An Experimental Study Of Repairing Long And Tubular Urethral Defect With Bladder Extracellular Matrix Seeding With Transgenic EPCs And Other Seeding Cells

Objectives: To construct novel urethral repair materials in vitro using methods of Tissue Engineering, Molecular Biotechnology and stem cells for repairing long and tubular urethral defect. Looking for better ways to reconstruct the severely damaged urethra in vitro. To construct the lentiviral vectors including antimicrobial peptide LL-37, i.e. pGC-FU-LL37-GFP. To investigate the methods of how to obtain mononuclear cells from adult New Zealand rabbit's peripherial blood (PBMCs), and then culture PBMCs in vitro, inducing them differentiated into the endothelial progenitor cells (EPCs). To transfect the cultured EPCs with the constructed lentiviral vectors pGC-FU-LL37-GFP, and analyze the transfection efficiency. To investigate the methods of how to obtain urothelial cells from bladder tissue. To investigate the mutualistic symbiosis feasibility of bladder acellular matrix graft with various seeding cells by subcutaneous model and the effect in repairing long and tubular urethral defect.Methods:(1)To perform lentivirus packaging and detective the lentivirus titer. To extract target gene LL37from plasmid using PCR, and directed exchange with the already enzyme-digested lentiviral vector, then transform into competent Escherichia coli. To identify the clonal colonies using PCR, sequence and comparative analysis the positive clones.(2)To prepare recombinant virus vector encoding Lentiviral kernel including the Antimicrobial Peptide LL-37. All plasmids were extracted with endotoxin-free and high-purity methods to comply with experimental standards.(3)To withdraw10ml adult New Zealand rabbit's peripherial blood (arteria femoralis) and obtain the peripherial blood mononuclear cells (PBMCs) by density centrifugation with Ficoll solution. The PBMCs were induced into EPCs with b-FGF, VEGF-C and other growth factors.(4) To transfect the EPCs with the recombinant virus vector pGC-FU-LL37-GFP, and detect the protein expression of LL-37by Western Blot.(5) Bladder urothelial cells and bladder smooth muscle cells were choose as seeding cells. Urothelial cells were originated from New Zealand rabbit's bladder urothelium, smooth muscle cells were obtained from bladder muscle layer by biopsy methods, and primary cells culture and purify system were built to proliferate the above seeding cells. The proliferation characteristic of these seeding cells were tested by cell counting, MTT cell proliferation assay and immunofluorescence technique.(6) Bladder acellular matrix graft (BAMG) were prepared by ourselves which were got rid of cell component completely.(7)The prepared stable passaging seeding cells were planted onto both sides of bladder acellular matrix graft respectively, and then put the seeding cells-extracellular matrix composite materials into the abdominal subcutaneous of adult New Zealand rabbits for further growth.(8)To identify the composite materials periodically by histological methods. To perform the reconstruction of long and tubular urethral defect by the tabularized seeding cells-BAMG composite materials.Results:(1) The recombinant lentivirus vector pGC-FU-LL37-GFP wre well constructed. In virus titer detection, there were significant differences between group1.00E-04ul and control group, but no significant differences between group1.00E-04ul and group1.00E-05ul, so we concluded that there were viral particles in group1.00E-04ul. Supposing there was at least one viral particle in group1.00E-04ul, the virus titer was:2.00E+8TU/ml(1/(1.00E-04)*20=2.00E+5TU/ul=2.00E+8TU/ml), thus we concluded the lentiviral vector pGC-FU-LL37-GFP we constructed could be available for our next experiments.(2)Rabbit urothelial cells, smooth muscle cells and endothelial progenitor cells were successfully cultured. The primary cultured EPCs from peripheral blood tended to attach to the substratum of the culture flasks after24hours or so. Most EPCs appeared as round or oval shapes and started to stretch to be spindle shapes after about3days. As time went by, the number of the spindle cells increased more and more, and cell colonies started to appear. At about14days, the substratum of the flasks would be almost covered with EPCs, and presented to be typical slabstones appearance. Immunofluorescene staining by the Dil-Ac-LDL and FITC-UEA-1to identify the well-cultured EPCs were performed after about14days. The fluorescence staining showed that nearly80%cells appeared as saffron yellow (double staining positive), indicating that we achieved sufficient number of EPCs.(3) Immunofluorescence detection showed that the transfection efficency of target gene pGC-FU-LL37-GFP transfected cultured EPCs was about90%. Western Blot detection using Mouse Anti-GFP and Goat Anti-Mouse IgG showed that sample group OE (normal target transfected EPCs) had characteristic stripe in36-55KDa, the same as fusion protein LL37(18KDa+28KDa=46KDa). So we concluded that the cultured EPCs transfected by pGC-FU-LL37-GFP could secrete protein LL37effectively.(4) The bladder acellular matrix graft made by ourselves showed good biocompability when co-cultured with urothelial cells, transfected EPCs and smooth muscle cells in vitro and abdominal subcutaneous, and also had the ability to promote proliferation of the seeding cells and well cell distribution.Conclusions: We could successfully construct the recombinant lentiviral vector pGC-FU-LL37-GFP under experimental conditions. The constructed lentiviral vector could transfect the cultured EPCs obtained from the adult rabbit's peripherial blood efficiently, and the genetically modified EPCs we constructed would likely be used to participate in making up newly materials of tissue engineering to benefit the vascularization and anti-infection abilities. Combined with methods of abdominal subcutaneous embedding, we could obtain novel cell-acellular matrix composite tissue engineering materials which would be likely to repair long and tubular urethral defect in order to achieve genuine reproduction.