Glucagon - like peptide-1 and its analogs gene therapy for diabetes research
|Keywords||SEAP Reporter gene Enzyme activity standards Glucagon - like peptide-1 and its analogues Diabetes Gene therapy|
The reporter gene is commonly used in animal models, one can mark the desired gene the transcriptional activity of genes, including the luciferase, GFP. However, like luciferase while having a high sensitivity, but the detection must be cleavage of the reporter gene of the animal cell or organ, discomfort for the animal experiments. Therefore, we need a either facilitate the detection, nor the endogenous expression of the reporter gene system services in gene therapy research to be undertaken. Repeat the detection of secreted alkaline phosphatase SEAP not only be able to meet this need, and can not destroy cells of the sample under conditions. Experimental animals, using only a small amount of blood can be often convenient to carry out the detection of the target gene, the animal injury minimal However, the current widespread use with a microplate reader detects SEAP absorbance value of the test method is difficult in the two samples of comparison between the efficiency of target gene expression differences, therefore, an urgent need to establish an accurate quantitative method to measure the amount of SEAP expression. We have established the absorbance value of the SEAP expression of the true value with the corresponding model, and come up with a way to improve the the SEAP detection system sensitivity, SEAP quantified as a reporter gene system for cell, tissue or transgenic animal experiments more accurately compare different target genes and the same target gene expression differences between gene therapy experiments provide support. Methods: SEAP substrate by establishing a standard curve, to make the formula, in terms of each other in the absorbance value of the enzyme activity of the SEAP protein between the absolute absorbance values ??between power constant from the enzyme, the enzyme reaction time, various amounts of mRNA and enzyme corresponding relations verified. Results: To establish at a wavelength of 405 nm, SEAP the absorbance value of the enzyme activity between the mutual conversion formula: y (enzyme activity) = 0.1913 * x (absorbance values) -0.0028; mutual conversion between the absolute values ??of the enzyme activity and protein formula: 1U (enzyme activity) = 5.035g (protein absolute amount). Made in cell experiments, and can be applied to the mice in vivo experiments, and proposes to improve the method of the SEAP detection system sensitivity, the success of a comparison between the efficiency of the different methods of the import target gene, the sensitivity reached 10 -5ng/ml. Conclusion: SEAP as a reporter gene system can be quantified from the qualitative level to rise to the quantitative level, can be used for cells, tissues or transferred gene in animal experiments, and provide support for future experiments. The second part of glucagon-like peptide-1 and its analogs diabetes gene therapy research since the eighties of the last century, the worldwide prevalence of diabetes showed significant growth trend. Up to 2011, recently published in the New England Journal of Chinese Diabetes large-scale epidemiological studies have shown that the prevalence of diabetes in China has reached 9.7%, the total number of diabetes has reached 92.4 million, the highest in the world the forefront; the prediabetes number nearly 150 million, or 15.5 percent. Prevention and control of diabetes without delay. Glucagon-like peptide -1 (glucagons-like peptide-1, GLP-1) as the the glucagon gene encoding a gastrointestinal hormone, its most significant feature is by promoting the regeneration of pancreatic β cell function and prevent apoptosis, to significantly improve glucose levels in people with diabetes. This type Ⅰ and type II diabetes treatment is important. However, the natural GLP-1 in the body will soon be DPP-IV (dipeptidyl protease IV, DPP-IV) degradation. Eng et al  in 1992 from the southwestern United States, the Gila monster (Heloderma suspectum) saliva isolated short peptide Exendin-4 and found it to have exactly the same functionality with the GLP-1 and DPP-IV has high tolerated. Now for the gene therapy for diabetes generally anti-DPP-IV degradation of GLP-1 mutant gene and the like, exendin-4 is based, mediated by the carrier, designed to achieve a sustained and efficient expression in vivo and extended half-life of the double effect. STZ-induced type 1 diabetes in mice fed a high-cholesterol diet and high fat and sugar type 2 diabetes mouse model built electroporation import GLP-1 and its long-acting analogues exendin-4 carrier gene therapy research is intended to explore ways and means of a more effective diabetes gene therapy can be applied to their final clinical support. Methods: 1.GLP-1 and its analogues in the in vitro model of the treatment construct a GLP-1-pSEAP, Exendin-4-pSEAP Exendin-4-ALB-pSEAP expression vector. Select concentration 45μg/ml PA, 9 days of induction conditions to create a mouse myoblast C2C12 insulin resistance model, the expression vector of this model for the role of GLP-1 and its analogues, and from the cell viability glucose consumption, cell fat content verified. 2.GLP-1 and its analogues in the treatment in vivo model: 1) the treatment of type I diabetes model: selection 150mg/kg STZ induced by injection at one time to build a type I diabetes mouse model has mouse model exendin-4 gene injection 30μgDNA, 60V electric shock once the method into two treatment studies, using Real-time PCR method to verify the expression of the target gene in mice, and from fasting plasma glucose, random blood glucose, oral glucose tolerance test, serum triglycerides, fasting insulin, pancreatic tissue immunohistochemistry, weight and other aspects of evaluation of the effects of gene therapy. 2) the treatment of type II diabetes model: selection of high fat, sugar cholesterol diet for eight weeks to build a type II diabetic mouse model of exendin-4 injection 30μgDNA, 60V electric shock method into the model mice genes were first treatment, from fasting plasma glucose, serum triglycerides, fasting insulin, body weight and other aspects of evaluation of the effects of gene therapy. Results: 1. Vitro results show that transfected with GLP-1, exendin-4 plasmid the C2C12 model cells compared to untreated model cells, cell viability increased by 50% and 140%, respectively, glucose utilization increased by 80% and 100%, intracellular TG content decreased by 125% and 200%. After treatment, the indicators of the model cell compared with normal cells there was no significant difference. Treatment of type I diabetes mouse model results show that the experimental mice after one treatment, glucose tolerance was significantly improved, fasting glucose decreased from 25 mmol / 1 to 10 mmol / 1, fasting insulin levels from 13.02μIU/ml increased to 22.97μIU/ml TG values ??decreased from 2.23 mmol / 1 to 1.05 mmol / 1, 10% of the weight gain, about to maintain a normal level of about 20 days. Treatment of 12 days, the results of immunohistochemistry showed that the treated mice islets can be restored to 1/3 the size of normal mice, but the structure is relatively loose; Real-time PCR results showed that at this time exendin-4 gene in mice treated pancreas, muscle, liver and other organs in normal mice compared to about 120 times the level of expression. After the second treatment, the experimental mice fasting glucose decreased from 30 mmol / l to 10mmol / l or less, fasting insulin levels from 17.56μIU/ml improve to 26.47/μIU/ml TG value decreased to 1.05 from 2.76 mmol / l mmol / l, body weight increased by 10%, and to maintain a normal level of about 35 days or so. Treatment of type II diabetes mouse model results indicate that, after treatment, the experimental mice fasting glucose decreased from 12.48 mmol / l to 10 mmol / l or less, fasting insulin levels decreased from 29.82μIU/ml 17.97/μIU/ml , TG value decreased from 1.57 mmol / l to 0.81 mmol / l, the weight decreased about 10%, can be maintained about the normal level of about 40 days. Conclusion: type Ⅰ the body and type II in vivo and in vitro model of research and experiments prove that this gene therapy method can effectively reduce fasting blood glucose levels of diabetic mice, and to improve the model animals fasting insulin, serum triglyceride content and glucose tolerance and other to detect indicators, provides new evidence and new ideas for future diabetes gene therapy research.