便携式在体机械灌注设备运行稳定性及保护无心跳供体器官的实验研究

Experimental study on operation stability and protection effect of portable in situ machine perfusion device for non-heart-beating donor organs

目的探讨便携式在体机械灌注设备(IMPD)运行稳定性和保护无心跳大动物供器官的作用。方法6只健康清洁巴马小型猪麻醉后开腹,分别经腹主动脉、下腔静脉插管并连接到自主研发的便携式IMPD,静脉注射氯化钾骤停心脏,观察5 min无复跳,启动设备对动物器官进行常温(37℃)机械灌注。调整离心泵转速和灌注流量以控制静脉血氧饱和度维持在60%~70%,持续灌注12 h,每2小时为观察时间点,记录设备管路流量、离心泵转速和流量/转速比值,检测肝、肾功能和血液内环境指标,灌注结束后获取胆总管、肝、肾和小肠组织,HE染色后光镜下评估病理改变情况。采用重复测量资料单因素方差分析比较灌注过程中各时间点运行参数、肝肾功能和血液内环境指标,组内两两比较采用LSD法,P<0.05为差异有统计学意义。结果全部实验均完成12 h灌注,灌注过程中设备运行稳定,无故障或停机意外,管路流量、离心泵转速和流量/转速比值均稳定。ALT、总胆红素(TBIL)和谷氨酰转移酶(GGT)稳定,灌注开始至结束各时间点与术前相比,差异均无统计学意义(P均>0.05)。灌注开始后AST缓慢上升,灌注第8、10和12小时AST分别为(73±21)、(90±30)和(114±48)(U/L),与术前相比,差异均有统计学意义(P均<0.05)。在实验猪心脏停跳的5 min内,乳酸在灌注开始时便处于峰值,自灌注第8小时起,乳酸数值与峰值相比差异均有统计学意义(P均<0.05)。K+浓度水平自灌注第2小时起各时间点与灌注开始时相比,差异均有统计学意义(P均<0.05)。ALT、TBIL、GGT、血尿素氮、血清肌酐、血气分析指标、Na^(+)、Ca^(2+)和Cl^(-)浓度水平稳定,灌注开始至结束各时间点数值与灌注开始时相比,差异均无统计学意义(P均>0.05)。自灌注开始后持续有胆汁生成、尿量产生。供肝灌注12 h的过程中,肝脏始终保持外观红润、质地柔软、边缘锐利。灌注结束后,胆总管、肝、肾和小肠组织病理结果示组织结构完整,无淤血和大面积坏死征象。结论便携式IMPD可以稳定灌注无心跳猪供器官12 h,可以较好维护胆总管、肝、肾和小肠器官。

Objective To investigate the operation stability and protective effect of portable in situ mechanical perfusion device(IMPD)on donor organs from large animals without heartbeat.Methods Six healthy and clean Bama miniature pigs got open surgery through a midline incision after anesthesia,and were spiled through the abdominal aorta and inferior vena cave respectively,and connected to self-developed portable IMPD.The heart was suddenly arrested by intravenous injection of potassium chloride,and no relapse was observed in 5 min.The mechanical perfusion of animal organs was performed by starting the device at room temperature(37℃).The rotational speed and perfusion flow of the blood pump were adjusted to maintain the venous blood oxygen saturation at 60%-70%.The Bama miniature pigs were got perfusion for 12 h(every 2 h for the observation point in time),during which the equipment pipeline flow,pump rotation speed and flow rate/speed ratio operation parameters were recorded and the liver and kidney function and arterial blood gas analysis were detected.The common bile duct,liver,kidney and intestine tissues were obtained and observed in HE staining to assess the pathological changes.One-way repeated measures analysis of variance was used to compare the operating parameters at different time points,hepatic and renal function,and internal environment indexes between the 2 groups,and that between groups with Least Significant Difference.P<0.05 was considered statistically significant.Results The 12 h perfusion was expected to be completed in all experiments.During the perfusion process,equipment operated stably without failure or shutdown accident.Pipeline flow,blood pump speed and flow/speed ratio were all stable.ALT,total bilirubin(TBIL)and gamma glutamyltransferase(GGT)were stable,and the preoperative physiological values were no significant difference from other time points(beginning to the end of perfusion)(all P>0.05).AST increased slowly after the beginning of perfusion,and AST was(73±21),(90±30)and(114±48)U/L at 8,10 and 12 h after perfusion,respectively,with statistical significance compared with the preoperative physiological values(all P<0.05).Within 5 min of cardiac arrest,lactic acid reached the peak at the beginning of perfusion,and there was a significant difference between the value of lactic acid and the peak value from 8th hour of perfusion(all P<0.05).There were significant differences in K+concentration levels at each time point from the 2nd hour of perfusion compared with the beginning of perfusion(all P<0.05).ALT,TBIL,GGT,BUN,creatinine,blood gas analysis indexes,concentrations of Na^(+),Ca^(2+)and Cl^(-)concentrations were stable,and there was no statistical significance in the values from the beginning to the end of perfusion compared with the preoperative physiological values(all P>0.05).Bile formation and urine production continued from the beginning of perfusion.The liver remained ruddy in appearance,soft in texture and sharp edge during 12 h of donor liver perfusion.After the perfusion,the histological results of the common bile duct,liver,kidney and small intestine showed that the histological structure was intact,without congestion,and no signs of substantial area necrosis.Conclusions Portable IMPD can stably perfuse the donor organs of non-heartbeat pigs for 12 h,and can maintain the common bile duct,liver,kidney and small intestine well.