强力霉素对糖尿病小鼠肌肉转染人胰岛素基因的调控作用

Regulated human proinsulin expression in vivo using Tet-inducible vectors

关键词:  四环素调控系统  胰岛素  糖尿病  基因治疗

Key words:  tetracycline regulatory system  insulin  diabetes mellitus  gene therapy

摘要:

［目的］研究强力霉素对糖尿病小鼠肌肉转染人胰岛素基因表达的调控作用。［方法］构建prTA-tet4-rhINS质粒；以链脲佐菌素（streptozotocin，STZ）诱导昆明小鼠成糖尿病模型。采用电穿孔法转移质粒prTA-tet4-rhINS，通过强力霉素（doxcycline，Dox）水给药/撤除以评价四环素系统调控人胰岛素表达的可行性。检测末梢血糖，血清人真胰岛素及小鼠肌肉组织人胰岛素原基因mRNA表达情况。［结果］电穿孔转染目的质粒胰岛素后，立即予饮Dox水，可在小鼠肌肉组织中检测到mRNA表达，第2周降糖效果最佳，撤除强力霉素后，血糖回升，于48 h升至转染前水平，再次予饮Dox水后，血糖逐渐下降，72 h内达到撤药前水平。重复给药／撤药两次结果一致。［结论］通过给药/撤除Dox可以实现对人胰岛素原基因表达的开关调控。

Abstract:

［Objective］To evaluate the hypoglycemic effect of a doxycycline regulated insulin expression vector electroporated to skeletal muscle of streptozotocin-induced diabetic mice.［Methods］The plasmid prTA-tet4-rhINS constructed previously was transferred to hindleg muscle of streptozotocin-induced diabetic mice of Kunming species,and fed in the drinking water with doxycycline immediately.The tail blood glucose was measured with glucose oxidase method daily.Human true insulin was analyzed with ELISA.Total mRNA were extracted from muscles and liver tissues.RT-PCR was used to analyze the human proinsulin mRNA levels. ［Results］Following intramuscular injection of the vector,the insulin mRNA expressions in mice muscles were examined and repeated induction of serum insulin protrein following doxycycline administration were observed.The best hypoglycemic effect was showed in second week after transfer,with withdrawal of inducer doxycycline,blood glucose began to increase to the level before transfer in 48 hours; and then with doxycycline administration,blood glucose of the mice decreased to the level gradully before withdrawal in 72 hours.Two cycles of regulation with the same results were achieved over 34 days.［Conclusion］The tet-on system is a useful tool for controlling the expression of human insulin electropotated in mice.