筛选p53靶向药物的细胞模型的构建  

Establishing reporter Cell lines as a model system for p53-targeted drug screening

作　　者：胡琳珊[1] 张海波[1] 童英[1] 张渝君[1] 

机构地区：[1]四川大学生命科学学院生长代谢衰老研究中心,成都610064

出　　处：《四川大学学报（自然科学版）》2013年第4期849-855,共7页Journal of Sichuan University(Natural Science Edition)

基　　金：国家自然科学基金(31170729)

摘　　要：为了筛选可以恢复肿瘤细胞中p53功能的小分子,作者用表达野生型p53的人类直肠癌细胞HCT116建立了一株能够应答激活p53信号通路的荧光素酶报告基因的稳定细胞系,同时用表达野生型p53的人类骨肉瘤细胞U2-OS建立了一株能够应答激活p53信号通路的mCherry红色荧光蛋白报告基因的稳定细胞系.为了检测筛选p53靶向药物的有效性,利用三种已知的以p53为靶点的小分子药物(cisplatin,doxorubicin以及Nutlin-3)处理这两种稳定细胞系,结果显示p53信号通路在这两个稳定细胞系中均能够被激活.为了探索小分子RNA作为恢复p53功能的靶标药物,并进一步验证这两种细胞模型用于药物筛选的可行性,分别检测了MDM2和MDMX的5个不同shRNA.通过比较HCT116稳定细胞的荧光素酶活性和U2-OS稳定细胞中荧光蛋白的荧光强度,我们筛选出了有效沉默MDM2或MDMX的shRNA.数据表明,这两种细胞模型不仅可用作筛选激活p53的小分子化合物的平台,而且可用于筛选激活p53信号通路的小分子RNA.To screen for small molecules that could restore the functions of p53 in tumor cells, a HCT116 reporter cell line （derived from human colorectal carcinoma carrying the wild type p53 gene） stably expressing luciferase under the control of promoter that containing a p53-responsive element, and a U2-OS reporter cell line （derived from human osteosarcoma carrying the wild type p53 gene） stably expressing mCherry red fluorescent protein under the control of promoter that also containing a p53-responsive element have been established. To validate these two stable reporter cell lines for p53-targeted drug screening, three known p53-targeted small molecules, Cisplatin, Doxorubicin and Nutlin-3 have been used to examine these cells, the results shown that p53 signal pathway was able to be activated in both stable reporter cell lines. To exploring small RNA molecules as targeted drug for restoring p53 functions and to further examine the usability of these two cell lines, five different shRNAs of human MDM2 and MDMX have been expressed in these cell lines, respectively. By comparing the lueiferase activity from HCT116 reporter cells and the fluorescent intensity from U2-OS reporter cells, the effective vs. non-effective shRNAs of MDM2 or MDMX were able to be distinguished. Thus, these newly established cell lines could be used as platforms for screening both p53-targeted small molecules and p53-targeted small RNA molecules.

关 键 词：p53靶向药物 报告基因系统 药物筛选 细胞模型 

分 类 号：Q78[生物学—分子生物学]