Tet调控STGC3基因表达CNE2细胞系的建立及其功能初步研究

Tet Regulating Expression System Establishment and Functional Analysis ofNovel Gene STGC3 in Nasopharyngeal Carcionma Cell Line CNE2

利用Tet-on调控系统,建立受强力霉素诱导STGC3基因表达的CNE2细胞系,为进一步研究STGC3的功能提供了一个理想的实验平台.先后将调控质粒pTet-on和反应质粒pTRE-STGC3转染入CNE2细胞,并用G418和潮霉素分别进行两轮筛选,运用RT-PCR选择对强力霉素诱导敏感的细胞克隆.用不同浓度强力霉素诱导CNE2/Tet/pTRE-STGC3细胞,RT-PCR检测STGC3的表达,确定强力霉素的最佳诱导浓度.采用此浓度的强力霉素分别诱导CNE2、CNE2/Tet/pTRE、CNE2/Tet/pTRE-STGC3三组细胞,测定细胞的生长曲线、克隆形成率和细胞周期分布.诱导STGC3基因高表达,CNE2细胞增殖速度显著减慢(P<0.05),克隆形成能力显著降低(P<0.01);流式细胞仪检测结果显示,瘤细胞群体中处于G0/G1期细胞数增加,细胞阻滞于G0/G1期.Tet调控STGC3基因表达CNE2细胞系的成功建立,为进一步研究STGC3基因的功能提供一个理想的细胞模型.

In an attampt to establish the functional expression of STGC3 with doxycycline （Dox） induced Tet-on regulating system in human nasopharyngeal carcinoma cell line CNE2, an ideal experimental platform was provided for further studies of STGC3. pTet-on regulating plasmid was transfected into CNE2, and stable expression of Tet-on was established in CNE2 through G418 select. Then the response plasmid of recombinant pTRE-STGC3 was steadily transfected into positive CNE2/Tet-on cells with hygromycin screen. Dox was used to induce the expression of STGC3 and a cell clone sensitive to Dox was selected. The best-induced concentration was determined with different concentration of Dox induction. Growth curves, clone formation rate and cell cycle distribution were detected after STGC3 gene up-regulated expression with Dox induction. The growth capacity and clone formation potential of CNE2/Tet/pTRE-STGC3 was significantly suppressed, compared with the controls （P〈0.05）. FCM analysis indicated that G0/G1 phrase cell rate of CNE2/Tet/pTRE-STGC3 was markedly higher than that of the controls and CNE2/Tet/pTRE-STGC3 cells were arrested in G0/G1 phase of cell cycle. Functional expression of STGC3 under Dox induced Tet-on regulation system was successfully established in CNE2, which provided an ideal experimental platform for further functional study of STGC3.