Fluorescent Labelling Of The Canine Bone Endothelial Progenitor Cells And Marrow Mesenchymal Stem Cells And Tissue-Engineered Patch Construction

In previous tissue engineering studies,it has been demonstrated that tissue engineered constructs constructed from canine bone marrow endothelial progenitor cells(EPCs)and bone marrow mesenchymal stem cells(MSCs)can effectively repair defects.And diseased tissue.However,different studies,the etiology of the disease,the timing of cell transplantation,the dose of the transplant,the route of delivery,and the choice of the end point of the transplant may all lead to different results.Therefore,the tissue engineering constructs completed after the in vitro labeling of the cells are applied to the post-transplantation,which can help the stem cell tissue engineering to achieve the tracing of the differentiation in vivo and better understand the repair behavior of the stem cells.At present,there have been many achievements in the research and application of in vitro cell labeling technology at home and abroad.However,the long-term tracing effect of labeling cells in vivo and the difficulty of fluorescent labeling of EPCs have not yet been effectively solved.To deal with these issues,this study transfected EPCs and MSCs with fluorescent dyes and lentiviruses carrying fluorescent protein genes.Overcoming the characteristics of fluorescent proteins that are difficult to label EPCs while ensuring long-term traceability of cells.To investigate the proliferative activity of RFP-EPCs and GFP-BMSCs after transfection.Exploring a method for preparing human amniotic membrane acellular matrix,Detecting biocompatibility of decellularized amniotic membrane matrix using histology and scanning electron microscopy.To investigate the feasibility of growth of RFP-EPCs and GFP-BMSCs on decellularized amniotic membranes and determine the characteristics of tissue engineeringamniotic membrane patch.The results were as follows:1.Comparing the efficiency of four types of lentivirus-mediated fluorescent protein gene transfection in canine bone marrow EPCs,employing pLVX-mcherry-IRES-puro to transfect EPCs,RFP-EPCs with a positive rate of fluorescence markers above 90% and stably inherited were screened out.2.Through “Repeated Freezing and Thawing-SDS-DNA Enzymatic” method,human decellularized amniotic membrane matrix with no cell residue,complete three-dimensional structure,high flexibility,and good biocompatibility can be obtained as an ideal tissue engineering scaffold material.3.The tissue-engineered amniotic membrane patch was constructed by combining RFP-EPCs and GFP-BMSCs with high positive rate and suitable fluorescence intensity,combined with acellular amniotic membrane matrix.Obtained tissue-engineered amniotic membranes that have high cell proliferation activity,good adhesion,uniform growth distribution,and can be applied to repair traces in vivo.In summary,this study obtained a fluorescent protein gene pLVX-mcherry-IRES-puro that successfully transfected canine bone marrow EPCs and a transfection protocol suitable for canine bone marrow EPCs without affecting its proliferative activity,and the transfection rate was obtained.Very high,RFP-EPCs and GFP-BMSCs with appropriate fluorescence intensity;combined with decellularized amniotic membrane matrix prepared by repeated freeze-thawing-SDS-DNA enzymatic method,they have high proliferation activity,good adhesion,and uniform distribution of mixed growth.In vivo repair of traced tissue engineered amniotic membrane patches.