The Basic Research Of Multi-way Preparation And Application Of Beta-like Cell

Diabetes has already become a chronic disease which does enormous harm to the human health only inferior to the malignant tumor. The traditional pharmacological treatment can not change diabetes’ advancement. Transplantation of pancreatic gland or the island of langerhans has brought the dawn for cure of diabetes, however, the shortage of donor cells and the immunologic rejection has hindered its further promotion. It is very important to seek new source for transplantation. The bone-marrowed mesenchymal stem cells (BMSCs) have become the ideal seed cells because they were easy to get, had multi-direction differential potential, easy to dye by the exogenetic gene. Therefore, the BMSCs separated from the adult body have been raised in vitro and increased massively, then were transformed to beta-like cells by the organization project. The beta-like cells were autotransplanted to diabetes individual, which would become a new way to solve the shortage of donor cells and the immunologic rejection effectively.In this research, the mouse’s BMSCs were taken out and raised in vitro, the multi cell factors induction, co-cultured induction, the transfer of insulin gene and transfers of PDX-1, NeuroD1 and the MafA gene were used to prepare beta-like cells. Then, the beta-like cells were transplanted to the different organs of diabetes model mouse; the best spot for transplantation was selected out. Moreover, the BMSCs of beagle canines were taken out and transformed to beta-like cells, then were autotransplanted to diabetes modle of beagle canines through artery intervention in diabetic Beagle canines. The long-term curative effect and the biological security were observed. The entire research was divided into four parts. Objective:The BMSCs were induced to beta-like cells. Methods:The bone marrowed mesenchymal stem cells of mice were separated and raised in vitro. The multi cell factors induction and co-cultured induction were used to transform the BMSCs to beta-like cells, and its’biological characteristic were observed in vitro. Results:The beta-like cells were gained from two kinds of methods and could secret the insulin. The beta-like cells induced by multi-cell factor can response to the glucose concentration, but the outputs of two methods were low. Conclusion:The beta-like cells induced by multi-cell factor induction were more close to beta cells in the function.The second part:the research of preparation of beta-like cell from the bone marrowed mesenchymal stem cells by transgeneObjective:The beta-like cells were prepared from BMSCs by transgene. Methods: Exogenetic insulin gene was transfected to mBMSCs by a recombinant retrovirus MSCV and expressed in the cells. The insulin secreted beta-like cells were gained. Another way, PDX-1. NeuroD1 and the MafA genes were transfected to mBMSCs by the adenovirus. the endocardial insulin gene was stimulated to express, and the beta-like cells were obtained. The efficiency of the two ways was compared each other. Results:The first way could prepare insulin secreted beta-like cells and the efficiency of this way was high. However, this kind of beta-like cells can not response to the glucose concentration. The second way’s beta-like cells could not only secrete the insulin but also be able to response to the glucose concentration, but the efficiency of production was low. Conclusion:Two kind of transgene ways could product beta-like cells, but the beta-like cells’ function of the first way need to improve. The beta-like cells of the second way were closer to beta cells in the function than first way’s, but the efficiency of production need to be raised. The third part:the research the choice of transplantation spot for beta-like cells and its’distribution in vivoObjective:Find out the best spot for beta-like cells’transplantation, and study its’ distribution in vivo. Methods:The beta-like cells transfected with insulin gene were transplanted into the liver, the kidney, the pancreatic gland, the spleen and the tail vein of diabetes model mouse separately. After the transplantation, the blood glucose, the weights, IPGTT and the insulin level of diabetic mice were monitored and contrasted between groups. The acceptor organs were analysised by immunity of tissue slice, the best transplant spot was choosed. The BMSCs prepared from GFP mouse were transplanted to diabetes mouse through the tail vein, and the distributions of the cells were observed in vivo. Results:In the group which cells were transplanted in liver, the blood glucose were dropped down, the weights were increased, IPGTT and the insulin level of blood serum were better than other groups. After transplanted by the tail vein, BMSCs were distributed to pancrease and some other organs. Conclusion:The liver was the ideal spot for transplatation of beta-like cells. BMSCs had the tendency to distribute and nest to pancrease and some other organs.The fourth part:Research of long-term curative effect and the security of the autotransplantation of the beta-like cells to diabetes Beagle caninesObjective:To study the long-term curative effect and the security of the autotransplantation of the beta-like cells to diabetic Beagle canines. Methods:BcMSCs were isolated from the bone marrow of Beagle canines, expanded, and transfected by a recombinant retrovirus MSCV carrying the human insulin and EGFP genes. The hepatic artery involvement technology was utilized to autotransplant the beta-like cells to the live of diabetic Beagle canines. Each physiological norm was monitored before and after transplantation, and the long-term stable treatment result was estimated. Simultaneously, the liver physiology functions, the pathology and tectology were used to confirm the biological security. Results:The autotransplantation of beta-like cells to diabetes Beagle canines made the blood glucose. the body weight, and the insulin level of the canines improve obviously, and the curative effect continued more than 80 weeks. The liver functions of the diabetes Beagle canines were normal and the transplant spot of liver has not been discovered the formation of tumors. So it was a reliable biological security way. Conclusion:The autotransplantation of beta-like cells to diabetes Beagle canines has the long-term curative effect to the diabetes, and was a relatively reliable biological security way, and the hepatic artery involvement technology was an ideal way for transplantation.