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本研究应用RevTet On基因表达系统 ,通过四环素衍生物强力霉素 (doxcycline ,Dox)调控血小板生成素 (TPO)基因在NIH3T3细胞中的表达。首先应用基因重组技术构建含TPO基因的重组质粒 pRevTRE/TPO ,然后将RevTet On基因调控系统的pRevTet On和 pRevTRE/TPO分别转染PT67细胞 ,从而包装RevTet On和RevTRE/TPO重组逆转录病毒 ,同时将此两种病毒感染NIH3T3细胞 ,建立整合入RevTet On和RevTRE/TPO逆转录病毒的阳性细胞株 (RevTet On3T3/TPO)。通过Dox调节TPO基因在此细胞株中表达 ,培养基中加入 2mg/LDox或不加Dox ,然后用RT PCR ,Western印迹和ELISA方法检测TPO基因表达情况。结果发现 ,RevTet On3T3/TPO当培养基中不加Dox时用RT PCR和Western印迹检测未见TPO表达 ,用ELISA检测表达TPO量低 ;在培养基中加入Dox时 ,RT PCR和Western印迹检测可见TPO表达 ,且TPO表达量高 ,为不加Dox时的 91倍。本实验表明 ,通过建立逆转录病毒整合的NIH3T3细胞株 ,利用四环素及其衍生物Dox调节TPO基因的表达 ,为TPO基因治疗的进行提供了有用的工具
In this study, RevTet On gene expression system was used to regulate the expression of thrombopoietin (TPO) gene in NIH3T3 cells by tetracycline derivative doxcycline (Dox). Firstly, recombinant plasmid pRevTRE / TPO containing TPO gene was constructed by gene recombination technique. Then, pTvTet On and pRevTRE / TPO of RevTet On gene regulatory system were transfected into PT67 cells respectively to package RevTet On and RevTRE / TPO recombinant retroviruses, meanwhile, NIH3T3 cells were infected with both of these viruses, and a positive cell line (RevTet On3T3 / TPO) integrated into RevTet On and RevTRE / TPO retroviruses was established. The expression of TPO gene was regulated by Dox in this cell line. The medium was supplemented with 2 mg / L LDOX or without Dox. The expression of TPO gene was detected by RT PCR, Western blotting and ELISA. The results showed that no TPO expression was detected by RT PCR and Western blot in RevTet On3T3 / TPO without Dox, and the amount of TPO detected by ELISA was low. When Dox was added to culture medium, RT-PCR and Western blotting were used to detect TPO expression, and TPO expression was high, 91 times as without Dox. This experiment shows that through the establishment of NIH3T3 retrovirus integration cell lines, the use of tetracycline and its derivatives Dox TPO gene expression regulation, TPO gene therapy provides a useful tool