[1]胡冰心,叶健斌,邱晓媚,等.过表达DLL3促进人胃癌细胞的增殖[J].南方医科大学学报,2018,(01):14.
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过表达DLL3促进人胃癌细胞的增殖()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2018年01期
页码:
14
栏目:
出版日期:
2018-01-20

文章信息/Info

Title:
Over-expression of human Notch ligand Delta-like 3 promotes proliferation of human gastric cancer cells in vitro
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
关键词:
人DLL3真核表达胃癌细胞增殖
Keywords:
human Delta-like ligand 3 eukaryotic expression gastric cancer cell line proliferation
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DLL3 siRNA 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mRNA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。
Abstract:
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 HEK293T cells, in which the expressions of human DLL3 mRNA 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 qRT-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-siRNA 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 HEK293T 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.

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更新日期/Last Update: 1900-01-01