The Study On Decellularization And Recellularization Of Rat Pancreas With Induced Insulin-producing Cells

Objective Preparing rat decellularized pancreatic scaffolds and cultivating BM-mMSCs drived insulin-producing cells(IPCs)in rat decellularized pancreatic scaffolds through the circulation perfusion device and evaluating the situation of the cells for growth and function.And it provides the experimental and theoretical support for recellularizing insulin-secreting organs in pancreas engineering.Methods1.BM-mMSCs were differentiated into IPCs via infected by PDX-1,MafA and NeuroD1.Immunofluorescence and qRT-PCR were detected of gene expression of the pancreas markers of the IPCs.ELISA assay was used to evaluate the secretory volume of insulin at different concentration of glucose.2.Rat decellularized pancreatic scaffolds were obtained by perfusion of 1%TritonX-100/0.1%ammonia through the splenic artery.The scaffolds were evaluated by HE staining,Masson staining,scanning electron microscopy(SEM),GAG and DNA quantitative analysis,extracellular matrix staining and biological compatibility.3.The circulation perfusion device was established.The IPCs were recellularized into the decellularized rat pancreatic scaffolds,cultured in the circulation perfusion device.Histological examination,immunofluorescence and qRT-PCR were used to observe the situation of the cells for growth and function.Results1.The expression of β cell-related genes and insulin were showed through qRT-PCR and immunofluorescence after BM-mMSCs infected by PDX-1,MafA and NeuroD1.IF and qRT-PCR showed gene and protein expression of insulin 1,insulin 2,PDX-1,NeuroD1,MafA,Glut 2,Nkx6.1and islet-1.The abtained IPCs were glucose responsiveness showed by the results of ELISA assay.2.After perfused of 1%TritonX-100/0.1%ammonia,HE staining and Masoon staining showed residual cells were almost removed and the collagen fiber components were retained largely.IF staining showed collagen Ⅰ and fibronectin were largely retained.The ultrastructure was examed by SEM.GAG content was decreased slightly and DNA content of decellularized scaffolds was very low.Biocompatibility tests showed good biocompatibility of pancreatic acellular scaffolds.3.The differentiated cells with insulin secretion function were implanted in the acellular pancreatic scaffold of rats and cultured by circulating perfusion.Histological examination showed that the differentiated cells were colonized and grew in the interstitium and vascular of the scaffold.The expression of insulin was positive by immunofluorescence.The expression of insulin gene was detected by qRT-PCR.The level of gene expression was significantly improved.Conclusions1.Multiple pancreatic transcription factors can synergistically promote BM-mMSCs differentiation and achieve significant insulin synthesis and secretion.2.The acellular scaffold of rat pancreas perfused with 1%Triton X-100/0.1%ammonia not only preserved the basic vascular structure,but also had good physical and chemical properties and biocompatibility.3.Pancreatic acellular scaffold can provide a good environment for IPCs to grow and function.