血小板生成素基因在NIH/3T3细胞中的表达与调控

应用 Tet- On基因表达系统 ,调控血小板生成素 (TPO)基因在 NIH/3T3细胞中的表达时间与水平 .籍脂质体介导的基因转移方法 ,p Tet- On质粒转染 NIH/3T3细胞株 ,得到稳定细胞株NIH/3T3- Tet- On.p TRE/TPO与 p TK- Hyg质粒共转染 NIH/3T3- Tet- On细胞株 ,得到双稳定细胞株 NIH/3T3- Tet- On- TPO.在培养基中加入或不加强力霉素 ,RT- PCR、Western印迹及 ELISA法检测培养上清 TPO表达 .结果表明 ,当培养基中不加强力霉素时 ,TPO无明显表达 (0 .1 μg/L) ;当培养基中加入 2 mg/L强力霉素时 ,TPO表达明显增高 (1 0 .8μg/L) .TPO表达水平与强力霉素浓度有关 ,随强力霉素浓度增高 ,TPO表达增加 .TPO表达水平还与强力霉素作用时间有关 ,加入强力霉素 6 h后 ,TPO表达明显增加 (1 .2μg/L) ,随培养时间延长 ,TPO表达增加 ,2 4 h达到峰值(1 0 .8μg/L) ,而且这种诱导作用是可逆的 .为进一步进行 TPO基因表达调控的体内研究奠定基础 ,有望为 TPO基因治疗提供一条可控的安全途径

Regulation of Thrombopoietin Expression by Doxycycline in NIH/3T3 Cells *

Tet on gene expression system was used to regulate the level and time of thrombopoietin (TPO) expression by doxycycline in NIH/3T3 cells.pTet On was transfected into NIH/3T3 cells,in which cells resistant to G418 were cloned and named as NIH/3T3 Tet On.pTRE/TPO and pTK Hyg were cotransfected into NIH/3T3 Tet On cells,in which cells resistant to hygromycin were cloned and named as NIH/3T3 Tet On TPO.TPO expression was examined in the absence or in 2 mg/L of doxycycline,at different concentrations of doxycline,and in the time course after exposure to doxycycline,by means of RT PCR,Western blot and ELISA.The result showed that there was no significant TPO expression in the absence of doxycycline,and the basal level was only 0.1 μg/L.After exposure to 2 mg/L doxycycline,TPO expression was greatly increased,and the peak level reached 10.8 μg/L.TPO expression was affected by the concentration of doxycycline in the cell culture.The more doxycycline,the higher the TPO expression.TPO expression was also affected by the time course of doxycycline in the cell culture,it was greatly increased after 6 hours.The longer of the culture time,the higher the TPO expression,until it achieved peak value at the end of 24 hours.Meanwhile,the inducible effect was also reversible.Tet On gene expression system is a ready access to the tight regulated and high level gene expression system.It is likely to provide a safe and regulatable way for TPO gene therapy.