| Part Ⅰ Investigating the mechanisms of human umbilical cord mesenchymal stem cells differentiation into insulin secreting cellsInducing human umbilical cord mesenchymal stem cells(Uc-MSCs)into islet-like clusters capable of synthesizing and secreting insulin can solve clinical donor shortage,immune rejection and possible ethical issues.To explore the mechanisms that initiate the directed differentiation of Uc-MSCs into islet-like clusters is meaningful that is expected to identify potential targets for drug action,so as to promote the directed differentiation of autologous pancreatic MSCs,which will be of great significance for the effective remission and treatment of diabetes.Our subject using RNA-sequencing technology to study the changes in mRNA levels involved in the early stage of directed differentiation of Uc-MSCs that identified and confirmed the pathways and key genes associated with the initiation of early differentiation of Uc-MSCs,which may serve as potential drug targets for subsequent researches and therapy.Firstly,Uc-MSCs were isolated from neonatal umbilical cords,and were induced to differentiate into islet-like clusters,which could be specifically stained scarlet with dithizone,highly expressed islet specific genes,and secreted insulin under the stimulation of glucose in vitro.Transplanted the differentiated islet-like clusters into type Ⅰ diabetes mice under renal capsule can significantly reduce the blood glucose level.This implied that our research successfully established a directed differentiation method of Uc-MSCs into islet-like clusters.Secondly,using RNA-sequencing technology,we analyzed mRNA level change in samples of early differentiation cells from different time points(undifferentiated,differentiated 1.5 hours,differentiated 6 hours),data showed that the KEGG enrichment of significant difference genes enriched in the TGF-beta signaling pathway,the referred difference genes also contained in the cell differentiation related genes,which TGFB3 expression amount continues to increase.These suggested that TGF-beta signaling pathway and TGFB3 may play important role in the early stages of differentiation.Finally,inhibition of TGF-beta signaling pathway by pathway inhibitor LY2109761,our research observed the blockage of differentiation process,and inhibition of TGFB3 expression by sequentially specific shRNA also blocked the differentiation process.These suggested that TGF-beta signaling pathway and TGFB3 do play important role in the early stages of differentiation and may serve as potential drug targets.Part Ⅱ Investigating the role and mechanisms of human umbilical cord mesenchymal stem cells derived exosomes in islet regenerationIn recent years,exosomes from various cells have played an important role in the diagnosis and treatment of diseases.They can deliver their own proteins,nucleic acids and other substances,or serve as carriers that deliver other drugs to the target sites.Existing studies have shown the therapy effect of mesenchymal stem cell on diabetes and related complications,our study using human umbilical cord mesenchymal stem cells(Uc-MSCs)derived exosomes to treat type I diabetic mice which found the recovery effect of the pancreas islet integrity,promotion of islet regeneration effect.This study provide guiding significance for clinical use of exosomes as a safe and effective treatment for diabetes.Firstly,exosomes were isolated from Uc-MSCs culture supernatant by ultra-high speed centrifugation.The exosomes were round or elliptic in shape,with particle sizes ranging from 30-150nm,and specifically expressed the exosomes markers TSG101,CD9 and CD63.Secondly,in vitro experiments showed that the isolated exosomes could be infiltrated into pancreatic tissue with a thickness of about 1-2mm,and the integrity of the islet was recovered by promoting the differentiation of pancreatic ductal cells into islet β cells.Finally,in vivo experiments showed that the isolated exosomes can effectively regulate the blood glucose level of type I diabetes mice,improve β cells function to a certain extent.The treatment groups of Uc-MSCs and its exosomes showed a significant increase in the number of intact islets in the pancreatic tissue,possibly by promoting the differentiation of pancreatic ductal cells into islet β cells.In conclusion,this study has confirmed Uc-MSCs derived exosomes could promote type I diabetic mice pancreas islet regeneration,provide important guiding significance for the treatment of diabetes. |
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