Notch1-NICD胞内结构域真核表达载体的构建及其初步功能分析

Construction of eukaryotic expression vectors of Notch1-NICD and its preliminary functional study in esophageal squamous cell carcinoma cells

目的:构建包含Notch1胞内结构域的Notch1-NICD真核表达载体及绿色荧光蛋白载体并转染食管鳞状细胞癌细胞株EC9706及Eca109,并对Notch1-NICD1在食管鳞状细胞癌中的功能进行初步探索。方法:根据GenBank上的Notch1-NICD序列设计扩增引物,由RT-PCR扩增NICD片段,测序鉴定,并通过Blast进行序列比对,然后回收目的片段并与真核表达载体pcDNA3.1(+)相连,构建Notch1-NICD真核表达载体pNotch1-NICD1,同时将扩增的NICD与pEGFP-C1载体相连构建绿色荧光蛋白表达载体pENotch1-NICD1,并转染EC9706及Eca109,观察Notch1在细胞中的定位及其途径的激活状态。同时将pNotch1-NICD1真核表达载体转染EC9706及Eca109,用MTT法分别在24h、48h、72h、96h及120h观察外源性Notch1片段的导入对EC9706及Eca109细胞增殖的影响。结果:通过RT-PCR获得1个Notch1胞内区片段,并成功构建了包含Notch1胞内区结构域的绿色荧光蛋白载体及真核表达载体。转染pENotch1-NICD1后,Notch1在胞质和胞核内均有表达,表明Notch1信号通路被激活。此外,MTT结果表明转染Notch1-NICD1片段的食管鳞状细胞癌细胞的增殖受到明显抑制(P<0.05)。结论:成功构建了Notch1绿色荧光蛋白表达载体及pcDNA3.1(+)真核表达载体。外源Notch1片段的引入能够明显抑制食管鳞状细胞癌细胞的增殖,提示Notch1基因有可能成为治疗食管鳞状细胞癌的一个潜在的分子靶点。

Aim：To construct an eukaryotic expression vector （pNotchl-NICD1） and the green fluorescence protein vector pENotchl-NICD1 ,so as to investigate the function of the key domains of Notchl-NICD in esophageal squamous cell carcinoma. Methods：The pair of primer was designed according to Notchl-NICD gene sequences of GenBank database, and the fragment was amplified using RT-PCR method and sequenced. The result of sequencing was checked through Blast tools. In addition, the fragment of Notchl-NICD was cloned into eukaryotic expression vector pcDNA3.1 （ ＋ ） and green fluorescence protein vector pEGFP-CI to obtain recombinant eukaryotic expression vector pNotchl-NICD1 and pENotchl- NICDI , which were transfected into EC9706 and Ecal09 cells; the eukaryotic expression vector pcDNA3. 1 （ ＋ ） and pEGFP-CI vector was also transfected as control as well as the wild cell lines. The proliferation of EC9706 and Ecal09 cells was measured by MTT method after transfection with pNotchl-NICD1 vector. Results：A fragment （named Notchl- NICDI ） was obtained, pNotchl-NICD1 and pENotchl-NICD1 were successfully constructed. The results of MTT showed that the proliferations of EC9706 and Ecal09 cells were obviously inhibited （P 〈 0.05 ）. Conclusion： pNotchl-NICD1 re- combinant eukaryotic expression vector and the recombinant green fluorescence protein vector pENotchl-NICD1 were suc- cessfully constructed. The introducing of foreign Notchl can activate the Notchl signaling pathway and give rise to the apoptosis of esophageal squamous cell carcinoma cells, suggesting that the Notchl gene may be a new molecular target for therapy of esophageal squamous cell carcinoma.