| Backgroud and Objective:Diabetes has become a common chronic metabolic disease, which seriously threats to human health, particularly for cardiovascular complications. The loss of islet beta cell is an important cause of leading to diabetes, type1diabetes is caused by autoimmune destruction of pancreatic islet beta cells, whereas, type2diabetes is generated by insulin resistance. Therefore, islet transplantation will become the most effective treatment in diabetic treatment. But islet transplantation requires a lot of islet cells, immune inhibitors will be used to suppress autoimmune response in long-term after transplantation, as the result, islet transplantation application has been restricted. Recent studies have taken attention to induce the stem cells and their differentiation into pancreatic islet cells for transplantation to treat diabetes. Bone marrow mesenchymal stem cells are a class of non-hematopoietic stem cells, which are present in adult marrow of high self-renewal and differentiation potential. They are easy to obtain and cultivation, possess excessive amplification ability in vitro and immunoregulatory properties, which may become ideal alternate sources of beta cells.Bone marrow mesenchymal stem cells (BMSCs) are a class of nonhematopoietic stem cells with multidirectional differentiation potential, compared with other stem cell, BMSCs in vitro was easier to separate, develop, and easier to stable transfection and expression of exogenous gene, autologous bone marrow MSCs transplantation can prevent receptor from immune rejection, hence, BMSCs are regarded as ideal source of islet beta cells.Pancreatic duodenal homeobox-1(PDX-1) is Pancreatic specific transcription factor, which has been playing an important role in development of the pancreas and proliferation differentiation and in maintaining Pancreatic beta cell function.This study intends to transmit PDX-1into bone mesenchymal stem cells at passage5and3by lipid transfection technique, and which would be induced into insulin-producing cells, so that diabetic rats model was constructed. Giving ground insulin-producing cells to transplant into diabetic rats by tail vein in, and comparing the curative effect of two generations of BMSCs by analyzing the monitoring of blood glucose, insulin, C peptide and weight and glucose tolerance test and pancreas immunohistochemical method.Material and Methods:1. In vitro transfection induction and identification of the rat BMSCs1.1BMSCs were isolated and cultured by whole bone marrow adherent culture method1.2Identification of BMSCs cell by immunocytochemical analysis1.3Transfection of PDX-1into rat BMSCs by Liposome transfection technique1.4Bone mesenchymal stem cells at passage3and5transfection by PDX-1will have induced differentiated into insulin-producing cells2. Transplantation of BMSCs into diabetic rat2.1Construction of diabetic rats model2.2Rat BMSCs were transplanted into diabetic rats model through the tail vein2.3Monitoring blood sugar, insulin, C peptide and weight and glucose tolerance test2.4Immunofluorescence histochemical analysis of pancreatic tissueResults:1. Isolation and cultivation of rat BMSCs. Immunofluorescence results were consistent with characteristics of BMSCs.2. PDX-1had been transfated into bone mesenchymal stem cells at passage3and5by lipid transfection technique, RT-PCR results show that specific amplification bands were detected in bone mesenchymal stem cells at passage5and3,and specific amplification bands were not detected in the control group, the result showed that PDX-1has been successful transfected into rat BMSCs. Bone mesenchymal stem cells at passage3and5transferred by PDX-1had been induced into insulin-producing cells, dimethyl sulfoxide (DMSO) and high sugar as inducers, Bone mesenchymal stem cells at passage5and3transmitted by PDX-1gradually gathered and formated islet-like cell aggregates, the result have not appeared in the control group, and the islet-like cell aggregates had been identified by dithizone staining.3. Our study has setting up to developing a high-dose STZ diabetes model. The rat maintaining had been high of blood glucose (28.6±3.49mmol/L) upto4weeks of STZ treatment.4. Compared with diabetic control group, blood glucose levels significantly decreased in group A and group B6days after transplantation(P<0.05), and blood glucose levels of group B were significantly lower than group B9days after transplantation (P<0.01). At2weeks after transplantation, secrum insulin levels of group A and group B were significantly increased in comparison to control group(P<0.05), and secrum insulin levels of group B were significantly higher than that of group A at3weeks after transplantation (P<0.05). The diabetic rat in group A and group B showed significant improvement in the glucose tolerance test compared with diabetic control rat (P<0.05). Result of immunofluorescence histochemistry showed that insulin-producing cells were survived and secreted insulin after transplantation.Conclusion:PDX-1transfected bone mesenchymal stem cells at passage3and5been differentiated into insulin-producing cells,which have capability to secrete insulin。 Differentiation of PDX-1transfected bone mesenchymal stem cells at passage5into insulin-producing cells was better effective in treating diabetic rat than that of PDX-1transfected bone mesenchymal stem cells at passage3. |
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