冷缺血对大鼠小体积肝移植肝再生的影响

目的探讨冷缺血对大鼠小体积肝移植术后肝再生的影响.方法本组在2002年9月～2004年8月利用大鼠肝总量30%原位肝移植模型,实验分为肝总量70%肝切除组(C组)和冷缺血1、3、5 h组(E1、E2、E3组),观察1w生存率及肝重量/受体原肝重量比值(EGW/RLW),并检测术后1、2、3、7d肝细胞增殖活性及肝组织学变化.结果E1组1w生存率及EGW/RLW分别达100%和95%,均明显高于E2、E3组(P均＜0.05);E1、E 、E3组均于术后2d达到增殖高峰,其中E1组峰值显著高于E2、E3组(P＜0.001),且与C组峰值无差异(P＞0.05);组织学检查见E1组肝细胞核分裂明显活跃.结论冷缺血1 h的小体积供肝和70%肝切除后肝脏具有同样的增殖活性,仅增殖高峰稍晚;冷缺血超过3h严重影响小体积供肝的再生能力和受者的存活率.     

大鼠肝移植后肝再生的实验研究

目的：探讨部分肝移植术后移植肝的再生问题。方法：建立大鼠部分肝移植模型，实验分为肝切除组（PLR组）、全肝移植组（OLT组）和部分肝移植组（POLT组）3组，分别于术后不同时间段取外周血检测总胆红素和谷丙转氨酶水平；取肝组织行组织学检查及流式细胞仪检测移植肝的增殖活性。结果：移植术后1w，肝功能酶学指标增高，后逐步降低；组织学检查术后可见单核细胞浸润，特别在门静脉周围汇管区，肝实质可见点状坏死。术后1个月可见胆管增殖；PLR组和POLT组还可见二倍体和多倍体的肝细胞，中央小静脉、肝窦和叶间静脉轻度扩张。PLR组和POLT组肝细胞增生活跃，3组分别于术后1d、2d、4d达到增殖高峰。结论：部分移植肝和肝切除后肝脏具有同样的增殖活性，但增殖高峰POLT组及OLT组均要晚于肝切除后的肝脏，但移植组增殖周期长。这可能是由于手术操作及肝脏缺血再灌注损伤所致。而持续时间长可能与受体免疫系统产生的细胞因子和激素的调控相关。     

冷保存对大鼠部分移植肝再生的影响

目的探讨冷保存对大鼠部分肝移植术后肝再生的影响。方法健康SD大鼠分为Ⅰ组（肝切除组）、Ⅱ组（冷保存1h部分肝移植组）和Ⅲ组（冷保存8h部分肝移植组）。观察各实验组生存率，比较各组术后1、6、12、24、48、72、168h肝质量／体质量比率、肝再生率、有丝分裂指数及增殖细胞核抗原表达。结果Ⅰ、Ⅱ、Ⅲ组7d存活率分别为100％、90％、40％；Ⅲ组术后2～3d大鼠肝质量／体质量比率、肝再生率、有丝分裂指数较Ⅰ、Ⅱ组明显偏低（P〈0．05）；Ⅲ组术后12h内增殖细胞核抗原表达较其余两组明显偏低（P〈0．05），48h才达高峰，至第7天阳性表达仍处高水平。结论长时间冷保存降低了部分肝移植术后的肝再生能力和大鼠术后生存率。     

大鼠40％小体积肝移植模型建立及移植肝的病理观察

目的建立稳定的大鼠40％小体积肝移植模型，并观察术后移植肝功能与病理的变化。方法采用改良二袖套法建立大鼠40％小体积肝移植模型，观察1w生存率，并于术后1、2、4、7天检测肝功能、移植肝组织学变化和免疫组化法检测肝细胞的增殖细胞核抗原（PCNA）。结果手术成功率97．5％，1w生存率60％，无肝期平均（124±2．5）min，丙氨酸转氨酶（ALT）、总胆红素（TB）术后即第1天即明显增高，第2天最高，以后逐渐降低。组织学检查术后2天即可见较多二倍体和多倍体肝细胞。免疫组化检测移植肝PCNA表达于术后第2天最高。结论通过技术改进，简化了手术操作，提高了手术成功率及模型的稳定性。大鼠小体积移植肝仍具有较强的再生能力。     

冷缺血对大鼠部分肝移植肝再生的影响

目的　研究冷缺血对移植肝再生及肿瘤坏死因子 (TNF α)和白细胞介素 10 (IL 10 )表达的影响。方法　建立稳定的大鼠部分肝移植模型。实验分为 :5 0 %肝切除对照组、冷缺血 30min后部分肝移植组 (实验 1组 )和冷缺血 10h后部分肝移植组 (实验 2组 )。观察各组生存率 ,并分别于术后 1d、2d、4d取肝组织 ,免疫组织化学检测各组增殖细胞核抗原 (PCNA)、TNF α、IL 10的表达 ;对各组肝再生增殖及TNF α和IL 10的表达进行相关性分析 ;探讨TNF α和IL 10的变化对大鼠部分肝移植术后肝再生的影响。结果　 5 0 %肝切除组、冷缺血 30min后部分肝移植组和冷缺血 10h部分肝移植组存活率分别为 10 0 % ,79%、2 9% ;冷缺血 10h部分肝移植组与冷缺血 30min部分肝移植组相比 ,PCNA、TNF α表达降低 (P <0 .0 5 ) ,而IL 10表达增高 (P <0 .0 5 )。结论　随着冷缺血时间的延长 ,降低了部分肝移植术后的肝再生能力和生存率。TNF α和IL 10在移植肝肝再生过程中起重要的调控作用。     

骨髓间充质干细胞促进大鼠小体积肝移植后肝再生的实验研究

目的 探讨骨髓间充质干细胞能否促进大鼠小体积肝移植后移植肝的再生.方法 体外分离、培养、鉴定大鼠骨髓间充质干细胞.在30％大鼠部分肝移植的模型基础上,实验分为对照组(30％ PLT)和骨髓间充质干细胞干预组(30％PLT+ MSC).比较两组肝移植术后的存活率,分析肝功能的变化,通过免疫组化来观察移植肝标本Cyclin D1和PCNA的表达,并对移植肝组织结构进行电镜观察.结果 骨髓间充质干细胞的干预,能够改善小体积肝移植术后肝功能状况,减轻组织学损伤,并能够提高存活率,30％ PLT组与30％ PLT+ MSC组一周存活率分别为16.7％和58.3％.而在Cyclin D1和PCNA的免疫组化表达中,30％ PLT组表达明显抑制,30％ PLT+ MSC组表达上调.结论 骨髓间充质干细胞可以存进大鼠小体积肝移植后移植肝的再生,改善肝功能,并提高存活率.     

大鼠小体积肝脏移植后早期肝内细胞因子的变化

目的 研究不同冷缺血条件下大鼠小体积肝移植(30%标准体积)后早期肿瘤坏死因子α(TNF-α)及白细胞介素6(IL-6)的变化,及其与肝脏再生的关系.方法 建立Lewis大鼠30%标准体积的原位肝移植模型.根据供肝在UW液中冷保存时间的不同,将受者分为3组:冷缺血1 h组、冷缺血8 h组和冷缺血16 h组,每组均为20只.观察受者存活情况至术后第7天,并分别在移植肝恢复血流后90 min、1 h、2 h、4 h和7 d收集样本,检测移植肝组织中TNF-α和IL-6表达情况,肝细胞DNA的合成情况,进行移植肝的形态学观察.结果 大鼠肝移植手术成功率均为100%.移植后第7天,冷缺血1 h和8 h组受鼠的存活率均为100%.冷缺血16 h组受鼠的存活率较低,移植后第7天无受鼠存活.冷缺血1 h组TNF-α和IL-6的表达水平较低,冷缺血8 h组和冷缺血16 h组TNF-α和IL-6的表达则高于冷缺血1 h组(F=58.81和F=184.12,P<0.05).冷缺血8 h组和冷缺血16 h组间TNF-α和IL-6的表达的差异无统计学意义.冷缺血1 h组增殖细胞数目明显高于冷缺血8 h组,差异有统计学意义(t=5.59,P<0.05).移植术后24h,冷缺血1 h组移植肝有轻度的组织学损伤;冷缺血8 h组移植肝有轻度的窦状隙扩张和轻度的炎症;冷缺血16 h组移植肝有局部淤血,存在肝细胞崩解和坏死等改变.结论 在小体积肝移植后早期,TNF-α和IL-6的上调表达对肝脏再生有重要意义.不同冷缺血时间的小体积肝脏移植物内存在早期启动肝脏再生的信号.     

大鼠小体积肝移植后肝再生障碍的实验研究

目的探讨部分肝移植后肝再生状况和小体积移植物发生肝再生障碍的可能机制。方法实验分为大鼠全肝移植组（OLT）、50%部分肝移植组（50%PLT）和30%部分肝移植组（30%PLT）。分析各组术后肝功能的变化,通过免疫组化观察移植肝标本Cyclin D1和PCNA的表达,并对移植肝组织结构进行电镜观察。结果各组ALT和AST于术后24hr达到峰值,且30%PLT组上升显著。Cyclin D1和PCNA的免疫组化表达中,50%PLT组表达明显上调,而30%PLT组表达则明显抑制。电镜观察发现30%PLT组术后微观结构改变显著。结论 30%PLT组术后肝细胞增殖再生受到明显抑制,推测与肝细胞微环境的恶化和叠加的缺血再灌注损伤有关。     

大鼠小体积肝移植中肝动脉重建的意义探讨

目的 探讨肝动脉重建在大鼠小体积肝移植中的意义.方法 采用改良二袖套法建立大鼠40%小体积肝移植模型,实验分动脉重建组(A组)和非动脉重建组(B组),观察1周生存率,并于术后第1、2、4、7天检测肝功能、移植肝组织学变化和免疫组化法检测肝细胞的增殖细胞核抗原(PCNA).结果 A组1周生存率66.7%,B组1周生存率50.0%(P>0.05).ALT和TB术后第1天即开始明显增高,第2天达高峰,以后逐渐降低,B组和A组各时间点相比较,TB第2、7天高于A组,ALT第4、7天高于A脉组,差异有显著性意义(P<0.05).A组术后中央静脉及肝窦扩张程度相对较轻,可见较多二倍体和多倍体肝细胞.PCNA表达于术后第2天最高,A组术后第1天的移植肝细胞PCNA阳性表达率高于B组,而术后第7天移植肝细胞PCNA阳性表达率低于B组,差异有显著性意义(P<0.05).结论 肝动脉重建可明显改善大鼠小体积移植肝的功能,促进移植肝的再生,有效地保护移植肝的组织学结构,减轻术后移植肝的组织学改变.     

大鼠肝移植小移植物模型的研究

目的 建立大鼠肝移植小移植物模型，探讨移植物体积对受体生存的影响。方法 48只雄性SD大鼠随机分为4组：70％肝切除组(A组)、全肝移植组(B组)、中等移植物组(C组)、小移植物组(D组)。观察各组大鼠生存率。于术后不同时间点，采取外周静脉血检测肝功能；行肝组织常规组织学及透射电镜检测。结果 D组第3天生存率为56％，显著低于A、B、C组(P＜0．01)；各时间点D组各生化指标均显著高于其他3组(P＜0．01)；A、C和D组肝脏组织学以肝细胞再生的表现为主，B组以淋巴细胞浸润为主。D组术后第3天光镜下和透射电镜检查可见分裂肝细胞；第21天移植肝体积已达正常水平。结论 大鼠肝移植小移植物模型供肝量应超过全肝重量的45％。影响小移植物成活的主要因素是缺血—再灌注损伤和手术技术。     

Liver progenitor cells activated after 30% small-for-size liver transplantation in rats: a preliminary study

AIM: To explore whether liver progenitor cells were activated after 30% small-for-size liver transplantation in rats. METHODS: 200 rats were arranged in three groups: 70% partial hepatectomy (PH), whole liver transplantation (WLT), and 30% liver transplantation (SLT) group. On days 1, 2, 3, 5, 7 after operation, 6 rats were sacrificed in each group at each time. One week survivals were analyzed; while liver injury and regeneration index were estimated by serum ALT AST, H&E staining and proliferating cell nuclear antigen index. The oval cell markers, including CD90 and OV6, were detected in liver sections by immunohistochemistry. RESULTS: The 50% survival rate of the SLT group was significantly lower than those of the PH and the WLT groups. At each time after operation, the serum ALT and AST were much higher in the SLT group. Compared with the PH group on days 1, 2, and 3 postoperatively, the PCNA indices were lower among the SLT group. OV-6 positive and CD90 positive cells were detected in the SLT group from day 2 postoperatively. These progenitor cells were first dispersed in the liver but restricted to the periportal region over the following days. CONCLUSION: Liver progenitor cell activation after SLT may be related to the liver dysfunction caused by a small-for-size graft.     

表达HGF的骨髓间充质干细胞促进小移植肝早期肝再生

目的 建立大鼠小体积肝移植模型,输注表达人肝细胞生长因子(human hepatocyte growth factor,hHGF)的骨髓间充质干细胞(mesenchymal stem cells,MSCs),研究其在移植早期对小移植肝促再生作用.方法 将已建立的表达hHGF和绿色荧光蛋白(green fluorescence protein,GFP)的MSCs,分别命名为HGF/MSCs,GFP/Mscs.建立大鼠30%肝移植模型.受体分为4组,实验组输注5×106HGF/MSCs;对照组则分别输注相同体积的生理盐水(PS),5×106 GFP/MSCs或1.0×109 pfu含hHGF的重组腺病毒液(Ad-HGF).分别于术后1,3,5,7 d各组随机抽取5只大鼠处死.取血检测血清ALT和hHGF.记录移植物湿重.取肝组织检测hHGF、c-met表达,以及肝细胞凋亡和增殖活性.另每组15只,分组同上,用于观察生存期.结果 PS组大鼠7 d生存率33.3%;组织学及血清学检查示术后肝脏损伤重,汇管区单核细胞浸润多;而实验组大鼠7 d生存率为73.3%.肝脏损伤轻,炎性细胞浸润少;实验组移植肝再生较PS组明显增加.结论 大鼠部分肝移植后,输注HGF/MSCs能够保护小体积移植肝,促进小移植肝再生,提高7 d生存率.     

Impaired hepatic regeneration by ischemic preconditioning in a rat model of small-for-size liver transplantation

OBJECTIVE: Graft size is one of the major risk factors in adult-to-adult living donor liver transplantation and rapid regeneration is an essential post-operative requirement. Ischemic preconditioning (IPC) has been shown to be an effective strategy in the reduction of hepatic ischemia-reperfusion injury and stimulation of liver regeneration. This study was designed to evaluate the effects of IPC on liver regeneration in small-for-size liver grafts. METHODS: We employed a rat orthotopic liver transplantation model using small-for-size (30%) grafts, in the presence or absence (control) of IPC (10 min of ischemia followed by 15 min of reperfusion). Survival rate, graft injury, hepatocellular proliferation, cell cycle progression, Stat3 activation, as well as TNF-alpha and IL-6 expression were assessed. RESULTS: IPC significantly enhanced the extent of graft injury and hindered hepatic regeneration in small-for-size liver grafts. The 7-day survival rate was also reduced by IPC, but failed to reach statistical significance. IPC did not affect TNF-alpha levels, but significantly decreased the elevation of IL-6 after reperfusion. These findings were correlated with down-regulation of cyclin E and cyclin D1, and decreased numbers of PCNA-positive nuclei in IPC grafts. These results were inconsistent with Stat3 activation, as P-Stat3 exhibited a stronger and prolonged pattern of expression in the IPC group, compared to controls. CONCLUSIONS: Ischemic preconditioning may impair liver regeneration in small-for-size liver grafts by decreasing IL-6 and blunting cell cycle progression, through a mechanism at least partially independent of Stat3.     

Attenuation of acute phase shear stress by somatostatin improves small-for-size liver graft survival.

The major concern of living donor liver transplantation is small-for-size graft injury at the early phase after transplantation. Novel therapeutic strategies should be developed. To investigate the protective effect of somatostatin related to hemodynamic stress on small-for-size liver graft injury, we applied a treatment regimen of low-dose somatostatin in a rat orthotopic liver transplantation model using small-for-size grafts (median, 38.7%; range, 35-42%). Somatostatin was given at 5 minutes before total hepatectomy and immediately after reperfusion in the recipient (20 microg/kg). Graft survival, portal hemodynamics, intragraft gene expression and hepatic ultrastructural changes were compared between the rats with or without somatostatin treatment. Seven-day graft survival rates in the somatostatin treatment group were significantly improved compared to the control group (66.7% vs. 16.7%, P = 0.036). In the treatment group, portal pressure and hepatic surface blood flow were significantly decreased within the first 30 minutes after reperfusion, whereas in the control group, transient portal hypertension and excessive hepatic blood flow were observed. Intragraft expression (both messenger RNA and protein) of endothelin-1 was significantly downregulated accompanied with upregulation of heme oxygenase-1 and A20. Better preservation of liver function was found in the treatment group. Hepatic ultrastructure, especially the integrity of sinusoids, was well protected in the treatment group. In conclusion, low-dose somatostatin rescues small-for-size grafts from acute phase injury in liver transplantation by attenuation of acute-phase shear stress that resulted from transient portal hypertension.     

Rapamycin Attenuates Liver Graft Injury in Cirrhotic Recipient—The Significance of Down-Regulation of Rho-ROCK-VEGF Pathway

To investigate whether rapamycin could attenuate hepatic I/R injury in a cirrhotic rat liver transplantation model, we applied a rat orthotopic liver transplantation model using 100% or 50% of liver grafts and cirrhotic recipients. Rapamycin was given (0.2 mg/kg, i.v.) at 30 min before graft harvesting in the donor and 24 h before operation, 30 min before total hepatectomy and immediately after reperfusion in the recipient. Rapamycin significantly improved small-for-size graft survival from 8.3% (1/12) to 66.7% (8/12) (p = 0.027). It also increased 7-day survival rates of whole grafts (58.3%[7/12] vs. 83.3%[10/12], p = 0.371). Activation of hepatic stellate cells was mainly found in small-for-size grafts during the first 7 days after liver transplantation. Rapamycin suppressed expression of smooth muscle actin, which is a marker of hepatic stellate cell activation, especially in small-for-size grafts. Intragraft protein expression and mRNA levels of vascular endothelial growth factor (VEGF) were down-regulated by rapamycin at 48 h both in whole and small-for-size grafts. Consistently, mRNA levels and protein expression of Rho and ROCK I were decreased by rapamycin during the 48 h after liver transplantation. In conclusion, rapamycin attenuated graft injury in a cirrhotic rat liver transplantation model by suppression of hepatic stellate cell activation, related to down-regulation of Rho-ROCK-VEGF pathway.     

FK 409 ameliorates small-for-size liver graft injury by attenuation of portal hypertension and down-regulation of Egr-1 pathway

OBJECTIVE: To investigate whether low-dose nitric oxide donor FK 409 could attenuate small-for-size graft injury in liver transplantation using small-for-size grafts. SUMMARY BACKGROUND DATA: The major concern of live donor liver transplantation is small-for-size graft injury at the early phase after transplantation. Novel therapeutic strategies should be investigated. METHODS: We employed a rat orthotopic liver transplantation model using small-for-size (40%) graft. FK 409 was given at 30 minutes before graft harvesting (2 mg/kg) to the donor and immediately after reperfusion (1 mg/kg) to the recipient (FK group). Graft survival, intragraft genes expression, portal hemodynamics, and hepatic ultrastructural changes were compared between the 2 groups. RESULTS: Seven-day graft survival rates in the FK group were significantly improved compared with those of rats not receiving FK 409 (control group; 80% versus 28.6%, P = 0.018). In the FK group, portal pressure was significantly decreased within the first 60 minutes after reperfusion whereas in the control group, transient portal hypertension was observed. Intragraft expression (both mRNA and protein) of early growth response-1, endothelin-1, endothelin-1 receptor A, tumor necrosis factor-alpha, macrophage-inflammatory protein-2, and inducible nitric oxide synthase was significantly down-regulated accompanied with up-regulation of heme oxygenase-1, A20, interferon-gamma-inducible protein-10, and interleukin-10 during the first 24 hours after reperfusion. Hepatic ultrastructure, especially the integrity of sinusoids was well protected in the FK group. CONCLUSIONS: Low-dose FK 409 rescues small-for-size grafts in liver transplantation by attenuation of portal hypertension and amelioration of acute phase inflammatory response by down-regulation of Egr-1, together with prior induction of heat shock proteins.     

Effect of middle hepatic vein reconstruction in living donor liver transplantation using right lobe

BACKGROUND: This study reviewed the impact of middle hepatic vein (MHV) reconstruction on right lobe graft with regard to functional recovery and graft regeneration at 1 week after transplantation. MATERIALS AND METHODS: From January 1999 to September 2005. 211 adult living donor liver transplantations were performed using the right lobe. The reconstruction of hepatic venous tributaries from segment 5 or segment 8 or both was performed in every cases of sufficient size. The patency of graft vessels was evaluated with computed tomography (CT) angiography on postoperative day 7. We analyzed liver enzymes (aspartate transferase [AST], alanine transferase [ALT] and bilirubin) at 1 week postoperatively and evaluated regeneration activity by CT volumetry at 1 week postoperatively. RESULTS: Among 211 cases, 182 (86.3%) were reconstructed with interpositional MHV grafts. Among them, 51 cases (51.9%) were patent at 1 week postoperatively. The levels of AST and ALT in patent cases of all patients and small-for-size grafts were lower than among the occlusion cases, albeit not significantly. The mean graft regeneration at 1 week postoperatively among patent cases was 1.75 +/- 0.39 versus 1.64 +/- 0.24 in the occluded cases (P = .111), but among small-for-size grafts, there was a significant difference in graft regeneration between patent versus occluded cases (2.05 +/- 0.50 vs 1.66 +/- 0.17, P = .037). CONCLUSION: Functional recovery and graft regeneration in small-for-size grafts showed a beneficial effect in patent cases, compared with occluded cases. Our selection criteria for MHV reconstruction must include cases of small-for-size grafts not all cases.