| Diabetes(Diabetes mellitus, DM) is a kind of chronic disease, which shows hyperglycemia caused by endocrine and metabolic disorders. Animal models is very common in preclinical studies of diabetes. It can mimic pathological processes of type 2 diabetes, and provides a experimental object for drug target screening, drug development, and efficient treatment. In this study, we builded overexpressing vector which had three genes’ integration overexpressing 11β-HSD1 in the liver and overexpressing CHOP and h IAPP in pancreas. We used microinjection to get F0, F1 generation mice with three targeted genes, and then used high-fat and high-sucrose diet to induce positive transgenic C57BL/ 6 mouse models. We evaluated and analysised the transgenic mice by observaing individual physiological and biochemical parameters, histological and pathological patterns,and molecular biology research results relating the expression and regulation of functional genes.1) Preparation and characterization of multi-gene transferred mice. We constructed a three-gene vector, built and breed F0, F1 mice, and identificated them.We selected positive transgenic mice of 7 weeks and 33 weeks old and used Real-time QPCR to detect organ-specific expression. The results show that we identificated them to be positive when three PCR bands were positive at the same time. Comparing with the negative control, the overexpression of positive mice tissues were ideal(Multiples from a few to even hundreds).2) The exogenous genes’ copy number analysis of transgenic mice. We got three-gene copy numbers of three F0 pedigrees of transgenic mice by Real-time QPCR. F1 generation mice’s 11β-HSD1 gene copy numbers of the 6# and the 11# pedigree show no significant differences(P = 0.49> 0.05), the remaining differences between the results of pairwise comparisons were significant(P <0.05). All positive average values were 20.33±3.72(11β-HSD1),7.13±1.41(CHOP) and 3.76±1.10(h IAPP), reflecting the integrated ability(11β-HSD1> CHOP> h IAPP). The F0 11# gene copy numbers are 13.15(11β-HSD1), 3.51(CHOP) and 0.70(h IAPP), F1 generation copy numbers of F0 11# pedigree were 27.29±3.1(11β-HSD1), 5.42 ± 0.73(CHOP), 1.61 ± 0.26(h IAPP). It’s found that three gene copy numbers are inconsistent between individual mice and consistent in intergeneration. But some of the copy numbers of F1 are more than F0 generation,3) The phenotypic analysis of high-fat and high-sucrose induced transgenic mice. Transgenic and wild control mice of the same age were induced by high-fat high-sucrose tests(Each of the four treatment of male and female: Tg HF-HG,Ng HF-HG,Tg Control,Ng Control). The phenotypic data such as weight, blood glucose, feed consumption and water intake were monitored during this period, besides insulin resistance and glucose tolerance was tested regularlly. Results: In high-fat and high-sucrose transgenic male group, weight gain was significant(p<0.01), while water intake between the groups were not significantly different(p>0.05); Fasting blood glucose of high-fat and high-sucrose transgenic male mice group was slightly higher than the other groups, but fluctuating(190 day, p<0.05). Compared with the wild control group, glucose tolerance of high-fat and high-sucrose transgenic male group were descend,and the insulin resistance happened in advance, starting at 2 months. The phenotype will become more and more obvious if they were continully induced(120 day, p<0.01).4) Pathology and molecular biochemical analysis of mouse model. The results showed that: liver, fat and other organs of high fat, and sugar induction treatment group weight gain was higher than control groups(Tg>Nc:p<0.05); In high-fat and high-sucrose treatment, serological results showed that transgenic male mice glucose was higher than the control group. C peptide levels reduced as a result of impaired insulin secretion. Hyperinsulinemia and hypertriglyceridemia were also happened. HE slice shows islets’ changement, some of islet areas of high-fat and high-sucrose transgenic group increases because compensatory insulin secretion, some islets were apoptotic and condensate but high-fat and high-sucrose wild group wae just compensatory increaseing. The protein expression between high-fat and high-sucrose treatment and control was significantly different, with significant differences between male and female. But in the same treatment, transgenic and wild groups are not obvious. Immunohistochemistry find that the size of islets and insulin secretion are consistent with HE sections, h IAPP accumulation and pancreatic apoptosis measurement found that h IAPP accumulated obviously and apoptotic factor Caspase 3 expressed increasingly in transgenic high-fat and high-sucrose islets.We manufactured mice of hepatic 11β-HSD1 overexpression and h IAPP and CHOP overexpression in pancreas by 2A mediated polycistronic vector, coupling with the high-fat and high-sucrose diet induced. It caused insulin resistance, significantly lower glucose tolerance and mouse pancreatic β cell apoptosis stress-related pathways started. It lead to the lack of insulin secretion. Most transgenic mice of has been reached to pre-diabetes stage with the features such as centripetal obesity, hyperinsulinemia, decreasing glucose tolerance, insulin resistance, increaseing islet compensatory or excessive apoptosis.We made preparation and preliminary experiments for generating transgenic pig models in the future. |
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