常温机械灌注修复长时间冷保存供肝的大动物实验研究
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摘要
目的探索常温机械灌注(NMP)在挽救大动物边缘供肝中的价值。方法 6只雄性、10~12月龄巴马小型猪分为静态冷保存(SCS)6 h组和SCS 24 h组,每组3只,分别于供肝获取后SCS 6 h、24 h后进行2 h NMP复苏。基于荷兰Organ Assist公司Liver Assist系统和供体猪自身血液,整合四通道生理仪器及自制灌注管路搭建NMP平台。在NMP过程中分别于灌注0、15、60、90、120 min 5个时间点收集灌注液用于肝功能(ALT、AST)检测及血气分析(p H、氧分压)。灌注结束取肝左叶相同位置少许组织,以10%甲醛固定,用于后续HE染色。结果 NMP开始时,SCS 24 h组ALT、AST水平略高于SCS 6 h组,之后AST、ALT水平均缓慢上升。NMP开始时SCS 24 h组p H值中位数为7.28,氧分压中位数为46 mm Hg(1 mm Hg=0.133 k Pa,下同),均低于SCS 6 h组(7.36,52 mm Hg),经过2 h NMP后SCS 24 h组p H值和氧分压逐渐接近甚至优于SCS 6 h组。HE染色发现,灌注前SCS 24 h组肝脏充血严重,炎性细胞浸润明显;NMP 2 h后,肝脏充血明显改善、炎性细胞减少,与SCS 6 h组无明显差异。结论基于Liver Assist系统和供体猪自身血液成功搭建的NMP平台,可有效改善长时间冷保存的边缘供肝质量,提示NMP在修复边缘供肝、扩展供肝来源方面具有重要临床及基础研究价值。
Objective To explore the application value of normothermic machine perfusion( NMP) for marginal liver in a porcine model. Methods Six healthy male Ba-Ma mini pigs( 10-12 months old) were equally divided into two groups according to the experimental design: static cold storage 6 hours( SCS 6 h) followed by 2 h NMP( SCS 6 h + NMP 2 h) group and( SCS 24 h + NMP2 h) group. The NMP perfusion platform was set up based on Liver Assist System,blood of donor,four-channel physiological instruments and self-made perfusion cannula integration. The perfusate of the two groups were collected at 0,15,60,90 and 120 min during NMP,which were used for liver function test and arterial blood gas analysis. A little liver tissue( fixed by 10% formaldehyde) was obtained from the left lobe,which was used for HE staining. Results The level of ALT and AST were higher in( SCS 24 h + NMP 2 h) group than those of( SCS 6 h + NMP 2 h) group at the beginning of NMP,and both rise gradually in the two groups after perfusion. The median p H and oxygen partial pressure value of( SCS 24 h + NMP 2 h) group were 7. 28 and 46 mm Hg at the beginning of NMP,bothhigher than those in( SCS 6 h + NMP 2 h) group( 7. 36,52 mm Hg),but the above indexes in( SCS24 h + NMP 2 h) group are progressively closer to or even better than those of( SCS 6 h + NMP 2 h)group. Pathology results showed that liver congestion and inflammatory cell infiltration were serious in( SCS 24 h + NMP 2 h) group before NMP,whereas after perfusion,these symptoms had improved markedly without significant difference to( SCS 6 h + NMP 2 h) group. Conclusion The NMP perfusion platform based on Liver Assist System and blood of donor could effective improve the quality of long-time cold preservation marginal livers,which means that it had important clinical and fundamental research value for marginal livers repairment and donor liver pool enlargement.
Objective To explore the application value of normothermic machine perfusion( NMP) for marginal liver in a porcine model. Methods Six healthy male Ba-Ma mini pigs( 10-12 months old) were equally divided into two groups according to the experimental design: static cold storage 6 hours( SCS 6 h) followed by 2 h NMP( SCS 6 h + NMP 2 h) group and( SCS 24 h + NMP2 h) group. The NMP perfusion platform was set up based on Liver Assist System,blood of donor,four-channel physiological instruments and self-made perfusion cannula integration. The perfusate of the two groups were collected at 0,15,60,90 and 120 min during NMP,which were used for liver function test and arterial blood gas analysis. A little liver tissue( fixed by 10% formaldehyde) was obtained from the left lobe,which was used for HE staining. Results The level of ALT and AST were higher in( SCS 24 h + NMP 2 h) group than those of( SCS 6 h + NMP 2 h) group at the beginning of NMP,and both rise gradually in the two groups after perfusion. The median p H and oxygen partial pressure value of( SCS 24 h + NMP 2 h) group were 7. 28 and 46 mm Hg at the beginning of NMP,bothhigher than those in( SCS 6 h + NMP 2 h) group( 7. 36,52 mm Hg),but the above indexes in( SCS24 h + NMP 2 h) group are progressively closer to or even better than those of( SCS 6 h + NMP 2 h)group. Pathology results showed that liver congestion and inflammatory cell infiltration were serious in( SCS 24 h + NMP 2 h) group before NMP,whereas after perfusion,these symptoms had improved markedly without significant difference to( SCS 6 h + NMP 2 h) group. Conclusion The NMP perfusion platform based on Liver Assist System and blood of donor could effective improve the quality of long-time cold preservation marginal livers,which means that it had important clinical and fundamental research value for marginal livers repairment and donor liver pool enlargement.
引文
1 Fayek SA,Quintini C,Chavin KD,et al.The current state of liver transplantation in the United States:perspective from American Society of Transplant Surgeons(ASTS)scientific studies committee and endorsed by ASTS council[J].Am J Transplant,2016,16(11):3093-3104.
2 Torre LA,Bray F,Siegel RL,et al.Global cancer statistics,2012[J].CA Cancer J Clin,2015,65(2):87-108.
3 Jia JJ,Zhang J,Li JH,et al.Influence of perfusate on liver viability during hypothermic machine perfusion[J].World J Gastroenterol,2015,21(29):8848-8857.
4 Jia J,Li J,Jiang L,et al.Protective effect of remote limb ischemic perconditioning on the liver grafts of rats with a novel model[J].PLo S One,2015,10(3):e0121972.
5 Guarrera JV,Henry SD,Samstein B,et al.Hypothermic machine preservation in human liver transplantation:the first clinical series[J].Am J Transplant,2010,10(2):372-381.
6 Dutkowski P,Polak WG,Muiesan P,et al.First comparison of Hypothermic Oxygenated PErfusion versus static cold storage of human donation after cardiac death liver transplants:an internationalmatched case analysis[J].Ann Surg,2015,262(5):764-770.
7 Ravikumar R,Jassem W,Mergental H,et al.Liver transplantation after ex vivo normothermic machine preservation:a phase 1(first-inman)clinical trial[J].Am J Transplant,2016,16(6):1779-1787.
8 Nasralla D,Coussios CC,Mergental H,et al.A randomized trial of normothermic preservation in liver transplantation[J].Nature,2018,557(7703):50-56.
9 贾俊君,李建辉,姜骊,等.肝移植供肝保存新途径:机械灌注[J/CD].中华移植杂志:电子版,2014,8(3):164-168.
10 Bral M,Gala-Lopez B,Bigam D,et al.Preliminary single-center canadian experience of human normothermic ex vivo liver perfusion:results of a clinical trial[J].Am J Transplant,2017,17(4):1071-1080.
11 van Rijn R,Karimian N,Matton APM,et al.Dual hypothermic oxygenated machine perfusion in liver transplants donated after circulatory death[J].Br J Surg,2017,104(7):907-917.
12 Banan B,Xiao Z,Watson R,et al.Novel strategy to decrease reperfusion injuries and improve function of cold-preserved livers using normothermic ex vivo liver perfusion machine[J].Liver Transpl,2016,22(2):333-343.
13 Westerkamp AC,Karimian N,Matton AP,et al.Oxygenated hypothermic machine perfusion after static cold storage improves hepatobiliary function of extended criteria donor livers[J].Transplantation,2016,100(4):825-835.
14 Watson CJ,Kosmoliaptsis V,Randle LV,et al.Preimplant normothermic liver perfusion of a suboptimal liver donated after circulatory death[J].Am J Transplant,2016,16(1):353-357.
15 He X,Guo Z,Zhao Q,et al.The first case of ischemia-free organ transplantation in humans:a proof of concept[J].Am J Transplant,2018,18(3):737-744.
16 Watson CJE,Jochmans I.From“gut feeling”to objectivity:machine perservation of the liver as a tool to assess organ viability[J].Curr Transplant Rep,2018,5(1):72-81.
17 Banan B,Watson R,Xu M,et al.Development of a normothermic extracorporeal liver perfusion system toward improving viability and function of human extended criteria donor livers[J].Liver Transpl,2016,22(7):979-993.
18 Goldaracena N,Spetzler VN,Echeverri J,et al.Inducing hepatitis C virus resistance after pig liver transplantation-a proof of concept of liver graft modification using warm ex vivo perfusion[J].Am J Transplant,2017,17(4):970-978.
19 中国医师协会器官移植分会,中华医学会外科学分会器官移植学组,中国肝移植注册中心科学委员会.中国移植器官保护专家共识(2016版)[J/CD].中华移植杂志:电子版,2016,10(3):98-106.
2 Torre LA,Bray F,Siegel RL,et al.Global cancer statistics,2012[J].CA Cancer J Clin,2015,65(2):87-108.
3 Jia JJ,Zhang J,Li JH,et al.Influence of perfusate on liver viability during hypothermic machine perfusion[J].World J Gastroenterol,2015,21(29):8848-8857.
4 Jia J,Li J,Jiang L,et al.Protective effect of remote limb ischemic perconditioning on the liver grafts of rats with a novel model[J].PLo S One,2015,10(3):e0121972.
5 Guarrera JV,Henry SD,Samstein B,et al.Hypothermic machine preservation in human liver transplantation:the first clinical series[J].Am J Transplant,2010,10(2):372-381.
6 Dutkowski P,Polak WG,Muiesan P,et al.First comparison of Hypothermic Oxygenated PErfusion versus static cold storage of human donation after cardiac death liver transplants:an internationalmatched case analysis[J].Ann Surg,2015,262(5):764-770.
7 Ravikumar R,Jassem W,Mergental H,et al.Liver transplantation after ex vivo normothermic machine preservation:a phase 1(first-inman)clinical trial[J].Am J Transplant,2016,16(6):1779-1787.
8 Nasralla D,Coussios CC,Mergental H,et al.A randomized trial of normothermic preservation in liver transplantation[J].Nature,2018,557(7703):50-56.
9 贾俊君,李建辉,姜骊,等.肝移植供肝保存新途径:机械灌注[J/CD].中华移植杂志:电子版,2014,8(3):164-168.
10 Bral M,Gala-Lopez B,Bigam D,et al.Preliminary single-center canadian experience of human normothermic ex vivo liver perfusion:results of a clinical trial[J].Am J Transplant,2017,17(4):1071-1080.
11 van Rijn R,Karimian N,Matton APM,et al.Dual hypothermic oxygenated machine perfusion in liver transplants donated after circulatory death[J].Br J Surg,2017,104(7):907-917.
12 Banan B,Xiao Z,Watson R,et al.Novel strategy to decrease reperfusion injuries and improve function of cold-preserved livers using normothermic ex vivo liver perfusion machine[J].Liver Transpl,2016,22(2):333-343.
13 Westerkamp AC,Karimian N,Matton AP,et al.Oxygenated hypothermic machine perfusion after static cold storage improves hepatobiliary function of extended criteria donor livers[J].Transplantation,2016,100(4):825-835.
14 Watson CJ,Kosmoliaptsis V,Randle LV,et al.Preimplant normothermic liver perfusion of a suboptimal liver donated after circulatory death[J].Am J Transplant,2016,16(1):353-357.
15 He X,Guo Z,Zhao Q,et al.The first case of ischemia-free organ transplantation in humans:a proof of concept[J].Am J Transplant,2018,18(3):737-744.
16 Watson CJE,Jochmans I.From“gut feeling”to objectivity:machine perservation of the liver as a tool to assess organ viability[J].Curr Transplant Rep,2018,5(1):72-81.
17 Banan B,Watson R,Xu M,et al.Development of a normothermic extracorporeal liver perfusion system toward improving viability and function of human extended criteria donor livers[J].Liver Transpl,2016,22(7):979-993.
18 Goldaracena N,Spetzler VN,Echeverri J,et al.Inducing hepatitis C virus resistance after pig liver transplantation-a proof of concept of liver graft modification using warm ex vivo perfusion[J].Am J Transplant,2017,17(4):970-978.
19 中国医师协会器官移植分会,中华医学会外科学分会器官移植学组,中国肝移植注册中心科学委员会.中国移植器官保护专家共识(2016版)[J/CD].中华移植杂志:电子版,2016,10(3):98-106.