| Background:With the rapid development of lung transplantation in recent years,both single and bilateral lung transplantation and heart-lung transplantation had successed,so that lung transplantation became the effective means to treatment all end-stage lung disease now.But the shortage of donor lung is still a major obstacle to the development of lung transplantation.Although kidney and liver donors arerelatively plentiful,only 15%to 25%of lungs from donorsof at least one other organ aretransplanted which is the lowest graft acceptance rate of any solid organ.If the method of lung preservation is safe and effective,it is that can improve the quality of donor lung,increase the success rate of lung transplantation,and ease the shortage of donor lung.At present,the common method of lung preservation was single keeping vitro lung at the low temperature after single pulmonary perfusion.and the preservation time of vitro lung is with 4hours.The common preservation methods all contained lung perfusion,but only in the early stage.Vitro lung is single kept at low temperature after lung perfusion which limits the vitro lung to obtain energy,shorts the duration of vitro lung preservation and hinders the remote transport of vitro lung.EVLP?Ex vivo lung perfusion?has always been focused on lung transplantation technology.Although many scholarsat home and abroad had done a lot of research on direct perfusion,retrograde perfusion,low temperature perfusion.normal temperature perfusion,pretreatment and technology bronchial artery recanalization,current research on the injury mechanism of vitro lung has no breakthrough,and lacking a safe and effective and convenient protection strategy.The limitations and problems of the traditional lung protection are increasingly prominent.Objectives:By comparingthe protective effect of the three preservation methods in vitro canine lung,including continuous pulsatile perfusion of a heart-lung machine and discontinuous pressure perfusion and single low temperature,to investigate the mechanism of lung injury in vitro and to explore the best method of vitro lung preservation.Methods:1.Thirty Mongrel dogs were randomly divided into 3 groups.Bilateral lungs were completely obtained with mechanical ventilation.The vitro lungs were preserved by the way of the continuous perfusion of a extracoporeal circulation machine,discontinuous pressure perfusion and cold-storage preservation.And the specimens were collected in light of the time point.2.Hemodynamic,respiratory,and blood gas parameters were continuously monitored and digitally recorded hourly.Parenchymal biopsies were used for quantification of wet to dry weight ratio.3.Antiscorbutate method was used to measure phospholipid?PL?.Thin-layer chromatography was used to detect L-alpha-Dipalmitoylphosphatidylcholine?DPPC?.The real-time fluorescent quantitative PCR and Westblot were used to detect SP-B and SP-C.4.The apoptosis of the neutrophils in the bronchoalveolar lavage fluids in vitro lungs were assayed with flow cytometry.Immunohistoehemistry was used to detect the macrophages phagocytosis of the apoptotic neutrophils.The total protein concentration was measured with BCA.Interleukin?IL-la/2/6/8?were detected with ELISA.Results:1.Changes of pulmonary function in vitro by three different methods of lung preservation?1?EVLP was performed for 8 h.In each experimental group,several parameters ofpulmonary functionshowed different degrees of decline.On the one hand,some parametersdecreased gradually,including pulmonary oxygenation ability?PO2/FiO2?andglucose.On the other hand,some parameters increased gradually,such as PCO2,lactic acid and W/D.?2?In the extracorporeal circulation group and pressure perfusion group,the function of pulmonary in vitro canine were significantly better than those of the single low temperature group,in which oxygenation ability?PO2/Fi02?and glucose were significantly higher,but PCO2,lactic acid and W/D were more lower.?3?In the extracorporeal circulation group,the function of pulmonary in vitro canine were significantly better than the pressure perfusion group,in which Cdyn,oxygenation ability?PO2/FiO2?and glucose were significantly higher,but pulmonary vascular resistance,PCO2,lactic acid and W/D were more lower.2.The expression of pulmonary surfactant in three different methods of lung preservation?1?The expression of DPPC/PL:In each experimental group,the ratio of DPPC/PL showed a trend of decline in bronchoalveolar lavage fluid of vitro lung?P<0.05?.At each time point,the ratio of DPPC/PL in the extracorporeal circulation group was more higher than that of the othertwo groups?P<0.05?,and the pressure perfusion group was more higher than that of the single low temperature group.?P<0.05?.?2?The expression of SP-B mRNAand SP-C mRNA?The expression of SP-B mRNA:In theextracorporeal circulation group,the expression of SP-B mRNAhad significantly decreased from 1h to 7h?P<0.05?.In the pressure perfusion group the expression of SP-B mRNAhad significantly decreased from lh to 6h?P<0.05?.The expression of SP-B mRNA in the e single low temperature group displayed a trend of decline from 1h to 6h and from 7h to 8h?P<0.05?.At each time point,the expression of SP-B mRNA in the extracorporeal circulation group was more than that ofthe single low temperature group?P<0.05?At 2h?3h?5h?6h and 8h time point,the expression of SP-B mRNA in the extracorporeal circulation group was more than that of the pressure perfusion group?P<0.05?.The expression of SP-B mRNA in the pressure perfusion group was more than that of single low temperature group from 3h to 6h?P<0.05?.?The expression of SP-C mRNA:In both of the extracorporeal circulation and the pressure perfusion groups,the expression of SP-C mRNA displayed a trend of decline from 1h to 8h?P<0.05?.In the extracorporeal circulation group,the expression of SP-C mRNAhad significantly decreased from 1h to 7h?P<0.05?,but it was no significant difference from 7h to 8h?P>0.05?.The expression of SP-C mRNA in the extracorporeal circulation group was more than that ofthe single low temperature group from 3h to 8h?P<0.05?.At 3h?5h?6h and 8h time point,the expression of SP-C mRNA in the extracorporeal circulation group was more than that of the pressure perfusion group?P<0.05?.At 4h?5h?7h and 8h time point,the expression of SP-C mRNA in the pressure perfusion group was more than that of the single low temperature group?P<0.05?.?3?The expression of SP-B protein and SP-C protein?The expression of SP-B protein:In each experimental group,the expression of SP-B protein was no significant difference from 1h to 3h?P>0.05?,but it was significantly decreased from 4h to 8h?P<0.05?.At each time point,the expression of SP-B protein in the extracorporeal circulation group was more than that ofthe single low temperature group?P<0.05?.At 1h?2h and 4h?8h time point,the expression of SP-B protein in the extracorporeal circulation group was more than that of the pressure perfusion group?P<0.05?.The expression of SP-B protein in the pressure perfusion group was more than that of the single low temperature group from 4h to 8h?P<0.05?.?The expression of SP-C protein:In both of the extracorporeal circulation and the pressure perfusion groups,the expression of SP-C protein showed a trend of decline?P<0.05?.In the single low temperature group,the expression of SP-C protein was no significant difference from 1h to 2h?P>0.05?,but it was significantly decreased from 3h to 8h?P<0.05?.At each time point,the expression of SP-C protein in the extracorporeal circulation group was more than that ofthe single low temperature group?P<0.05?.At 3h and 5h?8h time point,the expression of SP-C protein in the extracorporeal circulation group was more than that ofthe pressure perfusion group?P<0.05?.At 1h and 3h?8h time point,the expression of SP-C protein in the pressure perfusion group was more than that of the single low temperature group?P<0.05?.3.Expression of inflammatory responseparameters in three different methods of lung preservation?1?In each experimental group,the apoptosis of the neutrophils showed a trend of decline.In each group,there was no significant change of neutrophil apoptosis from 1h to 3h?P>0.05?,but the apoptosis of neutrophils decreased significantly from 4h to 8h?P<0.05?.At each time point,the number of apoptotic neutrophils were no significant difference from 1h to 3h among three groups?P>0.05?;At the 4hpoint,the apoptotic neutrophils in the both ofextracorporeal circulation group and the pressure perfusion group were significantly more than that of single low temperature preservation group?P<0.05?;At the 5h-8h point,the number of apoptotic neutrophils had a significant difference among three groups?P<0.05?,which the apoptosis of the neutrophils in the extracorporeal circulation group were more than that of the pressure perfusion group,and the pressure perfusion group was more than that of single low temperature preservation group.?2?In each experimental group,the macrophages phagocytosis of apoptotic neutrophils displayed a trend of decline?P<0.05?.At the same time point,the macrophages phagocytosis of apoptotic neutrophils in extracorporeal circulation group was better than the other two groups?P<0.05?.At the 1 h?2h point,there were not significant difference between the discontinuous pressure perfusion group and single low temperature group?P>0.05?,but the macrophages phagocytosis of apoptotic neutrophils inthe discontinuous pressure perfusion group was better than thesingle low temperature group?P<0.05?.?3?In each experimental group,there was a significant positive correlation between the apoptosis of neutrophils and phagocytosis of macrophagesat each time point?r=0.98,P<0.01;r=0.988,P<001;r=0.964,P<0.01?.?4?In each experimental group,the expression of IL-la,IL-2,IL-6 and IL-8 displayed a trend of increase?P>0.05?.At many time point,the expression of IL-la,IL-2,IL-6 and IL-8 had a significant difference among three groups?P<0.05?,which the expression of IL in the extracorporeal circulation group was less than that of the pressure perfusion group,and the pressure perfusion group was less than that of single low temperature.Conclusions:The safety time of lung preservation was extended to 6?8h by the extracorporeal circulation perfusion.With extracorporeal circulation perfusion,the effect of lungpreservation are significant better than that of pressure perfusion preservation and single low temperaturepreservation.In addition,the effect of lungpreservation with pressure perfusion preservation was more better than single low temperature preservation. |
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