肝脏低温保存-再灌注后的变化及机制

目的　研究肝脏低温保存 再灌注期间 ,肝细胞糖原含量与肝细胞凋亡之间的关系及其相关机制。方法　制备四组肝糖原含量显著不同的兔肝脏模型 ,肝糖原含量 (mg/ g)分别为 :A组 :15 .42± 2 .6 0 ;B组 :5 1.83± 6 .6 1;C组 :6 3 .2 2± 5 .84;D组 :78.5 8± 8.46。检测各组肝脏低温保存 再灌注过程中肝细胞凋亡及组织内氧自由基相关指标 (SOD、GSH、MDA)的变化情况。结果　各组肝脏低温保存 9h后 ,再灌注 6 0min时 ,组织内可见明显的肝实质细胞凋亡现象 ,其凋亡细胞数量依次为A组 >B组 >C组 >D组 ,四组间差异均有显著性 ;且此时各组肝脏组织中SOD、GSH或MDA水平差异也均有显著性。结论　肝脏低温保存 再灌注过程中 ,肝细胞内糖原能显著拮抗肝实质细胞凋亡的发生 ;其内在机制可能在于肝细胞糖原通过抑制氧自由基的产生而达到拮抗肝实质细胞凋亡的发生。     

肝脏低温保存-再灌注期间肝细胞凋亡及其与Bax基因蛋白相关性的研究

目的　研究临床肝移植过程中供肝低温保存-再灌注期间肝细胞凋亡及其相关机制。方法　应用细胞凋亡原位末端标记法并结合电镜观察,检测低温保存时间分别为0(A组)、3(B组)、6(C组)、9(D组)h的4组兔肝脏在低温保存-再灌注过程中肝细胞凋亡情况。同时,对各组肝脏再灌注前后肝细胞内Bax基因蛋白表达进行测定。结果　A、B、C、D各组肝脏于低温保存后的再灌注60min时,组织内可见明显的肝实质细胞凋亡；其凋亡细胞数量(1×10     

低温保存-再灌注肝脏糖原含量与肝细胞凋亡的关系

目的　研究肝脏低温保存 再灌注期间肝糖原含量与肝细胞凋亡之间的关系及其相关机制。方法　制备糖原含量分别为 (1 5 .42± 2 .60 )mg/ g(A组 )、(51 .83± 6 .61 )mg/ g(B组 )及(63 .2 2± 5 .84)mg/ g(C组 )的 3组兔肝脏模型 ,检测各组肝脏低温保存 再灌注过程中肝细胞凋亡及肝细胞内游离Ca2 + 浓度变化情况。结果　各组肝脏于低温保存 9h后再灌注 60min时 ,组织内可见明显的肝实质细胞凋亡现象 ;其凋亡细胞数量分别为 (× 1 0 - 2 个 / μm2 ) :A组 :1 7.58± 3 .64 ,B组 :1 2 .61± 2 .95 ,C组 :8.2 7± 2 .60 ,3组间差异均有显著性 (P <0 .0 1 ) ;并且此时 ,3组肝实质细胞内游离Ca2 + 浓度 (nmol/L)依次为 :A组 :379.1 8± 50 .2 6 ,B组 :331 .59± 43 .56 ,C组 :2 81 .47±40 .1 2 ,各组间差异均有显著性 (P <0 .0 1 )。结论　肝脏低温保存 再灌注过程中 ,肝细胞内高糖原含量能显著拮抗再灌注期间肝实质细胞凋亡的发生 ;其可能与高糖原含量的肝实质细胞内ATP充裕 ,细胞膜上Ca2 + ATP酶功能完善 ,细胞内外Ca2 + 平衡相对稳定有关     

肝糖原贮备对热缺血再灌注大鼠肝细胞凋亡的影响

目的探讨肝糖原贮备对热缺血再灌注肝细胞凋亡及肝损伤的影响。方法建立大鼠肝热缺血模型。实验分组:术前24h静脉注射25%葡萄糖组,2ml/只,每6h1次,高糖饮食(H组);术前禁食24h,饮水不限(L组);正常饮食对照组(N组)和假手术组(S组)。缺血45min,再灌注2、24h取材。流式细胞术检测细胞凋亡及Bcl-2、Bax蛋白。同时进行肝酶学检测、肝组织形态学观察。结果1.细胞凋亡及蛋白表达:(1)24h细胞凋亡百分率。S组N组(P<0.01)。2.肝酶学指标:各时相点4组间比较差异有统计学意义(P<0.05),S组相似文献   

离体肝脏低温保存-再灌注期间肝细胞凋亡现象观察

目的 探讨临床肝移植过程中离体供肝低温保存－再灌注损伤与肝细胞凋亡的关系。方法 采用细胞凋亡原位末端标记法并结合电镜观察,检测低温保存时间分别为０、３、６、９小时的４组免肝脏在低温保存－再灌注过程中肝细胞凋亡发生情况。结果 在低温保存后的再灌注期间,４组动物的肝组织内均可见明显的肝实质细胞凋亡现象,而且低温保存时间越长的肝脏,其再灌注后产生的肝实质凋亡细胞数量越多,但各组肝脏于低温保存末,其组织内     

肝糖原贮备对热缺血再灌注大鼠肝细胞凋亡的影响

目的探讨肝糖原贮备对热缺血再灌注肝细胞凋亡及肝损伤的影响。方法建立大鼠肝热缺血模型。实验分组：术前24h静脉注射25％葡萄糖组，2ml／只，每6h1次，高糖饮食（H组）；术前禁食24h，饮水不限（L组）；正常饮食对照组（N组）和假手术组（S组）。缺血45min，再灌注2、24h取材。流式细胞术检测细胞凋亡及Bcl-2、Bax蛋白。同时进行肝酶学检测、肝组织形态学观察。结果1．细胞凋亡及蛋白表达：（1）24h细胞凋亡百分率。s组〈H组〈N组〈L组（P〈0．01），各组24h凋亡率均高于2h（P〈0．01，S组除外）。（2）Bcl-2、Bax蛋白表达量。①再灌注24hBcl-2表达量：H组明显高于其余3组（P〈0．01），L组〈N组（P〈0．05），与2h比较H组表达升高（P〈0．01）；②再灌注24hBax表达量：S组明显低于其余3组（P〈0．01），L组〉N组（P〈0．01）。2．肝酶学指标：各时相点4组间比较差异有统计学意义（P〈0．05），S组〈H组〈N组〈L组。3．肝脏病理组织学改变：再灌注24hH组明显轻于N组及L组，并接近S组，L组最严重。结论预防性增加肝糖原贮备可抑制热缺血再灌注过程中肝细胞凋亡，减轻肝损伤，并可能通过影响Bcl-2、Bax的表达发挥作用。     

大鼠肝脏冷灌注保存中肝细胞凋亡及其调控基因的研究

目的 通过检测凋亡细胞计数及凋亡相关基因bax和Fas的表达，探讨细胞凋亡及其相关基因在冷灌注保存大鼠肝脏组织中的作用。方法 本研究采用脱氧核糖核苷酸末端转移酶介导的末端标记法（TUNEL法）及RT-PCR，按照供肝保存时间30、60、90、120、180、300min分为6组进行凋亡细胞计数及其相关基因的检测。结果 不同保存时间大鼠肝脏组织中均有凋亡细胞发生，随着时间延长，凋亡细胞计数呈进行性增加趋势；未检测到bcl-2的表达，但可明显检测到bax和Fas基因的表达。结论 在冷灌注保存肝组织中，细胞凋亡是早期细胞死亡的主要原因之一。细胞凋亡的发生可能与bax和Fas的高表达以及bcl-2的低表达有关。     

17-β雌二醇对大鼠肝切除肝缺血再灌注损伤肝细胞凋亡和Bcl-2、Bax表达的影响

目的 观察17-β雌二醇预处理对肝切除肝缺血再灌注损伤肝脏组织细胞凋亡及Bcl2、Bax表达的影响,并探讨其肝保护的机制.方法 建立大鼠肝切除肝缺血再灌注损伤模型,75只雄性SD大鼠随机分为3组:假手术组(Sham组)、缺血再灌注组(IR组)和17-β雌二醇预处理组(E2+ IR组).检测各组大鼠再灌注后lh、3h、6h、12 h、24 h肝功能变化.光镜下观察肝组织病理学改变.TUNEL法观察再灌注后12 h大鼠肝细胞凋亡情况、流式细胞学方法测定再灌注后12h肝细胞凋亡率.Western blot法检测再灌注后12 h Bcl-2和Bax的表达情况.结果 与Sham组相比,在IR组各时间点均可见ALT和AST增高,且在再灌注后的12h达到了最高值;病理学检查可见肝细胞肿胀,肝窦变窄,嗜中性粒细胞浸润和片状坏死等变化;在再灌注后12h,凋亡细胞增多及细胞凋亡率明显升高;肝脏组织Bcl 2表达减少,Bax的表达增加.17-β雌二醇预处理组在灌注后各时间点ALT和AST值明显下降,肝脏病理损伤改善;在再灌注后12h,凋亡细胞减少及细胞凋亡率明显降低,肝脏组织Bcl-2表达增加,Bax的表达减少.结论 17-β雌二醇对大鼠肝缺血再灌注损伤有明显的保护作用,其可能通过促进Bcl-2表达及抑制Bax表达,从而抑制肝细胞凋亡.     

三七总皂甙对大鼠肝脏低温保存再灌注期间肝细胞凋亡的影响

目的　研究三七总皂甙 (PNGS)对大鼠肝脏低温保存再灌注期间肝细胞凋亡的影响及其机制。方法　采用大鼠离体肝脏再灌注模型 (IPRL) ,用Fura 2法测定低温保存 2h后肝细胞内钙离子浓度 ;经乳酸林格氏液 (LR)低温保存 2 4h的肝脏再灌注 30min后进行肝脏功能检测、氧自由基代谢产物、肝细胞凋亡、Bcl 2蛋白表达及形态学观察。LR和DMEM液中加入不同浓度PNGS。结果　大鼠肝细胞内钙离子浓度、MDA、SOD、肝细胞凋亡及Bcl 2蛋白表达阳性率等项指标各实验组明显好于对照组 (P <0 0 1) ,PNGS对大鼠肝细胞凋亡的保护作用显示出剂量依赖性 ,在 2 0 0～ 6 0 0mg范围内随剂量增加保护作用随之增强 (P <0 0 1) ,在 80 0～ 10 0 0mg范围内虽有增强 ,但无明显差别 (P >0 0 5 )。结论　PNGS减轻了大鼠肝脏在低温保存再灌注期间肝细胞凋亡 ,可能与通过抑制钙超载、抗氧自由基损伤、提高Bcl 2蛋白表达作用有关。     

常温下缺血预处理对肝细胞凋亡调节基因表达影响的实验研究

目的 探讨常温下大鼠肝脏缺血预处理对细胞凋亡调节基因C-jun和Bcl-X_L的表达及其对肝细胞的保护作用。方法 将30只Wistar大鼠随机分成假手术组(S组,10只)、缺血再灌注组(IR组,10只)、缺血预处理组(IP组,10只)。S组在游离肝蒂后、IR组和IP组在再灌30 min后0、1、3、6、20 h点采集肝组织标本切片行肝细胞凋亡,C-jun和BcI-X_L基因表达及形态学改变等检测。各组均在3、6、20 h点采集血标本检测肝损害标记酶(ALT、AST、LDH)。结果 ALT、AST及LDH值各时间点IR组和IP组均明显高于S组(P<0.01),IP组明显低于IR组(P<0.01)。IR组和IP组与S组比较,细胞凋亡指数(AI)有显著性增加(P<0.01),IP组与IR组比较,AI明显减少(P<0.01)。C-jun基因在S组和IP组各时间点表达不明显;在IR组表达明显,3h点达高峰,并持续至6h点。Bcl-X_L基因在S组、IR组各时间点表达不明显,在IP组3、6、20h点表达明显(P<0.05或P<0.01)。形态学研究显示,IR组有肝组织大片坏死及不可逆超微结构损害;IP组未见明显肝细胞坏死,且超微结构损害为可逆性。结论 ①缺血预处理通过调节肝细胞凋亡调节基因C-jun和Bcl-X_L的表达,发挥其对大鼠常温下肝脏缺血再灌注损伤的保护作用;②IR损伤可能通过激活凋亡诱导基因C-jun表达而促发大鼠肝细胞的过度凋亡;③IP可能通     

Correlation of donor nutritional status with sinusoidal lining cell viability and liver function in the rat

We have demonstrated that the sinusoidal lining cell injury sustained by rat liver allografts during hypothermic storage is a critical determinant of graft viability. The present study was designed to examine the effect of donor nutritional status on hepatic microcirculation and graft function. Rat livers from four nutritional groups (group I, fasted; group II, fed; group III, intraperitoneal glucose; and group IV, fed plus intraperitoneal glucose) were excised and stored for 24 hr in Marshall's isotonic citrate solution. Then the livers were perfused under anoxic conditions with trypan blue. The percentage of nonviable SLC in each group was 26.7 +/- 8.1, 24.9 +/- 7.9, 17.6 +/- 6.9, and 5.9 +/- 1.9 in groups I, II, III, and IV respectively; i.e., there was a significant improvement in SLC viability with nutritional repletion in group IV. Electron microscopy was performed on livers from groups I and IV following 30-hr preservation in University of Wisconsin solution and after 16-hr preservation in Marshall's isotonic citrate solution. Biopsies were taken at the end of storage and after 1 hr of reperfusion at 37 degrees C. At the end of preservation group IV livers contained glycogen and had much more normal liver ultrastructure than group I livers. After reperfusion there was partial recovery of normal SLC morphology in both groups and depletion of glycogen in group IV. Liver function was studied on the isolated perfused rat liver system at 37 degrees C following 30-hr storage in UW solution. Transaminase release into the perfusate was significantly lower in nutritionally repleted livers than in livers from fasted animals. A significant reduction in perfusate platelet count occurred only in livers from fasted animals. The results show that nutritional repletion can reduce the injury of cold preservation to both hepatocytes and endothelial cells and improve liver function in the postpreservation period.     

The impairment of hepatocytes and sinusoidal endothelial cells during cold preservation in rat fatty liver induced by alcohol and the beneficial effect of hepatocyte growth factor

A fatty liver resulting from alcohol intake is often unattractive for grafting. In this study, we investigated the impairment of hepatocytes and sinusoidal endothelial cells (SECs) during cold preservation of alcohol-induced fatty liver and examined the efficacy of human recombinant hepatocyte growth factor (hrHGF). Rats were fed an alcohol diet. We performed histological examinations of the hepatocytes and observed the ultrastructural alteration of the SECs. Additionally, we measured hepatic transaminase and peroxidative lipids for hepatocellular injury and the hyaluronic acid uptake rate (HUR) to determine SEC injury. We added hrHGF to University of Wisconsin (UW) solution to assess the protective effect of the agent. Numerous fatty deposits were observed in ethanol-induced fatty livers. These grew with the duration of cold storage. Hepatic transaminases of the effluents increased during cold preservation in the livers of alcohol-treated rats. Additionally, peroxidative lipids in the effluents increased during cold preservation in the livers of alcohol-treated rats, whereas they were undetectable in non-alcohol-treated rat livers. The sinusoidal endothelium had severely deteriorated in the livers of alcohol-treated rats. Further, the HUR decreased with ethanol treatment and/or cold preservation. The addition of hrHGF suppressed the increase of hepatic transaminase in the effluent of cold-preserved alcohol-treated livers. Peroxidative lipids in the same effluents were undetectable. In fatty livers, both hepatocytes and SECs received severe damage during cold preservation. Furthermore, we demonstrated that hepatocellular injury was significantly inhibited by hrHGF.     

Glycogen storage of the liver: a determining factor of initial function of the hepatic graft]

In this study we have investigated the effects of hepatocytes glycogen storage on the quality of livers for transplantation. Rats were fed or fasted for 24 h and hepatocytes isolated and cold stored in UW solution for 24 and 48 hours. Viability of the cells was analyzed by LDH release after 2 hours incubation in L15 with O2. Also, rabbits were fed, fasted (48 h) or glucose fed (48 h) and livers cold stored for 6, 24 and 48 h in UW solution. Functions of the livers were analyzed by isolated perfusion for 2 hours. Hepatocytes from fasted rats released significantly more LDH than hepatocytes from fed rats after 24 and 48 h cold storage. In rabbit livers, fasting depleted glycogen by 85% but had no effect on ATP or glutathione concentration. Livers from fasted rabbits produced similar amount of bile, released similar concentrations of lactate dehydrogenase and aspartate transaminase into the perfusate, maintained similar concentrations of glutathione after 24 hours preservation when compared to fed animals. After 48 h preservation livers from fasted animals were less viable than livers from fed animals and the decrease of liver functions in livers from fasted animals preserved for 48 hours was prevented by feeding glucose. This study shows that liver glycogen storage in hepatocyte is an important metabolite for successful liver preservation. Glycogen may be a source for ATP and antioxydant synthesis during the early period of reperfusion.     

冷缺血与热缺血对大鼠肝细胞凋亡的影响

目的:观察冷保存与热缺血对大鼠肝细胞凋亡的影响。方法:用流式细胞检测技术，分组检测冷保存时和热缺血时的肝细胞凋亡率与增殖率。结果:冷保存时各组之间的肝细胞凋亡率与增殖率差异无显著性（P>0.05）；随着热缺血时间的延长，细胞凋亡率明显加重（P＜0.05），但细胞增殖率的改变不明显（P>0.05）。结论:大鼠肝脏冷保存/再灌注时肝细胞凋亡可能在有氧再灌注后加重；大鼠肝脏热缺血/再灌注时肝细胞凋亡可能在缺血时和再灌注后均可加重。     

钙离子阻滞剂对移植肝再灌注损伤的保护作用

目的：研究钙离子阻滞剂在大鼠移植肝中的保护作用。方法：采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记（TUNEL）法和逆转录-多聚酶链反应（RT-PCR）法，以尼莫地平来处理大鼠，将132只大鼠分为2组，观察肝组织中细胞凋亡及其相关基因。结果：非处理组的凋亡细胞计数分别为14.09％、15.79％、18.04％、21.02％、22.4％，处理组的凋亡细胞计数分别为12.07％、13.18％、14.68％、15.19％、16.14％。处理组Bcl-2的表达较非处理组再灌注前与再灌注后明显增加。结论：钙离子阻滞剂可以抑制细胞凋亡，可能通过诱导或抑制凋亡相关基因的表达而对细胞凋亡进行调控。     

HIV-1反式激活蛋白-血红素氧合酶-1融合蛋白对大鼠供肝冷保存期肝细胞凋亡的影响

Objective To study the effects of TAT-HO-1 fusion protein,HIV-1 transactiviting protein (TAT) and heme oxygenase-1 (HO-1),on hepatic cell apoptosis of rat donors in cold storage stage.Methods Forty-eight male SD rats were randomly divided into two groups.Rat livers were flushed and preserved with 4℃ HTK solution containing(group P) or uncontaining(group C) 50 mg/L of TAT-HO-1.The preserved solution and hepatic tissue were collected at 0,6,12,18 h of cold storage stage.TAT-HO-1 transducing into liver,alanine aminotransferase(ALT) level in preserved solution,hyaluronic acid(HA) level and the expression of caspase-3 in hepatic tissue,and the apoptotic index (AI) of hepatocytes and sinusoidal endothelial cells (SECs) were measured or detected.Results ALT level in preserved solution,HA level and the expression of caspase-3 in hepatic tissue,and the AI of hepatocytes and SECs increased time-dependently in cold storage stage in both groups (P＜0.05),with lower increasing extent in group P than that in group C (P＜0.05) at 6h,12h and 18h of cold storage stage.A stronger accumulation of HO-1 staining was also detected at the same time-points in group P than that in group C(P＜0.05).Conclusion TAT-HO-1 may transduce efficiently into rat livers,exerting protective effects on both hepatocytes and SECs during cold storage stage.Protein transduction technology may be a novel therapeutic means to reduce donor liver injury in preservation period for transplantion.     

Effect of intraportal glucose infusion on hepatic glycogen content and degradation, and outcome of liver transplantation.

Recent animal studies suggest that nutritional repletion may improve function of liver allografts, and the authors have found that intraportal glucose infusion in pigs produces rapid and substantial hepatic glycogenation. A controlled prospective randomized study in 32 patients was done to determine glycogen content and degradation in human livers during transplantation, and the effect of intraportal glucose-insulin infusions during the donor operation on these variables and on outcome of transplantation. Peripheral blood glucose concentrations were "clamped" at 14 mmol/L during the glucose-insulin infusion. Liver biopsies were taken at various stages of the procedure. Liver glycogen decreased 2.0 +/- 1.2 g/100 g dry weight liver (mean +/- standard error of the mean) in controls, but increased 6.8 +/- 1.8 g/100 g dry weight in glucose-infused donors. In both groups there was glycogen degradation during periods of cold preservation, anoxic rewarming, and after reperfusion with portal blood. Degradation rates were greater in the glucose-infused group than in controls in all three periods (p less than 0.05). Despite wide variation in postoperative aspartate aminotransferase (AST) levels among recipients in both groups, the difference in peak postoperative AST levels approached significance (p = 0.06). In addition, peak AST levels were closely correlated to anoxic rewarming time in both groups, but the slope of the relationship was much lower (3834 versus 734, p less than 0.01) in the glucose-infused group. Thus at anoxic rewarming times over 90 minutes, glycogenation was protective of liver function. Peak postoperative AST was significantly correlated to glycogen degradation in the cold preservation and rewarming periods in the glucose-infused group only. Intraoperative glucose infusions in humans can reglycogenate the liver, increase glycogen degradation, and improve certain outcome measures in liver transplantation.