Hypoglycemic Effect Of Direct Intramuscular Gene Transfer Of A Nude Doxycyline-Controlled Insulin Expression Plasmid In STZ-Induced Diabetic Mice

Objective: Diabetes mellitus is a chronic disease that afflicts the health of human being. It's caused by lack of insulin and/or deficient effect of insulin. Nowadays, multiple injection of insulin to patients is the common practice of treating diabetes, which in turn may cause hypoglycemia and inconvenience. Gene therapy is a molecular biological way to control diabetes mellitus, which foreign gene such as insulin gene is transferred into target tissue. This novel method is considered as a promising measure to conquer diabetes. According to this strategy, insulin gene transfer is expected to restore endogenous insulin production; the expression of insulin gene should get proper regulation. However, to achieve adequately regulated insulin production in response to change in blood glucose concentration remains a major huddle. Though several regulated expression systems have been proved effective, there do exist much limitation and cannot replace insulin injection. Tetracycline regulatory system is a "gene switch" system, in which target gene expression can be induced by administration or remove of tetracycline (Tet) or its derivatives such as doxycycline (Dox). It is now proved to be a safe, high-efficient regulatory system. The application of this system to insulin gene will open a new window of diabetes gene therapy.Methods: Plasmid prTA-tet4-rhINS was constructed. This plasmid contains two important parts: one is tetracycline regulatory system, which is composed of tetracycline operon sequence (ptet4) and rtTA gene sequence; the other is human proinsulin gene, which expressed under the control of tetracycline regulatory system. Balb/C mice were induced to diabetic with 150mg/kg body weight STZ, then 300 ug of prTA-tet4-rhINS was injected into the muscles of mouse hind legs and fed with Dox in drinking water with different concentration at the same time. Tail blood glucose was measured everyday with glucose oxidase method. Parts of mice were killed at the sixthday after plasmid transfer; blood samples, thigh muscles and liver were collected properly. Serum human insulin/pro-insulin was detected with a RIA kit. After isolation of RNA from muscles and liver tissues, RT-PCR was used to analyze the mRNA level of rhINS and mouse β-actin.Results: The blood glucose in diabetes mice was markedly reduced at the average 10mmol/l, diabetes mice gained body weight, and urine volume decreased. This effect can last for nearly two weeks. Furthermore, there is close correlation between the hypoglycemic effect and concentration of Dox in drinking water in a dose-dependent manner. Blood sugar in Dox 2mg/ml group was much lower than that in Dox 0.5mg/ml and 0mg/ml group (p<0.01). In muscle RNA sample (Dox 2mg/ml group), the expected 217bp band of human proinsulin mRNA was amplified with RT-PCR, but it is not detected in the liver total mRNA. Those results were not observed for mice without doxycycline in the drinking water. During the whole test, no hypoglycemia was observed.Conclusion: A single tetracycline controlled recombinant pro-insulin gene expression plasmid was constructed and it successfully decreased the glucose level of STZ-induced diabetic mice dose-dependently by doxycycline. Muscle can be selected as good target tissue for foreign insulin gene's expression. This research could provide a novel, simple and convenient strategy in diabetes gene therapy.