The Impact Of Adenovirus-mediated Ex Vivo Gene Transfer Of CGRP On The Preservation Of Donor Lung And The Ischemical Reperfusion Injury Of Rat Lung Allografts

The shortage of donor lung preservation time and the ischemical reperfusion injury(IRI)are still problems in lung transplantation. If these problems were solved, there will be ahigher operating successful rate. The common preservation time is only 4-6h in clinical lungtransplantation, which mainly involve in low temperature preservation and cold ischemicalinjury, and IRI is one of the main factors causing to lung transplantation failure. At present,gene therapy developed rapidly in the organ transplantation domain. Calcitonin gene-relatedpeptide(CGRP) has the function of vessel enlargement, anti-inflammatory andimmunomodulatory properties. In this topic, we constructed enhanced green fluorescentprotein (EGFP) labeled recombinant adenovirus (Ad)containing CGRP(Ad5-CGRP-EGFP),established rat model of donor lung preservation and orthotopic left lung allografttransplantation by "muff-like" anastomosis technique through improved technique, andobserved the expression of CGRP through fluorescence, to investigate the effect of genetransfer on subsequent of low temperature preservation of donor lung and cold ischemicalinjury and ischemical reperfusion injury after lung transplantation.Part1. Construction of Enhanced Green Fluorescent Protein (EGFP)Labeled Recombinant Adenovirus Mediated Calcitonin Gene-relatedPeptide (CGRP) Objective To investigate construction of enhanced green fluorescent protein (EGFP)labeled recombinant adenovirus containing calcitonin gene-related peptide (CGRP).Methods To identify CGRP, the recombinant adenovirus Ad5-CGRP-EGFP was constructedby us in Ad5Easy system based on the homologous recombination in bacteria. Results Theadenovirus Ad5-CGRP-EGFP was quickly constructed successfully by homologousrecombination bacteria using AdEasy system. Conclusion Ad5-CGRP-EGFP can besuccessfully constructed by homologous recombination in bacteria, which lay the foundationfor further research for gene transfer of CGRP.Part2. Construction Rat Models of Donor Lung Preservation andOrthotopie Left Lung Allograft TransplantationObjective To establish donor lung preservation and orthotopic left lung allografttransplantation rat model by improved technique. Methods This study improved thetraditional cuff technique in many aspects, including graft retrieval, cuff self-making,recipient pneumonoresection, "muff-like" vessel and trachea anastomosis techniques. In onegroup with 15 rats, the left lung allograft was stored at 4℃low-potassium dextran glucosesolution (LPDG solution) for 12 h, wet to dry weight ratio (W/D) was detected andhistological changes were observed under microscopy and electromicroscopy. In anothergroup with 30 clean-grade SD rats, the left lung allograft was stored at 4℃LPDG solutionfor 6 h, and then the left lung was transplanted into the recipient rat without microscope.When the transplanted lung had been reperfused for 4 h. Airway pressure, blood gas analysiswere detected before and after the right hilus of lung was block up, then left lung was cut,and W/D, histological changes under microscopy and electromicroscopy were determinedrespectfully. The reproducible ability of this model about donor lung preservation andischemical reperfusion injury was evaluated respectively. Results The warm ischemic timeof left donor lung before packed was nearly 0, time from perfusion to picked was (1±0.5)min. 30 rats with receiving transplantation were performed the anastomosis of pulmonaryartery, pulmonary vein and bronchus by single person without microscope, the time was (3±2) min, (7±2) min, (1.5±0.5) min respectively. The total graft ischemic time was(21±2)min, mean operation time was(55±6)min. The operation successful rate was 90%,survival rate arrived 100%. The experimental result demonstrated that the model couldduplicated the change of the lung preservation and the injury of ischemia-reperfusion.Conclusion The merit of this model corresponds with the construction of rat lungtransplantation model. All the manipulations were performed by single person withoutmicroscope. The harvesting of donor lung was so fast that ischemic time was nearly 0, thecuff structure and the anastomosis technique were simpler than before. The key point ofpneumonoresection was pulmonary vein protection, which increased the successful rate. Theimproved "muff-like" technique shortened the total operation time. This model duplicatedthe changes of lung preservation and the ischemia-reperfusion injury of lung allograft, anddemonstrated to be an ideal and suitable model for some research such as donor lungpreservation and IRI.Part3. Effect of Adenovirus-mediated Gene Transfer of CalcitoninGene-related Peptide on Preservation of Rat Donor Lung.Objective To investigate the effect of adenovirus-mediated CGRP gene transfer onpreservation of rat donor lung, and explore the mechanisms. Methods A formwork wasconstructed to preserve the rat donor lung using the left lung of SD rat as donor by improvedtechnique. The experiment was divided into four groups: group A, receiving sham operation;group B, receiving natural solution; group C, receiving empty adenovec; group D,receiving Ad- CGRP-EGFP gene transfection. In group B, the donor lung was ex vivoretroperfused by 4℃LPDG through pulmonary vein. In group C, the donor lung was exvivo retroperfused by empty adenovec which was diluted by 4℃LPDG fluid. In group D,the donor lung was retroperfused by Ad- CGRP-EGFP which was diluted by 4℃LPDG fluid.After being perfused, the donor lung was preserved for 12 hours in the 4℃LPDG fluid andW/D ratio, superoxide dismutase(SOD) activity, malondialdehyde(MDA) content andmyeloperoxidase(MPO) activity were detected. Graft pathologic histology was examined by light microscope and electron microscope. Specimen was examined by fluorescencemicroscope to examine gene transfection. The content of CGRP in lung tissue was measuredby radioactive immunoassay. The expression of TNF-a, IL-10 was detected byimmunohistochemical staining. The expression of TNF-a, IL-10 were quantified by ELISA.Apoptosis cell deaths were determined by in situ terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL). Results Afterthe donor lungs had been preserved for 12 hours, the samples of group C, receiving emptyadenovec and the samples of group D, receiving Ad-CGRP-EGFP gene transfection weredetected with a result of strong green fluorescence by fluorescence microscope; but thesamples of control group, group B, were detected without green fluorescence response. Thecontent of CGRP in group D was significantly higher than that in group B and group C byradioactive immunoassay (P＜0.01). This result indicated that donor lung in group Dsuccessfully express specific CGRP aim gene. Compared with control group, W/D ratio andMDA content decreased in the lung tissue of group D(P＜0.01),SOD activity increased in thelung tissue of group D(P＜0.01), but there was no significantly difference in MPO activity.Tissue impairment, cell infiltration, alveolar space effusion, interstitial edema in group Dwere minimal changes in histopathologic examination by light microscope. By electronmicroscope observation, the number of apoptosis cell and necrosis cell was less in group D,alveolar epithelium was nearly integrated and alveolar wall blood capillary impairment wasless in group D. Expression scores of TNF-a was attenuated(P＜0.01),but expression scores ofIL-10 enhanced (P＜0.01) through immunohistochemical staining. Detected apoptosis cellswere less by TUNEL (P＜0.01). Conclusion The experiment displayed adenovirus-mediatedgene transfer of CGRP expressed CGRP aim gene efficiently , lessened the donor lungimpairment which is induced by low temperature and cold ischemia effect, conservedendothelial cell, lessened graft disorganization, reduced the expression of TNF-a, increasedthe expression of IL-10,conserved the donor lung.Part4. The Impact of Adenovirus-mediated Gene Transfer of CGRP on theIschemical Reperfusion Injury of Rat Left Lung Allograft Objective To investigate the effect of adenovirus-mediated gene transfer of CGRP onischemia-reperfusion injury of rat left lung allograft and explore the mechanisms. MethodsA left lung transplantation formwork was constructed using SD rat as donor and recipient byimproved muff-like method. The experiment was divided into four groups: group A,receiving sham operation; group B, receiving natural solution; group C, receiving emptyadenovec; group D, receiving Ad- CGRP-EGFP gene transfection. In group B, the donorlung was ex vivo retroperfused by 4℃LPDG through pulmonary vein. In group C, thedonor lung was ex vivo retroperfused by empty adenovec which was diluted by 4℃LPDGfluid. In group D, the donor lung was retroperfused by Ad- CGRP-EGFP which was dilutedby 4℃LPDG fluid. The donor lung which had been perfused was perserved for 6 hours inthe 4℃LPDG fluid. Then the left lung orthotopic transplantation was performed. After leftlung allograft was reperfused for 4 hours, we blocked hilum of right lung, then measuredairway pressure and made arterial blood gas analysis to measure PaO₂ and PaCO₂. At last, ratwas executed. Lung allograft tissue was harvested to be detected. W/D ratio, superoxidedismutase(SOD) activity, malondialdehyde (MDA) contents and myeloperoxidase(MPO)activity were detected. Graft pathologic histology was examined under light microscope andelectron microscope. Specimen was detected under fluorescence microscope to examine thegreen fluorescence changes and get the message of gene transfection. CGRP in lung tissuewas measured by radioactive immunoassay. The expression of TNF-a and IL-10 weredetected by immunohistochemical staining. The expression of TNF-a, IL-10 were quantifiedby ELISA. Apoptosis cell death was determined by in TUNEL. Results After all allograftswere preserved for 6 hours before transplantation and assessed 4 hours after reperfusion.Specimen of group C and group D expressed strong green fluorescence under fluorescencemicroscope, group B expressed nothing. The contents of CGRP in group D was obviouslyhigher than B and C (P＜0.01) by radioactive immunoassay, which demonstrated thesuccessful transgene expression of CGRP in group D pulmonary allograft. Compared withcontrol group, the airway pressure, PaO₂. and the activity of SOD increased, W/D ratios,the content of MDA and the activity of MPO decreased (P＜0.01) in group D. Tissue edema, interstitial inflammation and exudation alleviated under light microscope, less apoptosisand necrosis cell was observed under electron microscope, and no significant interval thickerof alveolar capillary endothelium cell and alveolar epithelial cell, fewer cellular swelling,complete alveolar epithelium and almost no blood capillary injury were display. Lowerexpression of TNF-a and higher expression of IL-10 were detected byimmunohistochemistry (P＜0.01). Less apoptosis cells were found by TUNEL (P＜0.01).Conclusion The efficient expression of CGRP through adenovirus-mediated gene transferof AD-CGRP-EGFP was demonstrated by fluorescence observation. CGRP has the characterof blood vessel enlargement, anti-inflammatory and immunization, which can protectendothelial cell, reduce interstitial inflammation and graft inflammatory reaction, regulatedown the TNF-a expression, regulate up IL-10 expression, reduce apoptosis and ischemicalreperfusion injury of lung allografl, protect donor lung in IRI. So, this study demonstratedthat the expression of CGRP in lung isografts may become a new strategy, which can preventand reduce the injury of low temperature and cold ischemic and IRI.