Study On The Expression Of Nfic,Osteogenic And Adipogenic Related Genes In The Type1 Diabetic Rats And The Intervention Of Insulin

Diabetes mellitus (DM) is a metabolic diseases characterized by high blood glucose.It has insulin secretory defect or damage of biological effects, or even both. The long-standing high blood glucose can lead to a variety of organizations damages, especially the eyes, kidneys, chronic damage to the heart, blood vessels, nerves, dysfunction. Among them, type 1 diabetes is more often happened in children and adolescents, who must rely on insulin to lower the blood glucose. In 1948,Albright and Reifenstein first proposed the concept of diabetic osteoporosis (DOP), noted that the diabetic patients that suffering adverse long-term control of blood glucose were prone to the osteoporosis. Diabetic osteoporosis is a systemic metabolic disease based on the diabetes and has concurrent reduction of bone mass, bone fragility, and the microstructure also impaired. Bone tissue continue to grow and evolve through a complex reconstruction, which include bone formation and resorption. Studies have shown that, DM1 mainly due to reduced bone formation.Mesenchymal stem cells (MSCs) have abilities of osteogenic differentiation and adipogenic differentiation. Osteogenic differentiation influenced by numerous signaling pathways and related transcription factors, Nfic is one of the four members of the family nuclear factor ? (NFI)?Studies have shown that, expression of Nfic of osteoblasts in patients with osteoporosis was reduced, the overexpression of Nfic can contribute to the increase in osteoblast differentiation and new bone formation. In addition, Nfic inhibit adipogenic differentiation of BMSCs by inhibiting the expression of peroxisome proliferator-activated receptor y (PPARy),, showing age-related osteoporosis like phenotype.We established the experimental animal model of DM1 to observe the changes of blood glucose, weight, and in general, HE staining and immunohistochemistry experiments of the pancreatic tissue and the stability of model; The success rate of model establishment and the remedies for remodeling after the first failure and the effect contrast on rat pancreatic tissue; The expression of osteogenic genotype(Runx2, OSX, IGF-1, BMP-2) including Nfic of DM1 and bone specific marker genes (COL2A1, OC, ALP); The expression of adipogenic gene PPAR?; The effect of expressive change of Nfic on the expression of osteogenic genes and adipogenic gene PPAR?; The effect of insulin on osteogenic and adipogenic genetic expression in DM1 rats.Part1:Establishment of Type 1 Diabetic Rat Models, Effect of Remedial Measures for Failed Model and Effect of Insulin TreatmentObjective:To establish type 1 diabetic rat models by intraperitoneal injection of streptozotocin and observe the stability of diabetic changes in rats and the remedial measures for failed modeling rats and their results, and to explore the effect of insulin treatment in rats models.Methods:30 male Wistar rats(6?8-week-old) were randomly divided into 2 groups, including experiment [EG;n=25; 65 mg/kg of STZ, single i.p.] group and control [CG; n=5;citrate buffer solution i.p.] group.72h after injection with STZ, the blood glucose levels (BGLs) were evaluated to verify whether the type 1 diabetic rats model has been established successfully. The successful modeling rats were divided into 2 groups, including diabetes group and insulin treatment group. Insulin treatment group were injected with intermediate-acting insulin (4-6U/kg/d) after 4 days of modeling.The failed modeling rats were named remedy group which were normally fed for 1 week before the second intraperitoneal injection (65 mg/kg). Overt behavior, body weights and levels of blood glucose were evaluated at the 0th, 2nd,4th,6th,8th week respectively. All rats were executed at the 8th week to collect the samples of pancreas. HE staining method and immunohistochemical staining of insulin were used to observe the pathological changes. Results:1?Weight:The weight of diabetes group and remedy group decreased slightly, the weight of control group and insulin treatment group had a sharp increase.2?Blood glucose:72h after injection of STZ,21 diabetic models were established successfully out of 25 rats in this experiment (Random blood glucose levels?16.7mmol/L). Choose 20 rats randomly from the successful modeling group and then divided them equally into 2 groups. including diabetes group and insulin treatment group. After a second injection,4 failed modeling rats were successfully remodeled. There was no significant difference between diabetes group and remedy group. After the injection of insulin,the blood glucose level of insulin treatment group fell back to nomal.3?Pancreas histocytology: The pancreatic islets morphology of control group was nomal.Compared with control group rats, the pancreatic islets of diabetic rats including diabetes group?remedy group and insulin treatment group were irregular and shrinked obviously, and the endocrine cells of pancreatic islets were markedly decreased in experiment rats.The pancreatic islets morphology of remedy group was more irregular than the diabetes group. Compared with diabetes group rats, The pancreatic islets morphology of insulin treatment group was more regular, and the volum was larger.4?In immunohistochemical detection of pancreatic of insulin, we found that, compared with control group, insulin distribution area of diabetic rats significantly reduced. Compared with the dibetes group, the insulin distribution area of the remedy group and insulin treatment group was lager.Conclusion:With the dose of 65 mg/kg of STZ intraperitoneal injection, type 1 diabetic rat models can be established successfully with obvious symptoms and without conversion. By reinjection at the right moment, 4 failed modeling rats were successfully remodeled. Insulin treatment can make the type 1 diabetic rats maintain normal blood sugar level, it also have therapeutic effect on pancreatic cells to a certain extent.Part2:The study of expression of Nfic gene and its correlation with other osteogenesis related genes of diabetic rats.Objective:To explore the expression of Nfic gene and its correlation with other osteogenesis related genes obtained from diabetic rats. Effect of intervention of insulin on the pancreas and bone metabolism in diabetic rats Methods:Method of making type 1 diabetic rats model was the same as the first part. Insulin treatment group were injected with intermediate-acting insulin (4-6U/kg/d) after 4 days of modeling.Overt behavior, body weights and levels of blood glucose were evaluated at the 0th,2nd,4th,6th,8th week respectively. All rats were executed at the 8th week to collect the samples of femur and pancreatic tissue. Pancreatic tissue were used for HE staining and immunohistochemical staining of insulin. Q-PCR method was used for the measure of Nfic?Runx2?OSX?COL2A1?OC?ALP?BMP-2?IGF-1 mRNA expression. Results:8 weeks later, In Immunohistochemical detection of pancreatic of insulin,we found that, compared with normal control group, insulin distribution area of model group significantly reduced. Insulin distribution area of treatment group was between the other two groups. Q-PCR showed that, compared with control group,Runx2?OSX?OC?IGF-1mRNA of the model and treatment group declined significantly, there was no significant difference between the treatment group and the control group. COL2A1 mRNA, the model group was significantly lower than the control group and treatment group, the expression of the treatment group was significantly lower than the control group; meanwhile, ALP mRNA of the model group and the treatment group were significantly higher than the control group, model group was lower than the treatment group, but there is no significant difference. BMP-2?Nfic mRNA of model group and treatment group were significantly lower than the control group. There was no significant difference between the model group and treatment group. Conclusion:In type 1 diabetic rat model, Runx2, Nfic, OSX, BMP-2, IGF-1, OC, COL2A1 mRNA expression of the bone tissue was significantly decreased, ALP mRNA expression levels were significantly elevated, the decrease of Runx2, OSX, IGF-1, OC, COL2A1 mRNA expression was reversible by insulin application, thus would promote the bone formation.Part3:The study of expression of Nfic gene and its regulation of PPARy of diabetic rats and the intervention of insulinObjective:To explore the expression of Nfic gene and its regulation of PPARy obtained from diabetic rats and the effect of insulin intervention. Methods:Method of making type 1 diabetic rats model was the same as the second part. All rats were executed at the 8th week to collect the samples of femur. Q-PCR method was used for the measure of Nfic?PPARymRNA expression. Results:8 weeks later, Q-PCR showed that, compared with control group, the expression of Nfic mRNA of both two groups(B/C) declined significantly (p< 0.1) (p< 0.001), and there is no significant difference between B and C; Compared with control group, the expression of PPARy of other two group increased(p< 0.001)(p< 0.5). nevertheless, compared with group C, the expression of PPARy of group B decreased(p< 0.001). Conclusion:The declined expression of Nfic resulted in the elevated expression of PPARy, thus could promote the adipogenesis. The intervention of insulin could not Reverse the increase of PPARy and the decrease of Nfic.