基于CXCR4启动子的中药有效成分高通量筛选细胞模型构建和应用  

Establishment and Application of High-throughput Screening Model for Active Ingredients of Traditional Chinese Medicine Based on CXCR4 Promoter

作　　者：王强利[1] 国海东[1] 王奕[1] 吴心语 赵亮[2] 王莹[3] WANG Qiang-li;GUO Hai-dong;WANG Yi;WU Xin-yu;ZHAO Liang;WANG Ying(School of Basic Medical Sciences,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Shanghai Eastern Hepatobiliary Surgery Hospital,Shanghai 200438,China;School of Pharmacy,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学基础医学院,上海201203 [2]上海东方肝胆外科医院,上海200438 [3]上海中医药大学中药学院,上海201203

出　　处：《中国中医药信息杂志》2018年第11期52-56,共5页Chinese Journal of Information on Traditional Chinese Medicine

基　　金：国家自然科学基金青年基金(81303300;81703763);国家自然科学基金面上项目(81673729);上海中医药大学预算内项目(2015YSN04)

摘　　要：目的以CXCR4基因启动子为靶点,建立促干细胞归巢药物筛选细胞模型,并对中药单体进行筛选。方法将人CXCR4基因启动子序列(279 bp)插入荧光素酶报告基因载体p GL4.17-Basic中,构建重组质粒p GL4.17-CXCR4,与内参质粒p RL-TK共转染293细胞,经验证CXCR4启动子转录活性后,单细胞克隆培养以获得稳转细胞株,并用于候选中药单体1~10的筛选,最后应用Western blot验证所筛选出的中药单体促进间充质干细胞(MSCs)中CXCR4表达的作用。结果成功建立药物筛选细胞模型,筛选出候选单体6提高CXCR4启动子活性的作用最强,Western blot结果证实候选单体6可明显促进MSCs中CXCR4的表达。结论本研究建立的药物筛选细胞模型能实现对潜在促进干细胞归巢中药有效成分的高通量筛选。Objective To establish a cell model targeting the CXCR4 gene promoter to screen of drugs potentially promoting the stem cell homing,and to screen the monomers of traditional Chinese medicine.Methods The human CXCR4 gene promoter sequence(279 bp)was inserted into the luciferase reporter vector pGL4.17-Basic to construct the recombinant plasmid pGL4.17-CXCR4,which was co-transfected with the internal reference plasmid pRL-TK into 293 cells.After verifying transcriptional activity of CXCR4 promoter,single cell clones were cultured to obtain a stable transfectant cell line and used for screening of candidate Chinese herbal monomer(1 to 10).Finally,Western blot was used to verify the effect of the selected Chinese herbal monomer on the expression of CXCR4 in MSCs.Results The drug screening cell model was successfully established and the candidate monomer 6 was screened out because of the strongest effect on enhancing CXCR4 promoter activity.Western blot results verified that the candidate monomer 6 could promoted the expression of CXCR4 of MSCs.Conclusion The drug screening cell model established in this study was able to achieve high-throughput screening for potentially promoting the stem cell homing Chinese herbal monomer.

关 键 词：心肌梗死 干细胞移植 CXCR4/SDF-1轴 荧光素酶报告基因 细胞模型 

分 类 号：R285.5[医药卫生—中药学]