摘要
目的构建人全长DLL3的真核表达质粒,并分析上调和下调DLL3对人胃癌细胞增殖的影响。方法采用PCR扩增人全长DLL3基因并克隆至真核表达载体pCMV-Tag4中,通过酶切及测序鉴定后,瞬时转染HEK293T细胞,并以转染pCMV-Tag4质粒和无转染HEK293T细胞分别为阴性对照和空白对照,实时荧光定量PCR(qRT-PCR)和Western blot鉴定人全长DLL3基因的表达;进一步通过qRT-PCR和Western blot检测人DLL3在人正常口腔上皮细胞GES-1和AGS等3株胃癌细胞中的表达差异;当人DLL3/pCMV-Tag4瞬时转染3株胃癌细胞后,利用MTT检测DLL3过表达后胃癌细胞的增殖;同时,特异性人DLL3si RNA转染人胃癌细胞MGC803和MKN45,利用MTT检测DLL3下调后胃癌细胞的增殖。结果成功构建了人全长DLL3/pCMV-Tag4重组质粒,转染HEK293T细胞24 h后,qRT-PCR和Western blot表明DLL3在m RNA和蛋白水平上的表达显著高于对照组;MTT细胞增殖实验显示:过表达DLL3促进胃癌细胞的增殖,下调DLL3后抑制胃癌细胞的增殖。结论成功构建了人全长DLL3/pCMV-Tag4真核表达质粒并在HEK293T细胞中获得表达,过表达DLL3促进胃癌细胞的增殖,下调DLL3抑制胃癌细胞的增殖,本研究为以DLL3为新靶点对胃癌进行靶向治疗提供新思路。
Objective To construct a eukaryotic expression plasmid carrying human full-length Notch ligand Delta-like 3(DLL3)gene and study the effect of DLL3 knockdown and overexpression on the proliferation of gastric cancer cells in vitro. Methods Human full-length DLL3 gene was amplified by PCR and cloned into the eukaryotic expression vector pCMV-Tag4. After verification by restriction enzymes and sequencing, the recombinant DLL3/pCMV-Tag4 vector was transiently transfected into HEK293 T cells, in which the expressions of human DLL3 m RNA and protein were detected using real-time quantitative PCR and Western blotting, respectively. The expression of DLL3 in normal gastric epithelial cells and gastric cancer cell lines was detected by q RT-PCR and Western blotting. DLL3/pCMV-Tag4 was transfected into 3 gastric cancer cell lines, and their proliferation was assessed with MTT assay. Human gastric cancer cells MGC803 and MKN45 were also transfected with a specific human DLL3-si RNA to assess the effect of DLL3 down-expression on the cell proliferation. Results The recombinant eukaryotic expression vector DLL3/pCMV-Tag4 was successfully constructed and human full-length DLL3 was expressed in HEK293 T cells. MTT assay showed that DLL3 over-expression obviously promoted the proliferation and down-regulation of DLL3 inhibited the proliferation of the gastric cancer cells. Conclusion DLL3 overexpression can promote the proliferation of gastric cancer cells in vitro, and down-regulation of DLL3 inhibits the proliferation of gastrc cancer cells,which provides a novel strategy for targeted thrapy of gastric cancer.
作者
胡冰心
叶健斌
邱晓媚
林彦青
吴丹琳
温俊杰
罗美群
宁立军
李妍
宁云山
HU Bingxin1, YE Jianbin1, QIU Xiaomei2, LIN Yanqing1, WU Danlin1, WEN Junjie1, LUO Meiqun1, NING Lijun1, LI Yan1, NING Yunshan1(1.Biotherapy Institute, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou 510515, China; 2Zhuhai SMU Biomedicine Public Service Platform, Zhuhai 519090, Chin)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2018年第1期14-19,共6页
Journal of Southern Medical University
基金
国家自然科学基金(81470831)
国家自然科学基金-联合项目(U1401223)
广东省科技计划项目(2013B090800036
2015A040404021
2016B090919019)
广东省大学生创新创业项目(201512121122)
广东省大学生创新训练项目(201612121100
201612121243
201612121034)~~
关键词
人DLL3
真核表达
胃癌细胞
增殖
human Delta-like ligand 3
eukaryotic expression
gastric cancer cell line
proliferation