Experimental Study On The Effect Of Self-designed KYL Organ Preservation Solution On Macaque Liver Preservation

Experiment 1:Preparation of self-designed organ preservation solutionObjectives:To adjust osmotic pressure,reprepare liver preservation solution(KLY solution) and make full preparation for further experiment.Method:Concentration of Ingredients is changed into weight ratio according to formulation. After weighing ingredients accurately by using electronic scale,dissolving them with equal distilled water,filtering residue by using sterile filter paper,filtering them by using vacuum filter pump,and determining PH value,osmotic pressure over and over,we package the solution and storage it at cold temperature.All the manipulation are performed in sterile preparation room.Result:Through many times of experiments,we successfully prepared liver preservation solution which is slightly brown,semitransparent,not precipitated,not muddy and whose PH value is 7.4±0.05,osmotic pressure is 340±5mmol/L.Conclusion:We have grasped experimental method of preparing KYL solution,successfully finished preparing cheap and nature-stable organ preservation solution,established experimental base for further study.Experiment 2:Establishment of orthotopic liver transplantation modelObjectives:To establish macaque orthotopic liver transplantation model to found experimental base for further study.Methods:16 Macaque,averagely weighed 5～7kg,sex not defined,donor weight is slightly less than that of recipient.We establish macaque liver transplantation model through non vein bypass.The main experiment steps were divided into three parts:1 donor group operation,mainly including donor blood collection,liver perfusion through cannulation into portal vein and abdominal aortic artery and donor liver resection.2 donor liver repair.3 recipient liver resection and donor liver implantationResult:Success ratio of 8 cases of liver transplantation operation is 100%.5 cases of hepatic artery-abdominal aortic artery bypass were performed and 3 cases of hepatic artery end to end anastomosis were performed.Survival time is beyond 6 hours in all cases.The longest survival time is 9 hours.Conclusion:There are a lot of difficulties and influential factor in establishing macaque liver transplantation model.Proficient surgical technique,perfect perioperative treatment and patient manipulation is key to successful operation.Members in our experiment team have rich experience in rats liver transplantation and clinical liver transplantation,which is main cause for us to succeed in our experiment.However,poor experiment condition and difficulty with anesthesia lead to short macaque survival time posttransplantation.Experiment three:Liver transplantationObjectives:To study the protective effect of self-designed KYL solution on ischemia reperfusion injury in macaque donor liver.Method:According to former experiment,macaques were divided into two groups:1 experiment group:KLY solution group(n=4) 2 control group:UW solution group(n=4).After cryopreserved 4 hours ex vivo,donor liver was implanted into recipient.At 30 min and 6 hours after donor liver reperfusion,We recorded bile production and determined AST,ALT,NO,ET-1 and TNF-αby taking blood samples through femoral artery.After taking liver tissue,we performed morphological examination under light and electronic microscope.Meanwhile,we determined expression of some apoptosis related factors(BCL-2,Bax and Fas) by means of immunohistological method.Result:We found bile secretion in both groups.Bile secretion production increased as time went on.At 30 min and 6 hours after donor liver reperfusion,serum AST and ALT levels in KLY group are slightly lower than those in UW group.No significant difference was found with levels of Serum NO,ET-1 and TNF-αin two groups.Histological and morphological change of liver tissue observed under light and electronic microscope is similar in two groups.Significant expression of Bax and fax and weak Bcl-2 expression was noticed through immunohistological staining.Conclusion:Analyzed from bile production,liver enzyme levels(AST and ALT),ischemia reperfusion injury related factors(NO,ET-1 and TNF-α)levels,apoptosis related factors levels and histomorphological change of liver tissues,the protective effect of self-designed KYL solution on ischemia reperfusion injury in macaque donor liver are similar to that of UW solution. Experiment 4:Experimental study of donor liver cryopreservation with non circulation perfusion ex vivoObjective:To study the protective effect of self-designed KYL solution on macaque donor liverMethod:After livers from recipients in experiment 3 were resected,they were divided into KYL solution group and UW solution group randomly.We perfused livers fast with preservation solution through portal vein until liver color turned into shallow yellow.At the same time,we flushed hepatic artery and bile duct.After perfusion,slow down vein perfusion,cryopreserved liver and sorage it at 0～4℃.At 2^nd,4^th,8^th,16^th,24^th hour after cryopreservation,we recorded bile product.AST,ALT,NO,ET-1 and TNF-αin solution flushed out of liver were determined.We took liver tissue to observe histomorphological changes under light and electronic microscope,perform immunohistological staining and determine expression of apoptosis related factors BCL-2,Fas and Bax.Results:Result:We found bile secretion of cryopreserved livers in both groups.Bile production decreased,while,AST,ALT levels in solution flushed out of liver increased as time went on.At 2nd 4th 8th 16th,after transplantation,AST and ALT levels are similar,however, compared with those in KYL solution group,they increased significantly at the 24th hour after transplantation in UW solution group.NO level decreased as time went on.NO level in KYL solution group was slightly higher than that in UW solution.ET-1 level increased as time went on. ET-1 level in KYL solution group was slightly lower than that in UW solution.TNF-αlevel increased as time went on..No significance was found with TNF-αlevel in two groups. Histomorphological changes are similar in two groups.Conclusion:Analyzed from bile production,liver enzyme levels(AST and ALT) at 2nd 4th 8th 16th after transplantation,the protective effect of self-designed KYL solution on ischemia reperfusion injury in macaque donor liver is similar to that of UW solution.At 24th after transplantation,as regards to liver enzyme,KYL solution showed slight advantage over UW solution,the protective effect of our self-designed organ preservation solution(KYL solution) on donor liver is similar to that of UW solution.Analyzed from protective effect on liver microcirculation,liver sinusoid endothelial cells,KYL solution showed slight advantage over UW solution.In view of histological and morphological changes,cryopreserved effect of KYL solution is similar to that of UW solution.