自制KYL液和UW液保存大鼠肝脏效果的比较

目的比较自制的KYL液和UW液对大鼠肝脏的保存效果。方法采用大鼠肝脏非循环离体灌注模型(noncirculatedisolatedperfusionofratliver),随机以KYL液和UW液对大鼠肝脏保存0、4、8、16、24和48h,记录胆汁流出量,测定灌注流出液天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)、乳酸脱氢酶(LDH)和氧自由基代谢产物丙二醛(MDA)及超氧化物歧化酶(SOD)含量,检测肝细胞内钙离子浓度,观察肝脏组织形态学变化。同时设生理盐水保存阴性对照组,了解器官保存液对大鼠肝脏有无保护作用。结果KYL液保存的大鼠肝脏在保存16h以内各时相胆汁流出量均较UW液保存者高(P<0.01),灌注流出液AST、ALT和LDH含量与UW液保存者相近,肝细胞内钙离子浓度较UW液保存者低(P<0.01);KYL组保存24及48h时,MDA含量低于UW组,SOD含量高于UW组(P<0.01);光、电镜观察两者形态学变化基本一致。两组所有指标均较生理盐水保存组好,提示KYL保存与UW液一样对大鼠肝脏具有保护作用。结论自制的KYL液对大鼠肝脏的保存效果总体上与UW液相当,在再灌注后肝细胞胆汁分泌方面和钙拮抗方面略优于UW液,而在防止细胞水肿方面较UW液稍差。     

SWH液对大鼠肝脏保存中肝细胞凋亡的影响

目的　通过与UW液进行比较 ,观察SWH保存液对大鼠肝细胞凋亡及其对肝移植受者存活率的影响。方法　应用大鼠原位肝移植模型 ,通过HE染色、电镜和TUNEL法检测肝细胞凋亡以及观察移植术后 7d动物存活率。结果　随着保存时间的延长 ,两组的凋亡指数 (AI)均无明显升高 ,保存 18h后 ,两组的AI亦无明显改变 ,仅在行肝移植恢复血流循环后AI才明显上升 ,移植前后存在显著性差异 ,P <0 .0 1,SWH液组略低于UW液组 ;SWH液组移植术后 7d动物存活率有高于UW液组的趋势。结论　有效地防止肝细胞凋亡可以提高肝脏的保存效果 ,在防止肝细胞凋亡方面 ,SWH液优于UW液。     

环孢霉素A预处理对大鼠移植肝脏影响的实验研究

目的：研究环孢霉素A（CsA）预处理保存大鼠肝脏对原位移植后的植肝功能影响。方法：将大鼠随机分为A组（对照组，HTK液保存组）和B组（实验组，HTK液＋CsA保存组），两组均保存12h后行原位肝移植，分别于供肝保存0h、12h及植术后7d检测各组血清谷丙转氨酶（AST），谷草转氨酶（AST），乳酸脱氢酶（LDH）浓度及各时间段线粒体呼吸功能RCR及P／O值。通过HE染色及原位凋亡染色观察各组肝脏组织细胞形态学改变及凋亡情况。结果：B组血清ALT、AST、LDH活性于保存12h显著低于A组，B组各时间段RCR及P／O值于保存12h，移植术后0．5h及术后7d均高于A组。细胞形态学检查B组移植肝组织细胞结构改变轻微，凋亡指数于保存12h．术后0．5h均低于A组。结论：CsA预处理供肝主要通过保护线粒体功能减轻冷保存期和再灌注对大鼠供肝的损伤及抑制肝细胞的凋亡，使大鼠原位肝移植后存活率升高。     

自制肝脏保存液在大鼠原位肝移植模型中的应用

目的研制一种新型的专用于肝脏保存的溶液,以期达到价廉、专用、损伤小并能长时间保存的目标。方法自制肝脏保存液;建立大鼠原位肝移植模型;使用自制肝脏保存液(A组)、UW液(B组)和HC-A液(C组)保存大鼠肝脏2、8、24h后行大鼠原位肝移植,于移植后6 h比较各项肝脏功能。结果对于ALT、AST、HA,自制肝脏保存液组及其亚组与UW液组同步升高(P>0.05),与HC-A液组比较,前者的含量均低,差异有统计学意义(P<0.05)。对于LDH、STB,自制肝脏保存液组及其亚组与HC-A液组同步升高(P>0.05),与UW液组比较,前者的含量均高(P<0.05)。结论经大鼠原位肝移植模型证实,自制肝脏保存液与UW液在保护肝脏功能方面作用相当,优于HC-A液。     

自制CMU-1液保存大鼠肝脏的实验研究

目的　探讨CMU 1液保存大鼠肝脏的效果。方法　根据灌注液和保存液的种类将Wistar大鼠分为两组 :UW组和CMU 1组 ,每组分 6h、12h、2 4h 3个保存时限 ,每亚组 6只大鼠。采用离体循环灌注模型 ,研究CMU 1保存液对保存肝脏能量代谢、生化功能、胆汁分泌及形态学方面的影响。结果　随着保存时间延长 ,肝组织TAN含量及AEC逐渐降低 ,CMU 1组较UW组下降略缓慢 ,保存 2 4h后高于UW组 (P <0 0 5 )。再灌注 12 0min后CMU 1组的肝脏分泌胆汁量较UW组多 (P <0 0 5 )。相同时限相比 ,灌出液中ALT、LDH值两组之间无显著差异 (P >0 0 5 )。肝脏组织学变化两组间无明显差异。保存 6h后 ,保存液pH值无明显变化 ;保存 12h后pH值下降 ,两组无明显差异 ;保存2 4h后 ,UW组pH值下降较CMU 1组明显。结论　CMU 1保存液保存大鼠肝脏效果与UW液相似 ,在改善保存肝脏能量代谢、预防细胞内酸中毒、胆汁分泌方面略优于UW液。     

Celsior液与UW液对无心跳大鼠供肝保存效果的比较

目的 比较Celsior(CS)液与UW液对大鼠无心跳供者(NHBD)供肝的保存效果.方法 选取健康雄性SD大鼠作为肝移植的供、受者.通过阻断大鼠主动脉和膈上下腔静脉10 min的方法,制备和获取NHBD供肝,并采用不同的器官保存液灌注和冷保存供肝.随机将受者分为4组.CS8 h组:受者采用经CS液灌注和冷保存8 h的供肝移植;UW8 h组:受者采用经UW液灌注和冷保存8 h的供肝移植;CS16 h组:受者采用经CS液灌注和冷保存16 h的供肝移植;UW16 h组:受者采用经UW液灌注和冷保存16 h的供肝移植.受者门静脉开放前、开放后1、3及6 h,取各组受者的静脉血检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、内皮素1(ET-1)、白细胞介素1(IL-1)及肿瘤坏死因子α(TNF-α)水平;观察和比较各组受者的胆汁生成量、移植肝组织病理学改变及术后7 d内的存活率.结果 NHBD供肝经UW液灌注后呈"花斑"状,肝叶边缘灌注不良,经CS液灌注后肝叶边缘灌注良好.CS8 h组和UW8 h组受者的胆汁生成量分别为(0.21±0.01)ml和(0.10±0.02)ml(P<0.05).门静脉开放后1、3及6 h,CS8 h组受者的血清ALT及AST水平明显低于UW8 h组(P<0.05),门静脉开放后1、3h,CS8 h组受者的血清ET-1、IL-1及TNF-α水平均明显低于UW8 h组(P<0.05);CS8 h组受者移植肝肝窦扩张、门静脉充血及炎症细胞浸润等病理学改变明显轻于UW8 h组,CS8 h组和UW8 h组受者术后7 d的存活率分别为58.3%和25.0%(P<0.05).CS16 h组和UW16 h组受者各时点的胆汁分泌量、血清ALT、AST、ET-1、IL-1及TNF-α水平的比较,差异均无统计学意义(P>0.05),两组受者均在术后3 d内死亡,两组受者移植肝组织病理学改变无明显差异.结论 CS液对大鼠NHBD供肝的保存效果优于UW液,这可能与UW液较CS液粘稠及CS液能够减少枯否细胞的激活有关;NHBD供肝的冷保存时间不宜超过16 h.     

SX-1液对肝脏保存效果的实验研究

目的 应用改良后的Kamada二袖套法大鼠原位肝移植模型,检验SX-1液、HC-A液和UW液对肝脏保存的效果.方法 在无菌条件下配制肝脏保存液.建立大鼠原位肝移植模型.使用SX-1液、UW液和HC-A液保存大鼠肝脏2、8、24 h后行大鼠原位肝移植,于移植后6 h比较各项肝脏功能.结果 对于ALT、AST,SX-1液组(2、8、24 h)与UW液组同步升高,分析无统计学意义(P＞0.05),与HC-A液组相比,差异有统计学意义(P＜0.05).对于LDH,SX-1液组(2 h、8 h、24 h)与HC-A液组同步升高,差异无统计学意义(P＞0.05),与UW液组相比,差异有统计学意义(P＜0.05).对于分泌胆汁的肝脏个数,各组别与分泌胆汁的肝脏个数无差别(P＞0.05).本组内各时间点分泌胆汁个数有差别(P＜0.05).随肝脏保存时间增长,分泌胆汁的肝脏个数减少.结论 经大鼠原位肝移植模型证实,SX-1液在肝脏酶学方面与UW液作用相当,超过HC-A液水平.肝移植后6 h肝脏分泌胆汁的个数方面,SX-1液与HC-A液、UW液间无明显差别.     

磷酸肌酸对离体大鼠肝脏冷保存的保护作用

目的探讨磷酸肌酸(CP)对大鼠离体肝脏冷保存的保护作用。方法建立大鼠肝脏单纯冷保存离体灌注模型,对照组予单纯威斯康星大学保存液(UW液)灌注肝脏,低剂量组以UW液为基液加入1 g/100 ml CP灌注肝脏,中剂量组以UW液为基液加入2 g/100 ml CP灌注肝脏;高剂量组以UW液为基液加入3 g/100 ml CP灌注肝脏。各组大鼠肝脏分别于4℃相应灌注液中冷保存后0、6、12、18、24 h共5个时间点,分别检测肝下下腔静脉内保存液的丙氨酸转氨酶(ALT)、乳酸脱氢酶(LDH)含量,检测肝脏组织丙二醛(MDA)含量、髓过氧化物酶(MPO)活性,观察肝脏组织肝细胞的凋亡指数(AI)和肝脏组织核因子-κB阳性表达率,光学显微镜下观察肝脏组织的病理学变化。结果低、中、高剂量组大鼠肝脏在冷保存12 h后,ALT及LDH含量均低于对照组(均为P0.05);冷保存18 h后低、中、高剂量组大鼠肝脏组织的MDA、MPO含量均低于对照组(均为P0.05);在冷保存12 h及18 h时,低、中、高剂量组大鼠肝脏的肝细胞AI及核因子-κB阳性表达率均低于对照组(均为P0.05);冷保存24 h后,高剂量组保存液的ALT、MDA含量均明显高于对照组及低、中剂量组(均为P0.05)。病理检查结果显示,高、中、低剂量组大鼠肝脏的损伤明显轻于对照组,各剂量组之间比较无明显差别。结论在UW液中加入CP对大鼠离体肝脏冷保存有较好的保护作用,优于单纯应用UW液保存。     

保存温度对大鼠肝脏功能的影响

目的 研究温度对大鼠肝脏功能的影响,以确定大刀肝脏的是适保存温度。方法 采用单纯低温保存方法及建立大鼠原位肝移植模型研究保存温度对大鼠肝脏功能的影响。实验对0℃、4℃、8℃、12℃进行研究,保存时发为6,18及30h,保存18h后行肝移植术,检测指标包括肝脏酶学、能量指标及组织形态学改变。结果 ①保存18,30h的灌出液及移植后2h血清中AST、LDH水平为4℃＜0℃＜8℃＞12℃（P＜0.01或P＜0.05）。②保存18、30h及移植后20min的肝脏能量指标ATP、EC为4℃＞0℃＞8＞12℃（P＜0.01或P＜0.05）。③肝脏形态学示4℃组损害较其它组轻。结论 4℃对于大鼠肝脏是是适保存温度,它能够明显减轻大鼠肝脏的冷损伤程度。     

初始灌注液对离体低温保存大鼠肝脏的影响

目的 探讨缺血再灌注过程中初始灌注液对离体低温保存大鼠肝脏的影响。方法采用离体大鼠肝脏低温缺血保存再灌注模型90例,分别选择UW液、HC-A液和乳酸林格液作为不同的初始灌注液,观察大鼠肝脏在低温保存0、3、6、12、24 h后再灌注流出液中ALT、AST、LDH和ET的水平,同时比较3组之间肝脏细胞形态和细胞凋亡。结果 使用HC-A液作为初始灌注液的大鼠肝脏再灌注流出液中ALT、AST、LDH和ET水平较其他两组低(P<0.05),肝脏形态和细胞凋亡的发生3组差异无显著性。另外,上述指标均受低温保存时间的影响。结论 初始灌注液可影响离体低温保存大鼠肝脏的质量,细胞凋亡可能参与了肝脏的缺血再灌注损伤。     

Fructose 1-6 bisphosphate versus University of Wisconsin solution for rat liver preservation: does FBP prevent early mitochondrial injury?

Background

Fructose 1,6-biphosphate (FBP) has been shown to exert therapeutic effects in models of ischemia-reperfusion in organs other than the liver. This study compared FBP and University of Wisconsin (UW) solution during cold storage and reperfusion, among mitochondria of adult male Wistar rat livers.

Methods

Adult male Wistar rats were assigned to two groups according to the preservation solution used; UW or FBP Aspartate transaminase (AST), alanine transferase (ALT); and lactic dehydrogenase (LDH) were measured in samples of the storage solution obtained at 2, 4 and 6 hours of preservation. After 6 hours of cold storage, we reperfused the liver, taking blood samples to measure AST, ALT, LDH, and throbarbituric acid reactive substances (TBARS). Hepatic fragments were processed for histologic analysis; for determinations of TBARS, catalase, and nitric oxide as well as for mitochondrial evaluation by infrared spectroscopy.

Results

During cold preservation, levels of AST and LDH in the storage solution were lower among the FBP group, but after reperfusion, serum levels of AST, ALT, and LDH were higher in this group, as was catalase activity. TBARS and nitric oxide were comparable between the groups. In the UW group there was a higher amide I/amide II ratio than in the FBP group, suggesting an abnormal protein structure of the mitochondrial membrane. No signs of preservation injury were observed in any liver biopsy, but sinusoidal congestion was present in livers preserved with FBP.

Conclusion

FBP showed a protective effect for preservation during cold storage seeming to protect the mitochondrial membrane although it did not prevent reperfusion injury.     

自制HYD液对大鼠肝脏低温保存后生化功能影响的研究

目的 研制自制ＨＹＤ液对大鼠肝脏低温保存后生化功能的影响。方法 采用大鼠肝脏非循环离体灌注模型，比较ＨＹＤ液，ＵＷ液和乳酸林格液（ＬＲ液）对大鼠肝脏６，１２，２４，３０，３６ｈ保存后生化功能的影响。结果 保存１２ｈ的肝脏，其各项生化功能ＨＹＤ组明显优于ＬＲ组。三磷酸腺苷，磷酸腺苷含量及Ａｔｋｉｎｓｏｎ能荷（ＡＥＣ），ＨＹＤ组略高于ＵＷ组，且保存３６ｈ差异有显著性。     

Relationship between ischemia/reperfusion injury and the stimulus of fibrogenesis in an experimental model: comparison among different preservation solutions

Background and Aims

Orthotopic liver transplantation (OLT) has been the standard treatment for end-stage acute and chronic liver disease. Ischemia-reperfusion (I/R) injury is one of the major causes of poor graft function early after OLT, and adversely influencing graft and patient survivals. It is unknown whether I/R injury influences liver fibrogenesis.

Materials and Methods

Livers from 25 adult male Wistar rats were randomly assigned into 5 experimental groups according to the preservation solution: saline solution (SS); University of Wisconsin (UW) solution; Fructose 1, 6-biphosphate (FBP); S-Nitroso-N-Acetylcysteine (SNAC): or UW + SNAC (SNAC+UW). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) were determined in preservation solution samples at 2, 4, and 6 hours. After 6 hours of cold ischemia, ex situ reperfusion was applied to the liver for 15 minutes. Serum AST, ALT, LDH, and renin levels were determined. Fresh liver slices were processed for histological studies, determination of thiobarbituric acid reactive substances, catalase, and glutathione, and expression of TGF-β1 and angiotensin II AT1 receptor.

Results

AST was significantly lower during cold storage with UW than with the older media (P = .001); ALT was lower in the FBP group (P = .023) and LDH was lower in the FBP and SNAC groups (P = .007). After reperfusion, serum AST, ALT, LDH, and TBARS showed no significant differences among the groups. Catalase was significantly lower in the SS and FBP groups (P = .008 and P = .006, respectively). Compared with UW, glutathione concentrations were significantly higher in SS, FBP, and SNAC 200 (P = .004). Renin levels were significantly lower in the FBP group (P = .022). No histological signs of preservation injury were observed in the hepatic sample. No expressions were detected of TGF-β1 or AT1 receptor.

Conclusion

In this experimental model of early reperfusion injury, preservation changes related to higher levels of renin, which suggest its role in fibrogenesis. FBP was associated with lower renin levels than other solutions including UW.     

Comparison of University of Wisconsin (UW) and Eurocollins (EC) preservation solutions in a rat liver transplant model

Abstract. The Eurocollins (EC) and University of Wisconsin (UW) preservation solutions were compared in a rat liver transplant model. After hepatectomy, 48 rat livers were flushed with either EC or UW preservation solution and were randomly assigned to 1, 12, 24, and 30 h of preservation at 4C, resulting in eight groups each containing six livers. Following preservation, orthotopic liver transplantation with reconstruction of the hepatic artery was performed. The efficacy of the preservation solution was assessed at 48 h post-transplantation by survival histological features and aspartate transaminase assay (AST) values. None of the rats survived 30 h of liver preservation with EC whereas five out of six rats did with UW preservation. After 24 h of liver preservation, three of the six rats in the EC group survived, compared to all six rats in the UW group. Histological evidence of severe ischemia was found in both groups in all but one survivor (UW, 24 h). After 12 h of EC preservation, one rat died within 48 h and severe ischemic changes were found in the remaining five rats. Among the rats with 12 h of UW preservation, only two out of six showed ischemic changes, and all six rats survived beyond 48 h. Without preservation (1 h), ischemic damage was found in two out of six rats in each group and all rats survived. The median AST values were higher in the EC groups than in the UW groups; the difference became significant after 12-h preservation (EC 900 IU/1 versus UW 465 IU/1) and 24-h preservation (EC 5220 IU/1 versus UW 631 IU/1). However, the median AST value in the five surviving rats whose livers had been preserved for 30 h in UW climbed to 1880 (950–2240) IU/1. We conclude that UW solution provides better long-term preservation than EC solution. However, even with UW solution, the observed mortality, the severity of ischemic changes, and the pronounced increase in the median AST value cast doubt upon the safety of liver preservation beyond 24 h.     

Comparison of University of Wisconsin (UW) and Eurocollins (EC) preservation solutions in a rat liver transplant model

The Eurocollins (EC) and University of Wisconsin (UW) preservation solutions were compared in a rat liver transplant model. After hepatectomy, 48 rat livers were flushed with either EC or UW preservation solution and were randomly assigned to 1, 12, 24, and 30 h of preservation at 4°C, resulting in eight groups each containing six livers. Following preservation, orthotopic liver transplantation with reconstruction of the hepatic artery was performed. The efficacy of the preservation solution was assessed at 48 h post-transplantation by survival histological features and aspartate transaminase assay (AST) values. None of the rats survived 30 h of liver preservation with EC whereas five out of six rats did with UW preservation. After 24 h of liver preservation, three of the six rats in the EC group survived, compared to all six rats in the UW group. Histological evidence of severe ischemia was found in both groups in all but one survivor (UW, 24 h). After 12 h of EC preservation, one rat died within 48 h and severe ischemic changes were found in the remaining five rats. Among the rats with 12 h of UW preservation, only two out of six showed ischemic changes, and all six rats survived beyond 48 h. Without preservation (1 h), ischemic damage was found in two out of six rats in each group and all rats survived. The median AST values were higher in the EC groups than in the UW groups; the difference became significant after 12-h preservation (EC 900 IU/l versus UW 465 IU/l) and 24-h preservation (EC 5220 IU/l versus UW 631 IU/l). However, the median AST value in the five surviving rats whose livers had been preserved for 30 h in UW climbed to 1880 (950–2240) IU/l.. We conclude that UW solution provides better long-term preservation than EC solution. However, even with UW solution, the observed mortality, the severity of ischemic changes, and the pronounced increase in the median AST value cast doubt upon the safety of liver preservation beyond 24 h.     

Effect of fructose-1,6-bisphosphate in the cold storage solution after 12 and 36 hours of rat liver preservation

Fructose-1,6-bisphosphate (FBP) has been reported to have a protective effect on liver injury following ischemic/reperfusion periods because it maintains ATP levels during cold preservation. In the present study, we evaluated the effects of addition of FBP to storage solutions for cold liver preservation during 12 or 36 hours. Adult male Wistar rats were randomly divided into three experimental groups. The hepatic perfusion and preservation were performed with these solutions: UW; UW plus 10 mmol/L FBP; and FBP 10 mmol/L (FBPS) alone. The biochemical measurements of AST and ALT were performed on samples of the cold storage solution after 12- or 36-hour preservation. UW and FBPS solutions showed similar preservation grades at 12 hours. Addition of 10 mmol/L of FBP to UW solution induced liver injury and a poor preservation grade during 12 or 36 hours. UW solution was better than FBPS after 36 hours preservation. UW solution continues to offer a superior performance for liver preservation during long times; however, FBPS may be an alternative for short cold preservation times.     

大鼠供肝冷保存时间延长损伤移植肝肝细胞和肝窦内皮细胞

目的 探讨供肝冷保存时间与肝移植后肝细胞和肝窦内皮细胞（SEC）损伤的关系。方法 选取健康雄性SD大鼠作为供、受者，建立原位肝移植（OLT）模型。随机分为3组，冷保存1h组（H=48）：供肝获取后，置于4C的冷保存液中保存1h，再行OLT。冷保存12h组（n=48）：供肝获取后，置于4℃的UW液中保存12h，再行OLT。对照组（H=6）：大鼠只打开腹腔，不进行移植。前2组分别于术后1、6、12、24、48、72、96和168h采取血液及组织标本，对照组仅在开腹时取血液及组织标本，检测各组、各时点血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）及透明质酸（HA）的水平；观察移植肝的病理形态学变化，透射电镜观察其超微结构改变；原位末端脱氧核糖核酸转移酶标记法（TUNEL）检测移植肝细胞的凋亡情况；观察术后168h时的大鼠存活率。结果 冷保存1h和冷保存12h组肝移植后各时点血清ALT、AST及HA均较对照组明显升高（P〈0．05），并且冷保存12h组又明显高于冷保存1h组（P〈0．05）。冷保存12h组术后24h移植肝组织出现片状坏死，而冷保存1h组病理学改变不明显。冷保存12h组肝窦内皮细胞凋亡指数（AI）明显高于冷保存1h组（F=63．58，P〈0．01），两组大鼠移植肝组织均于术后6h出现凋亡高峰，且肝窦内皮细胞的凋亡指数明显高于肝细胞。冷保存1h组和冷保存12h组大鼠肝移植后168h时的存活率分别为100％和50％，两组比较，差异有统计学意义（F=6．39，P〈0．05）。结论 肝移植后肝细胞和肝窦内皮细胞的损伤程度与冷保存时间密切相关。肝窦内皮细胞对冷保存及再灌注损伤的敏感性高于肝细胞，其损伤方式以细胞凋亡为主。