Construction And Preliminary Evaduation Of Tissue Engieering Pancreastic Islet

Objective: To induce bmscs directional differentiation into Islet-like clusters with pancreatic tissue lysis fluid.The Islet-like clusters was identified and preliminarily evaluated,and the relationship between morphological changes and related gene expression was discussed.Methods: The pancreatic tissue lysis fluid was used as inducer to construct microenvironment,BMSCs as seed cells,tissue engineered islet was constructed,and morphological and structural changes were observed under inverted microscope.The tissue structure and cell distribution were observed by paraffin-embedded tissue section He staining,and the cell group was screened by DTZ staining.The cell composition was preliminarily verified by Mallory staining.The ultrastructure was observed by SEM and TEM.The related proteins(insulin,glucagon,C-peptide,somatostatin,VEGF,VWF,Nestin,CK-19,?-type collagen,?-type collagen,Pdx-1,Ngn3,MAFA)were detected by immunofluorescence histochemical assay.According to the morphological changes in the process of cell differentiation,the differentiation process was divided into three stages(induced cells,islet-like cells,Islet-like clusters),and three stages qRT-PCR,Pdx-1,Ngn3,Mafa Insulin and glucagon were used..Results: 1.The tissue engineered islets are encapsulated with connective tissue-like membranes containing alpha-like cells,beta-like cells and ?-like cells,and endothelial-like cells are visible in the structure,and ? collagen is detected.2.The regular distribution of ?-like cells and ?-like cells was similar to that of normal islet structures.3.TEM observation,the structure can detect four kinds of cells containing secretory particles,the cytoplasm is visible in the multi-level lysozyme and a large number of vacuoles.4.Immunofluorescence histochemical Staining assay: insulin,glucagon,C-peptide,somatostatin,VEGF,VWF,Nestin,CK-19,?-type collagen,?-type collagen,Pdx-1,Ngn3,Mafa with the prolongation of induction time,the positive cells increased gradually and the fluorescence intensity increased.5.QRT-PCR detection: Pdx-1,Ngn3,Mafa,insulin,glucagon gene expression regularity.Conclusion: The tissue engineered islet is composed of a variety of somatic cells,which can detect ?-like cells,?-like cells,?-like cells,endotheliallike cells,exocrine adenoid cells and collagen fibers containing collagen I.The tissue engineered islet has the primary function of secreting glandular cells.In the process of Bmscs differentiation into Islet-like clusters,the related gene expression of occurrence and differentiation is sequential,which is similar to the normal development process of pancreatic islet.Bmscs in a specific microenvironment,the natural differentiation trend is to form a variety of somatic cells and eventually develop into tissue-like structures.