Glucagon-like Peptide-1 And Its Analogues Gene Therapy For Diabetes Research

Reporter gene can be widely used in animal experiments to indicate the transcriptional activity of target gene, including luciferase, GFP, etc. However, like luciferase, which has highly sensitivity, must be crack animal cells or organs for testing, does not apply to animal experiments. Therefore, we need a reporter gene system which can detect the sample convenietly and has no endogenous expression to be carried out in our research of gene therapy. Secreted alkaline phosphatase(SEAP) is not only meet this demands,but also can test the sample without destroying the cells. For experimental animals, the test could be carried out conveniently with only a small amount of blood.But it is difficult to compare the differences of gene expression efficiency between two samples using the absorbance of SEAP which is detected by microplate reader. So we need to establish an accurate quantitative method to measure the expression efficiency of SEAP. We establish a model between the true value of SEAP and its absorbance, and develop a metrod to improve the sensitivity of the detection system. Our standard can make SEAP reporter system raised from qualitative level to quantitative level, supported the experiment of gene therapy.MethodEstablish the formula of SEAP reporter gene system:A standard curve for the product of SEAP was established, and the formula between the absorbance value and real value of SEAP was done, then we studied the formula from Michaelis constant of SEAP, reaction time, corresponding relationship between different amounts of mRNA and enzyme.ResultsWe established the formula between the absorbance value and activity of SEAP at 405nm wavelength:y(enzyme activity)=(x(OD value)+0.0028)/19.13*10; and the formula between the enzyme activity and absolute value of protein:lU(enzyme activity)=5.035ng(value of protein),which validated in the cell experiments and in vivo experiments of mice. We proposed the method of improving the sensitivity of SEAP, and make the comparison among the efficiency of different methods in gene therapy experiments successfully.ConclusionOur standard can make SEAP reporter system raised from qualitative level to quantitative level, which can used in cells, tissues, and transgenic animal studies, provide support for experiments in future. PartⅡGene therapy of diabetes using Glucagon-like Peptide-1 and its analogsThere is a significant increase in the number of diabetes all over the world from the beginning of 1980s. The latest report of the large scale epidemiological study of diabetes in China published in the "New England Journal of Medicine" showed the Chinese diabetes prevalence has reached 9.7%, while the total number of diabetes has met the of 92.4 million. The population of early diabetes has reached nearly 1.5 billion,15.5% of the total number.As a kind of gastrointestinal hormone encoded by glucagon gene, the most significant function of Glucagon-like peptide-1 (glucagons-like peptide-1, GLP-1) is to promote regeneration of pancreatic b cell and prevent apoptosis, so GLP-1 can improve blood glucose levels in patients with diabetes. GLP-1 means great significance in the treatment of typeⅠandⅡdiabetes. However, GLP-1 can be degradated by DPP-IV (dipeptidyl protease IV, DPP-IV) quckily.In 1992, Eng et al [4] discovered and purified the peptide Exendin-4 from Heloderma suspectum in the southwest of United States. They found it has the same funtion as GLP-1, and has high tolerance against the DPP-Ⅳ.At present, gene therapy for diabetes is based on mutated GLP-1 and exendin-4 which can generally anti-DPP-IV degradation, aimed to achieve sustainable and efficient expression and prolonged its half-life in vivo. We constructed the mice model of typeⅠand typeⅡdiabetes, using electroporated methord to carry on the study of gene therapy, intended to explore a more effective method in gene therapy of diabetes, provide experiment support for clinical application.Methord1. The role of GLP-1 and exendin-4 of insulin resistance in vitroWe constructed the expression vector include GLP-1-pSEAP,exendin-4-pSEAPand exendin-4-alb-pSEAP.45ug/ml PA was used to establish C2C12 cell model of insulin resistance for about 9 days, then studied the role of GLP-1 and exendin-4 in cell model of insulin resistance, and verified the results from cell viablity, glucose consumption and lipid content.2. The role of GLP-1 and exendin-4 of T1DM and T2DM in vivo(1) Treatment of T1DM:150mg/kg STZ was used to induced T1DM mice model. 30μgDNA,60V electric shock was used to treat the model. Verified the expression of exendin-4 by real-time PCR, and evaluated the effect of gene therapy treatment by blood glucose, oral glucose tolerance test, blood triglycerides, fasting insulin, body weight and so on.(2) Treatment of T2DM:high fat,sugar, and cholesterol diet was used to induced T2DM mice model for about 8 weeks.30μgDNA,60V electric shock was used to treat the type I and II diabetes model. Evaluated the effect of gene therapy treatment by blood glucose, blood triglycerides, fasting insulin, body weight and so on.Results1.Experiments in vitro showed that the C2C12 cell model transfected with GLP-1 and exendin-4 compared with untreated cells, cell activity increased by 50% and 140%, glucose utilization increased by 80% and 100%, the intracellular TG levels decreased by 125%and 200%. There is no significantly differences between the T2DM model cells and normal cells after treatment.2. The results after First treatment of T1DM mouse model indicated that, the glucose tolerance of experimental mice group was significantly improved, the fasting blood glucose decreased from 25mmol/l to 10 mmol/1, the fasting insulin level increased from 13.02μIU /ml to 22.97μIU/ml, TG values decreased from 2.23mmol/1 to 1.05 mmol/1, body weight increased 10%. The normal level of the treatment group can maintain for about 20days. 12days after treatment, the immunohistochemistry results showed that the the islet can restore to 1/3 of the normal size, but the structure is relatively loose, while the results of real-time PCR showed that expression of exendin-4 in pancreas, muscle, liver, and other organs has about 120 times than normal mice. After the Second treatment, the fasting blood glucose of experimental mice group was decreased from 39mmol/1 to 10 mmol/1, the fasting insulin level increased from 17.56μIU/ml to 26.947μIU/ml, TG values decreased from 2.76mmol/1 to 1.05 mmol/1, body weight increased 10%. The normal level of the treatment group can maintain for about 35days.3. The results after treatment of T2DM mouse model indicated that, the fasting blood glucose of experimental mice group was decreased from 12.48mmol/1 to 10 mmol/1, the fasting insulin level decreased from 29.82μIU/ml to 17.97μIU/ml, TG values decreased from 1.57mmol/1 to 0.81 mmol/1, body weight decreased 10%. The normal level of the treatment group can maintain for about 40days.ConclusionAfter the experiment of T1DM in vivo, and T2DM in vivo and in vitro, we show that this reseach of gene therapy can reduce the fasting blood glucose levels of the diabetic mice effectively, and improved the fasting insulin levels, serum TG levels, and glucose tolerance, a little better than other metrod in treating diabetes. It provieds new evidences and new ideas for gene therapy of diabetes in future.