| BackgroundWith the development of society and the improvement of living standard,more and more people are suffering from diabetes.In 2014,the number of adults with diabetes reached 422 million,diabetes has become a chronic noncommunicable disease that endangers human health.Although more and more mechanisms of diabetes have been revealed by scientists,however,there is still no effective treatment for this disease.The number of people who dies of diabetes every year reaches 1 million 500 thousand,in addition,2 million 200 thousand people died of the complications caused by diabetes.Diabetes is harmful to patients,at the same time,it causes great loss to the economy of various countries and receives more and more concern and attention.Laboratory animals play a vital role in the study of diabetes.The laboratory minipig is very suitable for diabetes research,because it is very similar to human in many aspects like organ morphology and physiology function.In the cultivation of laboratory minipig,we found a bama boar with hyperglycemic performance.This hyperglycemic bama boar was used to form a family by means of inbreeding,and the family has been bred to the fifth generation.After detecting,some of its offspring also shows hyperglycemia.We believe that this bama minipig hyperglycemia family is expected to be further bred and selected as an animal model,which could be used in the study of the mechanisms and treatment of diabetes.ObjectiveThe main purpose of this project is to detect and analyze a number of blood indexes of bama minipig hyperglycemia family,and try to establish diabetes model with hyperglycemic minipigs.Also,we try to find the gene mutation causing hyperglycemia by analyzing the related genes.Methods1.The indexes detection of hyperglycemic minipigs:ten fifth generation bama minipigs of hyperglycemia family and ten normal bama minipigs were selected,and respectively as the hyperglycemia group and the normal group.The experiment was carried out at six month old of the bama minipigs.The body weight,fasting plasma glucose,postprandial 2h blood glucose,insulin,glucagon,glycosylated hemoglobin,insulin resistance index,blood physiological and blood biochemical indexes of two groups were detected under fasting condition,and the glucose tolerance test was performed.The detection result of the two groups were compared and analyzed.2.High sucrose,high fat and high cholesterol diet induction:six fifh generation bama minipigs of hyperglycemia family were selected,which were given high sucrose,high fat and high cholesterol diet as the hyperglycemia+HHSFCD group.In addition,twelve normal bama minipigs were selected,six of them were given high sucrose,high fat and high cholesterol diet as the normal+HSFCD group,and the other six were given control diet as normal+CD group.The diet induction lasted for four months.During the diet induction,the body weight,fasting blood glucose,postprandial 2h blood glucose,insulin,glucagon,glycosylated hemoglobin,insulin resistance index,blood physiological and four blood lipids items of three groups were detected,and the glucose tolerance test was performed.The detection result of the three groups were compared andanalyzed.3.Polymorphism analysis of TCF7L2 gene and PPAR-a gene:fifteen fifth generation bama minipigs of hyperglycemia family and fifteen normal bama minipigs were selected,and respectively as the hyperglycemia group and the normal group.Ear tissues samples were collected from bama minipigs and genomic DNA were extracted.PCR test was performed to amplify the exon 8 fragment of TCF7L2 gene and exon 2 fragment of PPAR-a gene.The amplified products were sequenced after PCR,and single nucleotide polymorphisms of the target fragments were analyzed.The result of the two groups were compared and analyzed.Results1.The levels of FPG,2hPG,HbAlc,HOMA-IR,CHOL and LDL-CH in hyperglycemia group were higher than those in normal group,and the difference was statistically significant(P<0.05).In the results of IVGTT,FPG,30min blood glucose level and 120min blood glucose level in hyperglycemia group were higher than those in normal group,and the difference was statistically significant(P<0.05).There was no significant difference in blood physiological indexes and other blood biochemical indexes between the two groups.2.After four months diet induction,the body weight of the hyperglycemia+HSFCD group increased significantly and the body became obese.In the hyperglycemia+HSFCD group,the levels of FPG,2hPG,HbAlc,HOMA-IR and four blood lipid items increased,and the secretion of INS and GC decreased,while the glucose tolerance decreased.The normal+HSFCD group also became obese and showed a rise in the levels of four blood lipid items.In the normal+HSFCD group,the changes of FPG,2hPG,HbAlc,HOMA-IR and glucose tolerance were not obvious,the secretion of INS increased,and the mean corpuscular volume increased.The indexes in normal+CD group were remained at the normal level.3.TCF7L gene fragment sequencing results indicated that there existed three SNP loci,which were 118bp:A→T,220bp:G→A and 448bp:G→A.The 220A/A genotype and 448A/A genotype only appeared in hyperglycemia group,the genotype distribution in two groups was statistically significant(P<0.01).There existed three SNP loci in PPAR-a gene fragment,according to the sequencing results which were 117bp:C→T,381bp:A→G and 417bp:C→T,there was no significant difference in genotype distribution between the two groups.ConclusionThe individuals of the bama minipig hyperglycemia family showed hyperglycemic symptoms and abnormal lipid metabolism.After high sucrose,high fat and high cholesterol feed,the symptoms of its hyperglycemia were more serious,and impaired glucose tolerance was appeared,some individuals have developed diabetes.Compared with the normal bama minipigs,the hyperglycemic bama minipigs were more sensitive to the stimulation of a high calorie diet.The polymorphic mutations found in the TCF7L2 gene and the PPAR-a gene failed to determine whether they were associated with the heritability hyperglycemia of the bama minipig hyperglycemia family. |
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