Research On Effects And Mechanism Of Human Bone Marrow Mesenchymal Stem Cells Alone And With Glucagon-like Peptide1in Treatment Of Type-1Diabetes Mice

Objective:1、On the base of our previous work,we confirm further the effect of human bone marrow mesenchymal stem cells (hBMSC)transplantation in type1diabetes mice models of NOD/Ltj mouse and to explore the possible mechanism of hBMSCs in Type-1diabetes;2> Observe and compare the curative effects of hMBSCs alone and with GLP-1transplantation in treatment of NOD/Ltj mice to validate the cooperative effect of hBMSCs combined with GLP-1in treating Type-1diabetes, and investigate possible mechanism of this cooperative effect.3、Further confirm if hBMSCs could differentiate cross mesoderm into insulin-secreting cells.4、Observe the security of MSC transplantation(tumorigenicity et al).Methods:1、Establishing model of Type-1Diabetes mice:Purchased female NOD mice at the age of7-8weeks were injected with streptozotocin(STZ) at a daily dosage of40mg/kg for five consecutive days. Random blood glucose exceeding13.9mmol/1for2consecutive days indicates establishment of T1D model.2、Grouping:50mice established T1D model were divided into two groups.18mice for Control group(0W=4,2W=5,4W=5,6W=4),18mice for High-dose hBMSC Transplantation with GLP-1 injection group(HMSC+GLP-1)(2W=6,4W=7,6W=5)and12for High-dose hBMSC Transplantation group(HMSC)(2W=4,4W=4,6W=4).3、Observation period:The mice established T1D model in HMSC and HMSC+GLP-1group were injected hBMSC(2*106) via tail vein. The mice in HMSC+GLP-1group were injected liraglutide(0.2ug/mouse) intraperitoneally everyday after established T1D model until executed. Mice in both transplantation groups were executed respectively2/4/6weeks after established T1D model. Mice in control group were executed0/2/4/6weeks after established T1D model.4、Index and method:The mice were raised in clean environment. Blood glucose and weight of the mice were measured every week. Mice were executed at corresponding stage and blood from orbital venous was collected. The proportions of CD4+CD25+Foxp3+Treg and CD4+/CD8+T lymphocyte in the spleen mononuclear cell suspension were measured by flow cytometry. The contents of mouse insulin and human insulin in the mouse peripheral serum were measured by ELISA method. The level of inflammation cytokines in peripheral serum of the mice was determined by Luminex method. The pancreases were dyed by HE to observe the pancreatic structure and the content of insulin in the islets was measured using HC.Results:1、Effect on blood glucose:The blood glucose level of mice increased obviously after established T1D model; the blood glucose level of transplantation groups after treatment in1w,2w,3w and4w declined notably compared to the control group(P<0.01); when the two transplantation groups were compared with each other, the level of blood glucose in the HMSC+GLP-1group is significantly lower than the HMSC group in the4th week.2、Effect on weight:Since2weeks after established T1D model, the weight of mice in the HMSC group was higher than that in the control group(P<0.05) and has statistically significant difference on3W/4W(P<0.01). The weight of mice in the HMSC+GLP-1group was greatly higher than that in the control group on2W and3W(P<0.01). The weight of mice in the HMSC+GLP-1group was lower than that in the HMSC group on1W(P<0.05), and significantly lower on4W(P<0.01).3、Effect on immunomodulation:Compared with the control group, the proportion of Treg in transplantation groups rised greatly (P<0.01) on2W and rised on4W(P<0.05). Difference between the two transplantation groups showed no statistical significance in all observing weeks. The proportion of CD4+T lymphocytes in the HMSC+GLP-1group was higher than that in the HMSC group on2W/4W/6W(P<0.05).During the observation period, the difference of the proportion of CD8+T lymphocytes among three groups has no statistical significance(P>0.05).4、Effect on insulin:Compared with the control group, the content of mouse insulin in peripheral serum was higher on2W(P<0.05) in the two transplantation groups. The content of mouse insulin in the HMSC+GLP-1group was higher than that in the HMSC group on2W/6W(2W:P<0.01;6W:P<0.05). Besides, no human insulin was detected in peripheral serum of the mice in our research.5、Effect on inflammation cytokines:Proinflammatory factor:Level of IFN-γ:Compared with the control group, the level of IFN-γ in HMSC+GLP-1group fell on2W(P<0.05); the level of IFN-γ in both transplantation groups decreased on4W(P<0.05); Between the two transplantation groups, there was no statistical difference(P>0.05).Anti-inflammatory factor:Level of IL-4&IL-10:Compared with the control group, the level of IL-4in the HMSC+GLP-1group rised on both2W and4W(P<0.05); the level of IL-10in the HMSC+GLP-1group rised significantly on2W(P<0.01). During the observation period, the level of IL-4&IL-10in the HMSC group has no significant difference compared with the control group. The difference of the level of IL-4between HMSC and HMSC+GLP-1group has no statistical significance. The level of IL-10in the HMSC+GLP-1group was higher than that in the HMSC group on2W/4W(P<0.05).Level of TGF-β:Compared with the control group, the level of TGF-β in the HMSC+GLP-1group rised greatly on2W/4W(P<0.01) while that in the HMSC group was higher than the control group only on4W. The level of TGF-β in the HMSC+GLP-1group was significantly higher than that in the HMSC group on2W/4W(P<0.01).6、Effect on pancreatic structure and content of insulin in the islets:The mice’s pancreases were dyed by HE method. It is observed that compared with the control group, mice in the two transplantation groups has less lymphocyte infiltration, islet-like structure proliferation and alleviation of insulitis. Analysed with Image-ProPlus, the IHC results showed that the content of insulin in the islets of mice in the two transplantation groups was greatly higher than that of the control group(P<0.01); and the content in the HMSC+GLP-1group was again higher than that in the HMSC group(P<0.01).Conclusions:1. After hBMSC transplantation, level of blood glucose of NOD/Ltj mice decreased significantly; the content of insulin in both peripheral serum and the islets rises obviously, and damaged islet structure got repaired and proliferated to some extent.2. The proportions of Treg and Th increase after transplantation of hBMSC, while proportion of Tc decreases. Meanwhile, the level of IFN-γ falls and the levels of IL-4, IL10, and TGB-β rise.3. In the peripheral serum of NOD mice, we didn’t detect secretion of human insulin and thus no evidence of hBMSCs differentiating cross mesoderm into insulin-secreting cells is found in this research.4. Injection of GLP-1promotes proliferation of islet in peripheral serum, improves the content of cytokine(by elevating the level of IL-10and TGF-β) and promotes proliferation of islet B cells, but it may leads to weight loss of the mice.