄素、丹参酮ⅡA和人参皂苷Rb1三种药物在U251和Eca109肿瘤细胞中具有使p53活性增加的功能。
OBJECTIVE：To construct the Dual-Luciferase reporter vector with human p21 promoter to detect the re activation of p53 with traditional Chinese medicine （TCA）. METHODS： p21 promoter was amplified by using PCR from human blood genome, which could control the expression of Firefly gene. Renilla luciferase gene was controlled by the CMV promoter,and the EGFP gene connected to Renilla luciferase gene with the IRES. All the elements and genes men tioned above were cloned into the pLL3.7 vector. The recombinant vector pLL3.7 Dluc identificated by digestion and se quencing were transfected into the tumor cells of U251 and Ecal09 for constructing cell model to monitor the expression of p53. RESULTS： The dual-luciferase reporter vector with human p21 promoter gene was successfully constructed for screening anti-tumor medicine in cell model The ICs0 of 8 drugs which provided by MTT experiments were tested. The IC50values of curcumin were 20 μg/mL in U251 cells and 12 μg/mL in Ecal09 cells,respectively;The IC50 values of Tan- shinone II A were 8 μg/mL in U251 cells and 25 μg/mL in Ecal09 cells,respectively. Measurement of luciferase after 48 h and the data were analysised by SPSS 13.0 and P value was 0. 003 and 0. 002 when the cells were dealed with curcumin and with Tanshinone A respectively. The result was sinilar in Ecal09 cells（P=0. 001 ;P=0. 015）. There was a signifi cant difference with ginsenosideRbl in Ecal09 cells and the P value was 0. 022. CONCLUSION： Based on the p21 pathway and detected the expressions of dual-luciferase gene,drug screening results show that curcumin, tanshinone II A and gin- senosideRbl can strengthen the activity of p53 protein in tumor cells of U251 and Ecal09.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2013年第20期1565-1568,共4页
Chinese Journal of Cancer Prevention and Treatment
基金
教育部博士点基金(20124433120024)
广东省科技计划项目(2011B031800163)
