| Objective: To transfer the plasmid (prTA-tet4-rhINS) previously constructed in this laboratory into skeletal muscles of STZ-diabetic mice by electroporation (EP) and to study its regulation on blood glucose by doxycycline.Methods: (1) Plasmid prTA-tet4-rhINS and pLNCX were amplified by E.Coli and purified by polyethylene glycol method. (2) Kunming mice were induced to diabetes by streptozotocin (STZ). (3) The diabetic mice were divided into three groups. ①Group EP-ptetINS: In this group,150μg of plasmid prTA-tet4-rhINS was injected into each thigh muscle of the mice. Five minutes after the plasmid injection transcutaneous electric pulses were applied by two stainless steel needle electrodes placed about 5 mm apart in parallel of the injection area. The parameters of electroporation were field strength 200V/cm, wave length 40ms, 6 pulses, frequency 1 Hz. ②Group EP-pLNCX: The electroporation conditions were similar to group ET-ptetINS , but the plasmid pLNCX was devoid of the expression cassette of insulin. ③Group Nake-ptetINS: 150μg of plasmid prTA-tet4-rhINS was injected into each thigh muscle of the mice without electroporation enhancement. In all of the above group the mice were immediately provided with drinking water containing 2 mg/ml of doxycycline after transfer. (4) 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 reaction (RT-PCR). (5) Addition/withdrawal of doxycycline in the drinking water was taken to study the regulation of blood glucose.Results: (1)The diabetic mice in both group EP-ptetINS and group Nake-ptetINS reduced the blood glucose levels, normalized the body weight and decreased the urine volume of diabetic mice. In the above two groups serum true human insulin was significantly higher than group EP-pLNCX. A second injection of prTA-tet4-rhINS still reached to the same hypoglycemic effect as the first one after stopping of plasmid expression for the first injection. In contrast, group EP-pLNCX with plasmid pLNCX no decrease of glucose was observed. 80% of the diabetic mice in group EP-pLNCX died four weeks after the plasmids injection, while only 4.76% in group EP-ptetINS died.(2) In comparison to intramuscular injection of prTA-tet4-rhINS without electroporation, electroporation-mediated intramuscular transfer led to higher transfer efficiency (59.52% versus 12.50%, P<0.01), steadier hypoglycemic effect (reduction of standard deviation), longer duration of expression (9 weeks versus 7 weeks), higher but no significant level of human true insulin (55.67±1.37 versus 53.33±2.58μIU/ml, P>0.05), and mRNA level was slightly stronger than that of Nake-ptetINS. (3) The blood glucose levels of mice in both EP-ptetINS group and non-diabetic group were not different after 24-hour fasting (5.2±1.2 versus 4.9±0.2mmol/l, P>0.05) and no hypoglycemia was observed in group EP-ptetINS while the blood glucose in group EP-pLNCX remained high (16.1±1.2 mmol/l) even after 24-hour fasting. (4) The lowest level of blood glucose of the diabetic mice in group EP-ptetINS was recorded two weeks later. After withdrawal of Dox , blood glucose of diabetic mice began to increase in 48 hours and reached to the pretreatment level in 72 hours. Addition of Dox in drinking water the blood glucose gradually decreased again.Conclusions: (1) The injection of plasmid prTA-tet4-rhINS can successfully lead to the reduction of blood glucose, decreased urine volume, and normalization of body weight. (2) Electroporation can enhance the transfer efficacy and duration of the plasmid expression, and lead to steadier, longer duration of expression. (3) After successful transfer of the plasmid, the blood glucose levels can successfully be regulated by the addition/withdrawal of doxycycline in the drinking water, normalization of blood glucose can be reached after the 24-hour fasting without hypoglycemia .
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