人DLL1真核表达质粒的构建及过表达DLL1抑制人口腔鳞癌细胞SCC15的增殖  

Construction of eukaryotic expression vector of human Delta-like 1 and the overexpression of DLL1 inhibites proliferation of human oral squamous cell carcinoma SCC15 cells

作　　者：粟芃芃 叶健斌 邱晓媚 胡冰心[1] 李妍[1] 宁云山[1] 

机构地区：[1]南方医科大学检验与生物技术学院生物治疗研究所,广东广州510515 [2]珠海南医大生物医药公共服务平台有限公司,广东珠海519090

出　　处：《暨南大学学报（自然科学与医学版）》2017年第3期185-191,共7页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：国家高技术研究发展技术(863计划)项目(2014AA020909);国家自然科学基金-广东省联合基金重点项目(U1401223);国家自然科学基金面上项目(81470831);广东省科技计划创新载体建设项目(2013B090800036);广东省科技计划项目(2015A040404021;2016B090919019)

摘　　要：目的:构建人全长DLL1基因真核表达质粒及过表达DLL1对人口腔鳞癌细胞增殖的影响.方法:PCR扩增人全长DLL1基因,酶切和测序鉴定后克隆至真核表达载体pCMV-Tag4并构建真核重组质粒DLL1/pC MVTag4,瞬时转染HEK293T细胞并通过实时荧光定量PCR(Q-PCR)和Western blot鉴定DLL1的表达;进一步检测DLL1在SCC15等3株人口腔鳞癌细胞中的表达;DLL1/pCMV-Tag4瞬时转染SCC15细胞,Q-PCR和Western blot检测过表达及MTT检测DLL1过表达对细胞SCC15增殖的影响.结果:真核重组质粒DLL1/pCMV-Tag4在HEK293T细胞中成功表达;DLL1在口腔鳞癌细胞中的表达高于正常口腔细胞;DLL1/pCMV-Tag4在SCC15细胞中获得表达且过表达DLL1抑制SCC15细胞的增殖.结论:人全长DLL1分子在HEK293T及SCC15细胞中获得表达,过表达DLL1抑制口腔鳞癌细胞SCC15的增殖.Aim: To construct the eukaryotic expression plasmid of human DLL1 and study the effect of DLL1 overexpression on human oral squamous cell carcinoma(OSCC) proliferation. Methods: Human full-length DLL1 gene was amplified by PCR and cloned into eukaryotic expression vector pCMV-Tag4.The recombinant DLL1/pCMV-Tag4 plasmid was verified by enzyme restriction and sequencing and then transiently transfected into HEK293 T cells. The expression of human DLL1 was detected by quantitativereal-time PCR(Q-PCR) and Western blot. The expression of DLL1 in normal oral epithelial cells and OSCC cell lines was detected by Q-PCR and Western blot. DLL1/pCMV-Tag4 was transfected into SCC15 cell and the overexpression of DLL1 was detected by Q-PCR and Western blot. Then MTT was used to measure the effect of overpression DLL1 on the cell proliferation. Results: The eukaryotic expression plasmid of human DLL1/pCMV-Tag4 was successfully constructed and expressed in HEK293 T cells. DLL1 was higher expressed in OSCC cell lines than normal oral keratinocytes cell. DLL1/pCMVTag4 was successfully overexpressed in SCC15 cell and inhibited its proliferation. Conclusion: Human DLL1 was expressed in HEK293 T and SCC15 cells. The overexpression DLL1 inhibited SCC15 cells proliferation.

关 键 词：NOTCH信号通路 人DLL1 真核表达 pCMV-Tag4 SCC15细胞 

分 类 号：Q291[生物学—细胞生物学]