The Application Of Mesenchymal Stem Cell Conditioned Medium In Diabetic Animal Model And The Preliminary Exploration Of Optimization Measures

Diabetes mellitus is a common problem which is harmful to human health. There is still a high incidence of diabetes mellitus around the world. It is characteristic with the deficiency of beta cell in mass and the failure of islet function which will cause high glucose disorders. So it is the key to find ways to realize the beta cell regeneration for the treatment of diabetes and diabetes cure. Mesenchymal stem cells(MSCs) are adult stem cells which have the ability of self-renewal and multi-directional differentiation. The system of infusion can stimulate the pancreatic regeneration and then improve the high glucose disorders through paracrine actions.Based on the previous research, it has shown that the infusion of MSC-CM to the rats with the diabetes mellitus will make the regeneration of beta cells in pancreatic tissue in a short time. But it is limited in improving the high blood glucose of diabetic rats and the specific mechanism is unclear. In addition, many studies has shown that after the MSCs pretreatment in certain conditions could change the factor secreted.Then is there any possibility to improve the efficacy of MSCs in treating diabetes by changing the environment and make the glucose pretreatment of certain concentration?In this study, we induced the animal model of diabetes by the intraperitoneal injection of single high dose(65mg/kg) of STZ and after extracting 10 times of the ultrafitration of MSC-CM, we made the infusion. Then we analyzed the islet function recovery based on the results of serological indexes changes, and assessing regeneration ability of beta cell according to the pancreas immunofluorescence staining. In addition, we explored the mechanism of MSC-CM in promoting pancreas regeneration through insulin/ki67 double immunofluorescence. Furthermore, we preconditioned UC-MSCs with 200mg/dl,250mg/dl,350mg/dl,450mg/dl glucose and detected cell proliferation. And then we figured out the optimal glucose concentration and preconditioned time dependent on gene expression and the changes of secreted factors. We infusion HG-MSCs to diabetic rats to assess the therapeutic effect, preliminary exploration of MSCs therapy optimization methods.Results showed that MSC-CM infusion during early phase(<7 days) ameliorated the hyperglycemia significantly, increased serum insulin and C peptide level and the number of beta cells. The immunofluorescence staining of the cell proliferation index has shown that the MSC-CM treatment in the early stage(<7 days) Insulin/ki67 positive cells increased significantly. Moreover, preconditioning MSCs with 250mg/dl glucose could decrease cell senescence, increasing gene expression of FGF-19 and FGF-21, and also elevating the protein content of FGF-19 and FGF-21. Comparin g with normal MSCs, preconditioned MSCs improved bloo-d glucose and i slets function more significantly in diabetic rats, and enhan-cing the recove ry of islet function more effectively.This study confirmed that the infusion of MSC-CM to diabetic animal will play a role in the therapeutic effects after improving the beta cell proliferation. When the concentration of glucose pretreatment is 250mg/dl, the pretreatment time is 72 h, the UC-MSCs senescence is controlled and the stem cell secretion spectrum has improved significantly, it will further enhance the efficacy of UC-MSCs to the treatment of diabetes. This study has provided the new theory for the treatment of diabetes. Furthermore, it has provided a new way for improving the efficacy of the treatment of diabetes by high glucose pretreatment of MSCs.