大鼠边缘性体积供肝肝移植模型的建立和控制门脉压力对供肝保护作用研究

【作者】 卢景宁；

【导师】 彭民浩；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2008， 博士

【摘要】 第一部分大鼠边缘性体积供肝肝移植模型的建立目的本部分实验通过建立不同体积供肝大鼠原位肝移植模型,摸索出边缘性体积供肝大小的范围,探讨大鼠边缘性体积供肝肝移植模型建立的方法,为研究小肝综合征的发病机制及防治措施提供一个较为理想易于复制的小动物模型。材料和方法选用健康、9～12周龄、体重190～250g的雄性SD大鼠192只作为实验动物,取周龄相差1～3周,体重相差20～30克的大鼠,以小体重大鼠作为供体,采用离体减体积方式切除不同肝叶设计出不同体积供肝的大鼠原位肝移植模型。本部分实验设立4个实验组:A组,全肝移植组（n=24）;B组,50%部分肝移植组（切除肝左叶、双乳突叶,保留肝中叶、右后叶和右前叶作为供肝,使供肝与受体肝比值控制在50%左右,n=24）;C组,30%部分肝移植组（除保留肝中叶外,其余四叶均予以切除,使供肝与受体肝比值控制在30～35%之间,n=24）;D组:小于30%部分肝移植组（保留右前叶、右后叶和双乳突叶,切除肝中叶和左叶,使供肝与受体肝比值杏?0%,n=24）。供、受体均使用乙醚持续开放式吸入麻醉,大鼠原位肝脏移植模型建立采用的双袖套法,不进行肝动脉血管重建。观察各组术后肝功能ALT水平、门静脉压力和存活率,比较分析各组间ALT、门静脉压力及累积存活率之间的关系。结果全肝移植组（A组）7天累积存活率为100%,50%部分肝移植组（B组）7天累积存活率为83.3%,30%部分肝移植组（C组）7天累积存活率为16.7%,小于30%部分肝移植组（D组）受体均于术后48小时内死亡。各组组间存活率经log rank（mantel-cox）方法比较,A组与B组累积存活率组间差异无统计学意义,P=0.148;C组与A组累积存活率有显著性差异,P＜0.001;C组与B组累积存活率比较,P＜0.001,组间差异有显著的统计学意义,C组与D组两组存活率有显著性差异,P＜0.001。受体移植前基础门静脉压力各组间差异无统计学意义,P＞0.05。A组术中开放门静脉后1小时内门静脉压力稳定,B组开放门静脉后门脉压力虽有小幅上升,但门脉压力仍保持相对稳定,而C组和D组开放门静脉后门脉压力显著升高,15分钟达到峰值,两组门脉压力峰值分别较基础门脉压力升高65%和82%。两组门静脉压力30分钟后开始回落,至45分钟到60分钟逐渐稳定,但仍维持于一高位。将供肝体积大小与各时间点的门静脉压力进行Pearson相关分析后发现,供肝体积的大小与基础门静脉压力无相关性,相关系数r=0.249,P=0.241;而供肝体积的大小与开放门静脉后5、15、30、45和60分钟门静脉压力呈负相关性,（相关系数分别为r=-0.926,r=-0.936,r=-0.904,r=-0.903,r=-0.902,P＜0.001）。肝移植术后24小时各组ALT水平明显升高,四组中以D组水平最高,C组次之,B组和A组最低,各组间差异有统计学意义,P＜0.001。将供肝体积大小与各组移植术后24小时ALT水平进行Pearson相关分析后,结果显示供肝体积的大小与移植术后24小时ALT水平呈负相关性,相关系数r=-0.704,P＜0.001。结论1、大鼠减体积供肝肝移植模型供肝体积大小的安全界限为50%标准体积;体积介于30～35%标准体积的供肝应视为边缘性体积移植物,小于30%标准体积的供肝可视为超小体积移植物。上述标准可作为大鼠减体积供肝肝移植模型小体积移植物选择的参考标准。2、大鼠边缘性体积供肝肝移植模型的建立,可选取周龄相差1～3周,体重相差20～30克的大鼠,以小体重的大鼠作为供体,除保留肝中叶外,其余四叶均予以切除,可以使供肝与受体肝湿重比值控制在30～35%之间。3、大鼠小体积供肝肝移植模型中,供肝体积大小与开放门静脉后的门脉压力升高幅度及术后24小时ALT水平呈负相关性,小体积供肝肝移植术中开放门静脉后急剧升高的门静脉压力对供肝造成的应力性的损伤,是导致肝移植术后小体积移植物功能损害、受体存活率降低的主要原因。第二部分暂时性控制门脉压力对大鼠边缘性体积移植物保护作用的研究目的本部分实验拟对大鼠边缘性体积供肝肝移植术后小体积移植物早期损伤的机制进行初步研究,探讨在大鼠边缘性体积供肝肝移植术中使用三甘氨酸-赖氨酸-加压素,降低再灌注期间门静脉压力对边缘性体积供肝早期损伤的保护作用机制。材料和方法选用健康、体重相差20～30克的雄性SD大鼠120只,以小体重大鼠为供体,按第一部分介绍的方法建立大鼠边缘性体积供肝肝移植模型,设立三甘氨酸-赖氨酸-加压素实验组和生理盐水对照组,实验组的用药方案为:受体于肝脏切除前10分钟和供肝恢复门静脉灌注后立刻经股静脉缓慢推注三甘氨酸—赖氨酸—加压素50ug/kg/份0.5mL,对照组按同样的时间和径路注射生理盐水0.5mL。观察两组术后一般情况、门静脉压力和存活率,两组分别于术后6小时和24小时各取6只受体在无菌条件下采取下腔静脉血标本和肝组织标本。测定血清中ALT、AST水平、血浆中内毒素浓度、肝细胞匀浆内SOD活力和MDA含量、RT-PCR检测肝组织TNF-a mRNA表达情况及光镜下和电镜下观察肝组织形态学改变。结果1、实验组7天累积存活率为66.7%,对照组7天累积存活率为25.0%,经统计学log rank（mantel-cox）方法比较,P=0.044,两组累积存活率之间的差异有统计学意义。2、两组受体移植前基础门脉压力差异无统计学意义（10.20 vs 10.12,P=0.565）。实验组开放门静脉后门静脉压力维持相对稳定,波动于13.52～11.20cmH₂O之间,而对照组恢复门静脉灌注后门静脉压力显著升高,波动于16.07～11.85 cmH₂O之间。两组开放门静脉后门静脉压力的峰值出现在开放门静脉后15分钟,但实验组门脉压力峰值要明显低于对照组（13.52 vs16.07,P=0.003）,峰值较基础门脉压力升高幅度分别为33%和59%。开放门静脉30分钟之后两组门脉压力逐渐回落。实验组在开放门静脉后的5、15、30和45分钟各时间点门脉压力均明显低于对照组,而于60分钟两组门脉压力无统计学差异。3、两组肝移植术后血清ALT和AST水平显著升高。实验组ALT和AST水平在肝移植术后6小时和24小时较对照组明显降低,差异有统计学意义,P＜0.05。说明再灌注期间降低门脉压力可减轻小体积移植物肝实质细胞损伤的程度。4、实验组和对照组肝组织中SOD活性和MDA水平在移植术后6小时和24小时相比较差异无统计学意义,P＞0.05。提示实验组和对照组肝移植术后供肝均遭受相同程度的缺血再灌注损伤。5、实验组血浆内毒素水平术后6小时和24小时均低于对照组,各亚组间差异有统计学意义,P＜0.05。提示再灌注期间降低门脉压力可降低肝移植术后内毒素水平,减轻内毒素血症所引发的继发性供肝损伤。6、实验组术后6小时和24小时TNF-αmRNA表达水平均低于对照组,P＜0.05。提示再灌注期间降低门脉压力可降低供肝炎症反应的程度,减轻小体积移植物肝移植术后早期的损伤。7、光镜下可见实验组和对照组均出现不同程度的肝窦充血、局部肝窦扩张、血管内皮细胞完整性受损、肝板结构改变及肝细胞空泡变性等形态学异常的表现,与实验组相比较对照组在术后6小时和24小时形态学异常的表现均较为明显且程度严重。8、电镜观察结果显示,两组均出现不同程度的肝窦内皮细胞线性结构完整性破坏、线粒体肿胀等的形态学异常。实验组在采取降低再灌注期间门静脉压力的保护措施之后,肝细胞的超显微结构保持得相对完整。结论1、大鼠边缘性体积供肝肝移植术中开放门静脉后瞬间急剧升高的门静脉压力对肝窦内皮细胞造成不可逆的应力性损伤是导致肝移植术后移植物丧失的主要原因。2、大鼠边缘性体积供肝肝移植术中使用三甘氨酸-赖氨酸-加压素降低再灌注期间门静脉压力,可明显的减轻边缘性体积供肝早期损伤的程度。第三部分暂时性控制门脉压力对大鼠边缘性体积移植物再生影响的研究目的本部分实验通过在大鼠边缘性体积供肝肝移植术中使用三甘氨酸-赖氨酸-加压素降低再灌注期间门静脉压力,减轻小体积移植物早期损伤的程度,初步探讨三甘氨酸-赖氨酸-加压素对大鼠边缘性体积供肝肝移植术后小体积供肝再生的影响。材料和方法选用健康、体重相差20～30克的雄性SD大鼠240只,以小体重大鼠为供体,按第一部分介绍的方法建立大鼠边缘性体积肝移植模型。设立三甘氨酸-赖氨酸-加压素实验组和生理盐水对照组,三甘氨酸-赖氨酸-加压素用药方案同第二部分。两组于术后1、2、4和7天各随机抽取6只存活受体在乙醚麻醉下取肝组织标本。分别测定供肝湿重、供肝细胞增殖指数和肝组织免疫组化ki-67标记指数,了解大鼠边缘性体积供肝肝移植术后供肝再生的初步情况。结果1、实验组肝移植术后供肝湿重（D₁）、肝移植术后大鼠供肝湿重与受体原肝脏湿重之比（D₁/R）和供肝再生率（D₁/R—D₀/R）在第1天和第2天明显高于对照组,P＜0.05,差异有统计学意义,而在第4天和第7天两组各亚组间差异均无统计学意义,P＞0.05。2、供肝细胞增殖指数（PI）结果显示,两组供肝细胞增殖指数的高峰均出现在术后的第2天。实验组肝移植术后的第1天和第2天供肝细胞增殖指数较对照组明显增高,差异有统计学意义,P＜0.05。而两组肝移植术后第4天和第7天的供肝细胞增殖指数则无统计学差异,P＞0.05。3、免疫组化ki-67标记指数结果显示,两组Ki-67标记指数的高峰均出现在术后第2天。实验组术后第1天和第2天的Ki-67标记指数均高于对照组,各亚组间比较差异有统计学意义,P＜0.05。而肝移植术后第4天和第7天两组各亚组间Ki-67标记指数的差异无统计学意义,P＞0.05。结论三甘氨酸-赖氨酸-加压素对大鼠边缘性体积移植物术后肝细胞的再生没有直接的促进作用,其只是通过降低门静脉压力,减轻边缘性体积供肝的损伤程度,间接的维护供肝细胞再生的顺利进行。更多还原

【Abstract】 Objective Our aim in this part is through different kind of reduced size liver transplantation in rats,to establish an animal model of orthotopic liver transplantation using marginal size graft.And it provides a suitable and stable animal model of marginal size graft liver transplantation model for studying small for size syndrome after liver transplantation.It is the base to understand the mechanism of grafts injury in small-for-size syndrome.Methods Male Sprague-Dawley rats,weighting from 190 to 250 grams,weight differs by 20 to 30 grams,the little one were chosen as donors.The experiment compared 4 groups of liver transplantation with designated ratios of donor and recipient liver weight.group A,whole graft group（n=24）;group B,50%ratio liver transplantation（The median lobe and right lobe of the liver was selected to be the graft,n=24）;group C, 30%ratio liver transplantation（The median lobe of the liver was selected to be the graft,n=24）;group D,less than 30%ratio liver transplantation （The median lobe and left lobe of the liver was reduced,and remained lobe was selected to the graft,n=24）.A rat model of nonarterialized orthotopic liver transplantation without veno-venous bypass was used.In the small-for-size graft group,the lobe ligation technique was used to reduce the graft size on the backtable.Operation parameters,portal pressure,liver function and survival rate were observed and analyzed in all group.Results One week survival rate of the group A,B,C and D was 100%,83.3%,16.7%and 0 respectively.The portal pressures of the whole graft group were relative stable after reperfusion.But there was a significant increase in portal pressure in the reduce size graft group at 5 and 15 minutes after reperfusion compared with that before transplantation.This increase in portal pressure reached a peak at 15 minutes（increased by 65%～82%,group C,16.47cmH₂O vs 9.97 cmH₂O,P＜0.05;group D,18.25 cmH₂O vs 10.05 cmH₂O,P＜0.05）and gradually subsided after 30 minutes of reperfusion.Each group ALT levels increased significantly at 24 hours after liver transplantation,the four groups in Group D to the highest level,C group followed,and then B group,Group A lowest.Each group of differences have statistics significance,P＜0.001.Conclusions1.The minimum safely graft volume in rat liver transplantation is 50%, The marginal size graft volume ratio is between30～35%,and the ratio less than 30%should be considered as extra-small-for-size liver transplantation in the rat.2.To establish an animal model of orthotopic liver transplantation using marginal size graft in rat,we can use weigh from 190 to 250 grams rat, and weight differs by 20 to 30 grams,the little one were chosen as donors.The median lobe of the liver was selected to be the graft and can let the ratio of the graft between 30～35%.3.In a rat liver transplantation model using marginal size graft,the portal pressure changes in marginal size grafts are transient.Progressive damage of the graft may result from microcirculatory failure due to irreversible endothelial injury after reperfusion. Objective The aim of this study is to investigate the protective effect of terlipressin on decrease portal pressure in a rat liver transplantation model using marginal size graft and to clarify the possible mechanism of the marginal size graft injure after acute phase of reperfusion.Methods The previously described rat model of nonarterialized liver transplantation using marginal size grafts was used.Survival recipients were divided into two groups:experimental group and control group.In experimental group,50ug/kg rat body weight of terlipressin was infused via the femoral vein before portal vein clamping and immediately after reperfusion,respectively.In control group,same vehicle of saline was administered as control.Six rats in each group were sacrificed randomly at 6hr and 24hr after reperfusion respectively.The survival rate of animals and portal pressure was investigated.The animals in experimental group and control group were sacrificed and blood samples were collected for endotoxin level and liver function measurement.Partly Liver specimens were morphologically examined under light microscopy and electron microscopy.The content of SOD and MDA in liver tissue was measured. The mRNA expression of TNF-αwas detected using real-time PCR （RT-PCR）.6 rats at each time pointing each group were studied.Results1.Survival study showed the 7-day survival rate of 66.7%in terlipressin treatment group and 25.0%in control group,respectively.The significant difference was investigated between two groups（P＜0.05）.2.There was a significant increase in portal pressure in both groups at 5 and 15 minutes after reperfusion compared with that before transplantation.This increase in portal pressure reached a peak at 15 minutes（increased by 59%in control group and 33%in experimental group,13.52 vs 16.07,P=0.003）and gradually subsided after 30 minutes of reperfusion.The portal pressures of the experimental group were relative stable after reperfusion.3.Biochemical data showed that the level of hepatic enzymes（ALT and AST）reached the peak at 6 hours after reperfusion in each group.At 6 and 24 hours after reperfusion,the levels of ALT and AST in experimental group were significantly lower than those in control group （P＜0.05）.4.Morphological investigations using light microscopy and electron microscopy showed the well-preserved morphology and ultrastructural integrity in experimental group,5.RT-PCR results showed that the intragraft expression of TNF-αmRNA in terlipressin treatment group was down-regulated at each time point after reperfusion.6.The level of plasma endotoxin after reperfusion was increasing by time. At 6 and 24 hours after reperfusion the levels of plasma endotoxin in experimental group were significantly lower than those in control group respectively（P＜0.05）.7.The content of MDA after reperfusion was increased by time,inversely; the level of SOD was decreasing by time.But There was no statistical significance（P＞0.05）Conclusions1.In the rat liver transplantation model using marginal size graft,the distinct hepatic injury of marginal size liver grafts was characterized by irreversible endothelial injury that resulted from severe hepatic sinusoid shear stress induced by transient significant increase portal hypertension at the early phase after reperfusion.2.Terlipressin can rescue marginal size graft from acute phase injury after reperfusion by attenuated the acute phase shear stress that resulted from transient portal hypertension. Objective The aim of this study is to evaluate the effects of control portal pressure on liver regeneration in marginal size liver transplantation in rat.Methods The previously described rat model of nonarterialized liver transplantation using marginal size grafts was used.Survival recipients were divided into two groups:experimental group and control group.In experimental group,50ug/kg rat body weight of terlipressin was infused via the femoral vein before portal vein clamping and immediately after reperfusion,respectively.In control group,same vehicle of saline was administered as control.Six rats in each group were sacrificed randomly at 1、2、4 and 7 days after transplantation respectively.Each graft was removed and weighed.The regenerative response of liver graft was evaluated by Flow Cytometry.Expression of Ki-67 antigen in hepatocyte was measured with immunohistochemistry assay. Results1.On 1 and 2 days after transplantation the wet weight of graft and liver regeneration rate in experimental group was significantly larger than that of the grafts in control group.But on 4 and 7 days after transplantation there are no different in both groups.2.The results of the hepatocyte proliferate index and Ki-67 labeling index of the graft show,that the hepatocyte proliferate index and Ki-67 labeling index of the graft reach a peak at 2 day after transplantation in both group.The hepatocyte proliferate index and Ki-67 labeling index of the graft of experiment group was higher than that in control group on 1 day and 2 day after transplantation.While there was no significant difference between two group on 4 day and 7 day.ConclusionsTerlipressin have no directly effect on hepatocyte regeneration in rat liver transplantation model using marginal size graft.It only by reducing portal pressure,protect the marginal size graft and indirectly for the maintenance the liver regeneration smoothly.更多还原