| Background/Aim:Diabetes mellitus (DM) is a complicated metabolic disease, with the fundamental treatment nowadays being diet control, insulin injections, islet or pancreas transplantation, which is limited because exogenous insulin injections fail to simulate normal insulin secretion in islet beta cells successfully and islet transplantation lacks organ donors. So far, stem cells with highly self-renewal and multi-directional differentiation potential have become a new hope for the treatment of diabetes. In this research, the effects of Adipose-derived stem cells (ADSCs) for treating type I diabetes in rats (Rattus norvegicus), and the expression changes of mRNA and protein of relevant signaling pathways in induction into islet-like beta cells of ADSCs and the mechanism of treating type I diabetes were investigated, so that to provide experimental and theoretical basis for the treatment of diabetes based on adult stem cells in the future.Methods:Rat ADSCs were separated and cultivated in vitro, carrying on the morphology observation, surface marker identification, and induction into islet p-cells with observation and identification using dithizone staining, immunohistochemistry, PCR, q-PCR and Western Blotting to detect insulin and islet-like cells related gene expression, compared with pancreas adult stem cells (PASCs). Then molecular signals involved in the process of ADSCs induction into islet beta cells were detected. The time dependent mRNA and protein levels change of key factor Dvl2, Gsk3 beta and beta-catenin in Wnt signaling pathways and Insulin were tested. T1DM rat model was built with Streptozocin (STZ), then PASCs, ADSCs and islet β-cells differentiated from ADSCs labeled by Dil were transplanted respectively. Combined with clinical examination system analysis, the changes in blood glucose, body weight, glucose tolerance, urine glucose, the output of urine, amount of water drinking before and after cell transplantation were observed. The islet morphology change was detected with pancreatic tissue paraffin section of HE staining. The distribution of cells labeled by Dil was detected with frozen section. The changes in expression of Wnt signaling pathway moleculars before and after the treatment of diabetic rats were detected with Western Blotting. Finally, the effect of ADSCs for treating type Ⅰ diabetes rats was concluded.Results:1. Pure ADSCs was separated and differentiated into islet β-cells successfully, and could form islet-like round cell clusters, which presented brown red with dithizone staining, were positive in PDX1, CK19, Nestin and Insulin with immumohistochemical staining, expressed insulin, PDX1 and glucagon detected with RT-PCR. The expression quantity of insulin, Gcg, Gck, GLUT2, Irs1 and Irs2 mRNA in islet p-cells differentiated from ADSCs were 0.69 times (P<0.05),2.12 times (P <0.01),1.15 times (P>0.05),0.58 times (P<0.01),1.38 times (P<0.01),0.83 times (P<0.01) than that of in PASCs using q-PCR, respectively. At the same time, the expression of insulin protein in islet P-cells differentiated from ADSCs was corresponding.2. In the process of ADSCs differentiation into islet β-cells, the expression quantity of insulin mRNA and protein were increased (P<0.01); the expression quantity of Gcg and Gck mRNA were increased (P<0.05); the expression quantity of Irs1 mRNA was decreased first and then increased (P<0.01); the expression quantity of Irs2 mRNA was increased first and then decreased (P<0.01). During this process, the expression quantity of Dv12, Lrp5 and GSK3β mRNA were increased (P< 0.05); the expression quantity of β-catenin mRNA was increased, but there was no significant difference (P>0.05); the expression quantity of Tcf712 mRNA was increased in 6-10 d (P>0.05). Correspondingly, the expression quantity of Dv12, GSK3β and p-GSK3β protein were increased (P <0.01), the change in expression quantity of non-p-β-catenin protein was not obvious (P>0.05), in accordance with the trend of changes in mRNA relative expression quantity.3. T1DM rat model was built successfully. After the transplantation of PASCs, ADSCs and islet β-cells differentiated from ADSCs, blood glucose levels, urine glucose levels, output of urine and amount of water drinking of T1DM rat model were decreasd, body weight was increased, glucose tolerance was improved, compared with diabetic control group and diabetic control group injected with PBS within 10 weeks. Besides, the treatment effects in cell transplantation group of islet β-cells differentiated from ADSCs was obvious better than that of other groups.4. The results of pancreatic tissue paraffin section of HE staining showed that β-cells in pancreatic islet were increased in cell transplantation groups, compared with PBS and diabetic control group, suggesting cell transplantation groups had repair function in islets of T1DM rats. The results of frozen section of Dil labeled red fluorescent cells showed that PASCs, ADSCs and islet β-cells differentiated from ADSCs had been transited to pancreas, and some of them been to spleen and kidney.5. The results of Western Blotting showed that the relative expression quantity of Dvl-2, non-p-β-Catenin, GSK3β, p-GSK3β and Insulin protein were decreased (P<0.01) in diabetic rats compared with normal rats; the relative expression quantity of Dvl-2, non-p-β-Catenin, GSK3β, p-GSK3β and Insulin protein were increased (P<0.05) in cell transplantation rats compared with diabetic rats.Conclusions:Based on our results, Wnt signaling pathway was activated in the process of ADSCs differentiation into islet β-cells, but it’s not obvious in accumulation of non-β-p-catenin protein. Cell transplantation groups of PASCs, ADSCs and islet β-cells differentiated from ADSCs had therapeutical effects to T1DM rats through transiting to pancreas, repairing injured islets. And the treatment effect in cell transplantation group of islet β-cells differentiated from ADSCs was more prominent than that of other groups. Wnt signaling pathway was inhibited in T1DM rats induced with STZ, the inhibition of Wnt signaling pathway had been relieved in cell transplantation groups. The above research conclusions provide theoretical basis, cell selection and good prospect for the treatment of diabetes. |
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