| Research BackgroundDiabetes is a common chronic disease.Due to its high incidence and serious damage to the body caused by complications,diabetes has become the third largest non-communicable disease in the world.More than 90%are diagnosed with type 2 diabetes.At present,both chemical drugs and biological drugs have defects in safety and efficacy in the treatment of type 2 diabetes.Gene therapy was born in the 1970s.During the 50 years of its development,new viral vectors have been used in a mature way,delivery methods have been continuously improved,and treatment ideas have been expanded.Therefore,using gene therapy technology to treat type 2 diabetes has attracted more and more attention.Glucagon-like peptide-1(GLP-1)is an important intestinal endocrine hormone.It has many functions such as reducing hyperglycemia,promoting insulin secretion,stimulating the proliferation of pancreatic β-cells,inhibiting glucagon secretion,reducing insulin resistance,delaying gastric emptying,suppressing appetite,protecting the cardiovascular and nervous system,and so on.Because of its multiple characteristics mentioned above as an ideal hypoglycemic drug,it has become the research hotspot in the field of diabetes treatment drugs.Since the active site of natural GLP-1 is sensitive to dipeptidyl-peptidase IV(DPP-4),the GLP-1 half-life in vivo is extremely short,which limits its clinical application.In order to promote the druggability of GLP-1,researchers pay attention to the modification of GLP-1.Human umbilical cord mesenchymal stem cells(hUC-MSCs)can not only differentiate into IPC to reduce blood sugar in diabetic animals,but also inhibit the immune system’s damage to islet cells and reduce insulin resistance.Without immune rejection and ethical issues,they are regarded as ideal drug carriers.Here,we used hUC-MSCs as drug carriers carrying GLP-1 adenovirus gene,then the type 2 diabetes model was applied to verify its activity and mechanism in the treatment of type 2 diabetes.Research purposesGenetic engineering technology prolongs the half-life of GLP-1 in vivo,which makes GLP-1 and hUC-MSCs play synergistic effects against type 2 diabetes.This result provides a new method to develop new anti-type 2 diabetes drugs and theoretical support for gene therapy of type 2 diabetes.Research methods1.Preparation of Type 2 Diabetes Model MiceThe method of streptozotocin combined with high-fat and high-sugar diet was used to prepare type 2 diabetes model mice.Changes in food intake,water intake and body weight were observed and compared.HK method was taken to detect blood glucose level in mouse serum.ELISA method was taken to detect mouse serum insulin level and calculate HOMA-IR value.Mouse pancreatic mitochondrial function was detected with O2K ability metabolism analysis system.HE method was applied to evaluate the pathological changes of mouse pancreas.2.Activity analysis of hUC-MSCs modified by GLP-1Fluorescence microscope was used to observe the effect of GLP-1 transfection on the morphology of hUC-MSCs;q-PCR method was used to detect GLP-1 mRNA content in cells after transfection;ELISA method was taken to detect GLP-1 content in mouse serum after intramuscular injection of new cell lines;Changes in body weight and food intake of mice were observed after the new cell line modified with intramuscular injection.3.Study on the hypoglycemic effect of hUC-MSCs modified by GLP-1Blood glucose changes in mouse serum was detected with HK method;ELISA method was used to detect insulin levels in mouse serum and calculate HOMA-IR value;Glucose tolerance was tested to detect mice’s glucose utilization;Insulin tolerance was tested to detect mice’s sensitivity to insulin.4.Study on the hypoglycemic mechanism of hUC-MSCs modified by GLP-1Mice pancreatic mitochondrial function was detected by O2K ability metabolism analysis system;CHOP,GRP-78 and SXBP-1 protein expression in pancreas were detected with Western blot;ELISA method was used to detect serum glucagon levels in mice;HE method was used to evaluate pancreatic pathological changes in mice.Research resultsCompared with the normal group,the weight change of the model group was not significant(p>0.05);food and water intake increased significantly(p<0.01);fasting blood glucose value increased significantly(p<0.01);serum insulin content was significantly decreased(p<0.01),while the HOMA-IR value increased(p<0.01);the minimum respiratory oxygen consumption of pancreatic mitochondria was not statistically different(p>0.05);the maximum respiratory oxygen consumption decreased(p<0.05);the basic respiratory difference was significant(p<0.01);the oxygen consumption of the model group after ADP stimulation was lower than that of the normal group(p<0.01),and the oxygen consumption of the model group was much higher than that of the normal group(p<0.01),which was unrelated to ADP stimulation;the respiratory control rate of model group was significantly reduced(p<0.01);ADP:O value was reduced(p<0.01);HE showed that the pancreatic structure of the model group changed significantly.After hUC-MSCs modified by GLP-1 gene,fluorescence microscope results showed that the cell morphology was normal;qPCR experiment results showed that GLP-1 mRNA expression increased in AD-GLP-1 which modified by gene engineer(p<0.01);After intramuscular injection of AD-GLP-1 cells(1×106/ml),GLP-1 in the blood of mice increased significantly(p<0.01);at the same time,the food intake(p<0.01)and body weight of mice decreased(p<0.01);There was no change of AD-GFP in the control group(p>0.05).After 3 weeks of administration of AD-GLP-1,the weight of the mice was significantly reduced(p<0.01)compared with the model group;the food intake was reduced(p<0.01);the fasting blood glucose value was significantly reduced(p<0.01);fasting insulin significantly increased(p<0.01);HOMA-IR decreased significantly(p<0.01);insulin resistance AUC decreased significantly(p<0.01);AUC decreased significantly in glucose tolerance test.Compared with the model group,after 3 weeks of administration of AD-GLP-1,the minimum respiratory oxygen consumption of mouse pancreatic mitochondria did not change significantly(p>0.05);the maximum respiratory oxygen consumption increased(p<0.05);the difference of basal respiration was significant(p<0.01);the oxygen consumption after ADP stimulation was significantly higher than that of the model group(p<0.01),and the oxygen consumption which was unrelated to ADP stimulation was much lower than that of the model group(p<0.01).The respiratory control rate in the AD-GLP-1 group was significantly increased(p<0.01),and ADP:O value was significantly increased(p<0.01);the membrane potential level was significantly increased(p<0.01);the reactive oxygen production rate was significantly reduced(p<0.01);the contents of SXBP1,GRP-78 and CHOP in the pancreas were significantly lower than that in the model group(p<0.01);the glucagon level was significantly lower(p<0.01);HE results showed that the morphology of the pancreas in model mice was improved.Analysis conclusionhUC-MSCs were modified with GLP-1 through genetic engineering technology to prepare a new cell line AD-GLP-1.For type 2 diabetes model mice,the fasting blood glucose level of the model mice decreased;the fasting insulin level increased;HOMA-IR decreased;insulin tolerance and glucose tolerance improved;body weight and food intake decreased.The results above showed ideal resistance effect to 2 Type 2 diabetes.The hypoglycemic mechanism may be related to enhancing the respiratory capacity of pancreatic mitochondria,improving mitochondrial function,and regulating plasma reticulum stress-related proteins in pancreatic mitochondria of type 2 diabetic mice.Thereby,insulin levels in the body was increased,and glucagon levels were reduced.And finally,resistance of blood glucose and insulin were improved in type 2 diabetic mice. |
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