The Study Of Exendin-4 Role In Bone Marrow-derived Mesenchymal Stem Cells Treating Diabetes

Type 2 diabetes mellitus (T2DM) is a clinical syndrome mainly represented glucose metabolic disorder caused by genetic and environmental elements. Insulin secretion, effect or both defects lead to metabolic disorders included carbohydrates, fats, protein, water, and electrolytes, et al. T2DM can cause multiple system organ damaged chronic complications which is the major cause of disability or death. The current treatment is mainly to lower blood sugar, to a extent, it can improve insulin resistance and metabolic syndrome, but can not cure diabetes, especially for multi-organ tissue included islet cells damage has little effect. Mesenchymal stem cells (MSCs) which have repair ability and multi-differentiation potential have become the research focus in the area of diabetes and its complications treatment.Studies have shown that bone marrow-derived mesenchymal stem cells (BMSCs) transplantation has fast onset of action in most tissue damage repair, with a direct repair capacity. Our previous research showed that BMSCs have a good direct repair in multiple organ tissue injury in T2DM, it is very clear to improve the complications and overall situation in T2DM, BMSCs also have a certain role in islet restoration, but not yet observed in the short term islet function fully restored, also not seen the same reports. At the same time, we have seen BMSCs have a certain degree of improving insulin resistance and the hypoglycemic effect, but their direct hypoglycemic effect is weaker in short-term. YANG Wei-hong's study has shown that high glucose can inhibit the proliferation of BMSCs and promote their apoptosis, our studies also show that high glucose and fat increase apoptosis of BMSCs, also can lead to (3-cell insulin secretion capacity decline. Therefore, if no supplementary treatment, the role of BMSCs in T2DM body may be influenced by etiology, under the influence of etiology of T2DM, it is not conducive for BMSCs to differentiate to islet cells, BMSCs alone treatment has some limitations.Exendin-4 is a GLP-1 (glucagon-like peptide-1) analogues extracted from lizard salivary glands, with a longer plasma half-life and strong biological activity. It can play a hypoglycemic effect through a number of pathophysiological targets for T2DM, improve pancreatic local micro-environment at the same time, it also can promote the regeneration of islet cells and stem cells differentiation to islet cells. However, it is relatively slow to play a role, it will take longer cycles to stimulate islet cell regeneration, but also for the improvement of systemic complications is an indirect role, dependent on the body's stem cell system, BMSCs and Exendin-4 has a very good complementary in the T2DM treatment. In order to explore a lower side effect, effective treatment method for pancreatic islet dysfunction in T2DM, this research will explore whether Exendin-4 helps BMSCs play a better role in the process of BMSCs transplantation, whether Exendin-4 in vitro can promote the differentiation of BMSCs to the insulin-producing-cells (IPCs), whether Exendin-4 in the body can promote BMSCs to differentiate into IPCs, whether BMSCs play a therapeutic effect is through the promotion of pancreaticβ-cell regeneration, reducingβ-cell apoptosis, the joint use of Exendin-4 is able to enhance the synergistic therapeutic effect of BMSCs. The various parts of the findings are summarized below, respectively.1.Isolation and identification of normal rats BMSCsBMSCs were isolated and cultured by whole bone marrow adherent culture method, the cells were observed under light microscope, their growth characteristics were recorded by growth curve, obtained BMSCs were induced to differentiate into the bone and fat cells, their surface markers (CD34, CD44, CD45, CD71, CD 105) were analysised by flow cytometry. The results showed that, BMSCs grew spindle-shaped whirlpool, they could make calcium deposits and lipid droplets in the course of osteogenic and adipogenic differentiation, BMSCs appeared to express osteoblast and fat cells characteristics, their identification of surface markers CD34, CD45 were negative, CD44, CD71 and CD105 were positive, the vast majority of obtained cells were BMSCs. This part we established a rat BMSCs cultured system, provided a stable cell model for the follow-up of stem cell research.2.Exendin-4 on rat BMSCs transdifferentiation into islet-like cells in vitroIn this section study, we used two-step method to induce BMSCs differentiate into islet-like cells, different concentrations of Exendin-4 (5,10,20,40,80nM) were added to observe its effect on differentiation. Dithizone staining were used to identify islet-like cell clusters, insulin-producing cells were identified through immune staining, the induced cells response to glucose stimulation were observed by radioimmunoassay, Real time-PCR were used to detect the expression of isletβ-cell-related genes Ngn3, PDX-1, Insl and Glut2. The results showed that islet-like cell transformation increased under Exendin-4, supernatant insulin levels were significantly higher, isletβ-cell-related genes Ngn3, PDX-1, Insl and Glut2 mRNA expression increased, within a certain range in a dose-dependent.3.The effect of Exendin-4 on BMSCs transplantation for treatment of T2DMT2DM rats were randomly divided into normal control group, T2DM model group, BMSCs transplantation group, Exendin-4 treatment group and BMSCs+Exendin-4 group, while Brdu labeled BMSCs transplanted from the rat tail vein to the rats body with T2DM, the effects of different interventions on the body weight, blood glucose, blood lipids, plasma insulin and adiponectin levels were observed, distribution and differentiation of BMSCs in rat pancreatic tissue were observed by immunohistochemistry and double-labeled immunofluorescence technique. The results showed that, BMSCs can migrate to the T2DM rat pancreatic tissue, and induce into insulin-producing cells. The use of Exendin-4 in BMSCs transplantation process can significantly reduce blood sugar, increase insulin positive area expression in rat pancreatic tissue, improveβ-cell function.4.Mechanism study of Exendin-4 on BMSCs transplantation for treatment of T2DMTo explore mechanism of BMSCs transplantation for the treatment of T2DM, TUNEL were observed in each group of islet cell apoptosis, Real-time PCR and Western blot methods were used to detect PDX-1 and insulin mRNA and protein expression in each group pancreatic tissues, islet Caspase3 and Bax protein levels were detected by immunohistochemistry and Western blot method. The results showed that, BMSCs transplantation increased islet PDX-1 and insulin mRNA and protein expression, promoted regeneration of islet P-cells, while able to reduce islet Caspase3 and Bax protein levels, reduce the apoptosis of islet cells, the joint use of Exendin-4 can enhance the therapeutic effect.Conclusions1.Through the whole bone marrow adherent methods, successfully isolated and cultured rat BMSCs, the obtained cells were confirmed as BMSCs through the biological characteristics and application of flow cytometry, for the follow-up experiments provided sufficient BMSCs products and a good experimental basis.2.In vitro studies showed that Exendin-4 can effectively promote BMSCs to differentiate into functional islet-like cells, these cells were positive in DTZ staining, they could secret insulin after glucose stimulation, they also express P-cell-related genes such as Ngn3,PDX-1,Ins1 and Glut2. The role of Exendin-4 on BMSCs differenciation was in a dose-dependent within a certain range.3.The use of high-sugar high-fat diet plus low-dose injection of STZ method successfully established rat model of T2DM, the T2DM model had obesity, hyperglycemia, dyslipidemia, hyperinsulinemia and insulin resistance characteristics, closed to human T2DM pathogenesis, they could be used to T2DM experimental research.4.Allogeneic BMSCs transplantation can treat rats T2DM, transplanted BMSCs can migrate to the T2DM rat pancreatic tissue and to differentiate into insulin-producing cells, while application of Exendin-4, can significantly reduce the T2DM rats blood glucose, increase insulin secretion, improve P-cell function.5. BMSCs transplantation increased islet PDX-1 and insulin mRNA and protein expression, promoted regeneration of islet P-cells, while able to reduce islet Caspase3 and Bax protein levels, reduce the apoptosis of islet cells, the joint use of Exendin-4 can enhance the therapeutic effect, this study gives a new insight into the cells treatment of T2DM.