电穿孔介导的可调控人胰岛素基因肌肉转染对糖尿病小鼠的降糖作用

Electroporation enhanced intramuscular transferring of a doxycycline-regulated human proinsulin expression plasmid and its hypoglycemic effect on STZ-induced diabetic mice

[目的]研究用电穿孔转染强力霉素调控的人胰岛素原基因表达对糖尿病小鼠的降糖作用。[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。成模小鼠分为3组:①ET-ptetINS组(42只):为强力霉素调控的胰岛素质粒INS(prTA-tet4-rhINS)电穿孔组,②ET-pLNCX组(35只):为不含INS基因的空质粒pLNCX电穿孔对照组;③Nake-ptetINS组(48只):为强力霉素调控的胰岛素质粒INS单纯肌肉注射组。各组转染后立即予饮浓度为2 mg/mL强力霉素(Doxycycline,Dox)水。检测末梢血糖,血清人真胰岛素及小鼠肌肉组织中人胰岛素原基因mRNA表达情况。[结果]胰岛素基因肌肉转移后使糖尿病小鼠血糖下降,体重稳定增加,尿量减少,血清中检测到较高水平真胰岛素;质粒停止表达后重复注射仍然有效。电穿孔转染降糖作用可达9周,而直接注射组约为7周。电穿孔组转染成功率较直接肌肉注射更高(59.52%vs12.50%),降糖作用更稳定(鼠间血糖变化差值变异更小),真胰岛素水平更高,为(55.67±1.37)μU/L vs(53.33±0.58)μU/L。采用电穿孔法小鼠的肌肉组织人胰岛素原基因mRNA的表达程度较强。禁食24 h后电穿孔法小鼠血糖水平接近非糖尿病小鼠(5.2±1.2)mmol/L vs(4.9±0.2)mmol/L,且没有出现低血糖。[结论]与质粒直接注射相比,电穿孔介导人胰岛素基因转移的降糖作用更持久,个体差异更低,转移有效率及胰岛素水平更高。

To study the hypoglycemic effect of a intramusclularly injected doxycycline regulated insulin expression.[Methods] A plasmid prTA-tet4-rhINS constructed previously was transferred to the hindleg muscle of streptozotocin-induced diabetic mice of Kunming species by direct intramuscular injection or together with electroporation,and doxycycline was given in the drinking water as a expression regulater.Random blood glucose levels were determined by glucose oxidase method at 8 AM and human true insulin was measured with ELISA.Human proinsulin gene mRNA level was detected by reverse transcript polymerase chain reaction（RT-PCR）.[Results] Both direct injection and electroporation decreased blood glucose level,increased serum human insulin level and normalization of body weight.For one injection,glycemia control could last nine weeks in electroporation group,while seven weeks in direct injection group.In comparison to intramuscular injection of prTA-tet4-rhINS without electroporation,electroporation-mediated intramuscular transfer led to higher transfer efficiency（59.52% vs 12.50%,P0.01）,higher but no significant level of human true insulin（55.67±1.37） μIU/mL versus（53.33±2.58） μIU/mL（P0.05）,and mRNA level was slightly stronger.24 hours fasting led to nearly normalization of glycemia in both of transfer methods without hypoglycemia.[Conclusion] The electroporation method can improve transfer efficiency and leads to longer expression duration and steadier control of blood glucose.