四川大学学报（自然科学版）
四川大學學報（自然科學版）
사천대학학보（자연과학판）
JOURNAL OF SICHUAN UNIVERSITY（NATURAL SCIENCE EDITION）
2013年
4期
849-855
,共7页
胡琳珊%张海波%童英%张渝君
鬍琳珊%張海波%童英%張渝君
호림산%장해파%동영%장투군
p53靶向药物%报告基因系统%药物筛选%细胞模型
p53靶嚮藥物%報告基因繫統%藥物篩選%細胞模型
p53파향약물%보고기인계통%약물사선%세포모형
p53-targeted drug%reporter cell line%drug screening%shRNA
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-O S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RN A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 。
為瞭篩選可以恢複腫瘤細胞中p53功能的小分子，作者用錶達野生型p53的人類直腸癌細胞HCT116建立瞭一株能夠應答激活p53信號通路的熒光素酶報告基因的穩定細胞繫，同時用錶達野生型p53的人類骨肉瘤細胞U2-O S建立瞭一株能夠應答激活p53信號通路的mCherry紅色熒光蛋白報告基因的穩定細胞繫。為瞭檢測篩選p53靶嚮藥物的有效性，利用三種已知的以p53為靶點的小分子藥物（cisplatin ，doxorubicin以及Nutlin-3）處理這兩種穩定細胞繫，結果顯示 p53信號通路在這兩箇穩定細胞繫中均能夠被激活。為瞭探索小分子RN A作為恢複p53功能的靶標藥物，併進一步驗證這兩種細胞模型用于藥物篩選的可行性，分彆檢測瞭MDM2和MDMX的5箇不同shRNA 。通過比較HCT116穩定細胞的熒光素酶活性和U2-OS穩定細胞中熒光蛋白的熒光彊度，我們篩選齣瞭有效沉默MDM2或MDMX的shRNA 。數據錶明，這兩種細胞模型不僅可用作篩選激活p53的小分子化閤物的平檯，而且可用于篩選激活p53信號通路的小分子RNA 。
위료사선가이회복종류세포중p53공능적소분자，작자용표체야생형p53적인류직장암세포HCT116건립료일주능구응답격활p53신호통로적형광소매보고기인적은정세포계，동시용표체야생형p53적인류골육류세포U2-O S건립료일주능구응답격활p53신호통로적mCherry홍색형광단백보고기인적은정세포계。위료검측사선p53파향약물적유효성，이용삼충이지적이p53위파점적소분자약물（cisplatin ，doxorubicin이급Nutlin-3）처리저량충은정세포계，결과현시 p53신호통로재저량개은정세포계중균능구피격활。위료탐색소분자RN A작위회복p53공능적파표약물，병진일보험증저량충세포모형용우약물사선적가행성，분별검측료MDM2화MDMX적5개불동shRNA 。통과비교HCT116은정세포적형광소매활성화U2-OS은정세포중형광단백적형광강도，아문사선출료유효침묵MDM2혹MDMX적shRNA 。수거표명，저량충세포모형불부가용작사선격활p53적소분자화합물적평태，이차가용우사선격활p53신호통로적소분자RNA 。
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 luciferase activity from HCT 116 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 .
