Preliminary Evaluation Of Secretory Function Of Islet Like Structures

Objective: The differentiation of BMSC were induced by pancreatic tissue lysate,pancreatic islet-like structures were formed and identified,explore morphological changes of the cells during differentiation.Aim to explore the relationship between structural morphological changes and secretory function in the formation of pancreatic islet-like structures.Methods: Pancreatic tissue lysate was used to induce BMSC to differentiate into pancreatic islet-like structures,changes of cells and structures' morphology were observed under inverted phase contrast microscope.The structures and cells' morphology were observed by HE staining in paraffin embedded,differentiated cell clusters were screened by dithizone staining,distribution of cells were identified by Mallory staining.Insulin,C-peptide,Glucagon,Somatostatin proteins were detected by immuno-cytochemical staining,expression of Pdx1,Ngn3,MaFA,Nkx6.1,Inulin,Glucagon,Somatostatin and GLUT-2 genes were detected by real-time fluorescence quantitative PCR.The typical structures in the induction system were selected.Inulin and C-peptide in the cell culture solution were detected by enzyme-linked immunosorbent assay to evaluate the secretion function of the islet-like structures.Results:1.The morphological changes of cells during induction and differentiation.In the early stage of induction,the scattered cells increased and gathered into cell clusters.Cell masess contracted and the cells density increased,structures surrounded by the membrane were gradually formed.2.Insulin,C-peptide,Glucagon,Somatostatin positive cells could be detected in pancreatic islet-like structures.Nkx6.1 expression was low in the early stage of induction,continued to high expression later.The expression of GLUT-2 was low in the early induction,began to express in the later stage,reached the peak when pancreatic islet-like structures were formed.3.Enzyme-linked immunosorbent assay showed that the concentrations of Insulin and C-peptide in the culture medium increased at first,then tended to be stable.Conclusion: The differentiation and development of bone marrow mesenchymal stem cells induced by pancreatic tissue lysate formed islet-like structures.The morphological changes showed obvious regularity: after the induction,the scattered cells increased and aggregated into cell clusters;then the cell clusters contracted,the cell density increased.Structures' surfaces were surrounded by membranes were gradually formed.Induction of early Nkx6.1began to express,continued to increase,displayed the states of cells differentiation;as structures became more complex,GLUT-2 was gradually higher,indicating that they differentiates into cells with a transporter of glucose transporter and remained stable higher levels;functional development of islet-like structures were initially assessed by examining the association of Insulin and C-peptide's secretion.