Tet调控STGC3基因表达CNE2细胞系的建立及其功能初步研究

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.

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

In an attampt to establish the functional expression of STGC3 with doxycycline (Dox) induced Tet-onregulating system in human nasopharyngeal carcinoma cell line CNE2, an ideal experimental platform wasprovided for further studies of STGC3. pTet-on regulating plasmid was transfected into CNE2, and stableexpression of Tet-on was established in CNE2 through G418 select. Then the response plasmid of recombinantpTRE-STGC3 was steadily transfected into positive CNE2/Tet-on cells with hygromycin screen. Dox was used toinduce the expression of STGC3 and a cell clone sensitive to Dox was selected. The best-induced concentrationwas determined with different concentration of Dox induction. Growth curves, clone formation rate and cell cycledistribution were detected after STGC3 gene up-regulated expression with Dox induction. The growth capacity andclone 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 higherthan that of the controls and CNE2/Tet/pTRE-STGC3 cells were arrested in G0/G1 phase of cell cycle. Functionalexpression of STGC3 under Dox induced Tet-on regulation system was successfully established in CNE2, whichprovided an ideal experimental platform for further functional study of STGC3.