Sorafenib prolongs liver regeneration after hepatic resection in rats

BackgroundThe multikinase inhibitor sorafenib inhibits angiogenesis and tumor cell proliferation. Sorafenib targets signaling pathways involved in liver regeneration. Previous works on regenerating mouse liver show differing results. We asked to which degree different lengths of sorafenib treatment would influence liver regeneration after hepatic resection in rats.MethodsFischer-344 rats received intragastric injections of sorafenib (5–15 mg/kg/d), underwent a two-thirds partial hepatectomy (PH), and were sacrificed at different time points thereafter. Sorafenib treatment was stopped 0, 3, or 14 d after PH. Serum levels of aminotransferases and labeling indices of S-phase nuclei (bromodeoxyuridine and MIB-5) were analyzed, body and liver weights measured, and levels of phospho-ERK determined by Western blot.ResultsSorafenib increased aminotransferases and the number of S-phase nuclei at baseline, but decreased liver weights and levels of phospho-ERK 24 h after PH. The number of S-phase nuclei and mitotic indices decreased 48 h after PH and increased 7 d after PH in animals on sorafenib treatment. Relative liver weights were restored 5 d after PH in control rats, at 7 d in animals receiving sorafenib prior to surgery, at 10 d in rats where sorafenib was stopped 3 d after surgery, and after 14 d in rats on continuous treatment.ConclusionsIn this rat model, the regenerating liver adapted to the proliferation-inhibitory effect of sorafenib during continuous treatment. Sorafenib given after hepatic resection did not completely inhibit liver regeneration, but it prolonged the regenerative phase in proportion to the length of treatment.     

肝切除联合肝动脉切除对梗阻性黄疸大鼠肝细胞再生和凋亡的影响

目的 探索在梗阻性黄疸时,不同范围肝切除联合肝动脉切除对肝细胞再生和凋亡的影响.方法 155只雄性SD大鼠行胆总管结扎制备梗阻性黄疽模型,5 d后二次手术分为:胆肠再通内引流组;肝切除(42%、70%)联合胆肠再通内引流组;肝切除(42%,70%)联合肝固有动脉切除、胆肠再通内引流组.动态观察二次手术后24 h、72 h、7 d肝组织HGF、bcl-2 mRNA含量及蛋白表达、肝细胞增殖和凋亡指数的变化,并统计各组死亡率.结果 高胆红素血症、行胆肠冉通内引流的同时,大鼠肝切除或肝切除联合肝动脉切除后,肝再生均受抑制,凋亡增多;较之肝切除组和42%肝切除联合肝固有动脉切除组.70%肝切除联合肝固有动脉切除组术后肝组织HGF、bcl-2 mRNA含量显著减少,肝细胞再生明显受抑而凋亡显著增多,死亡率显著增高(P<0.05).结论 高胆红素血症时,肝切除量是影响大鼠肝切除联合肝动脉切除实施安全性的重要因素,42%肝切除联合肝固有动脉切除、胆肠再通内引流,对肝细胞再生和凋亡影响较小,安全町行;70%肝切除联合肝动脉切除、胆肠再通内引流后肝细胞再生显著受抑制,凋亡增多,死亡率高,应避免实施.     

Prognostic factors affecting survival and recurrence after hepatic resection for hepatocellular carcinoma in cirrhotic liver

Aim  

Hepatic resections for hepatocellular carcinoma (HCC) in cirrhotic liver are characterized by early recurrence and poor survival. In this study, we analyzed several factors affecting both survival and recurrence after hepatic resection.     

nodosin促进大鼠部分肝移植肝细胞再生的实验研究

目的  探讨溪黄草有效成分nodosin对大鼠减体积肝移植术后肝细胞再生的影响。方法  以Wistar大鼠作为供体, SD大鼠作为受体, 采用改良的二袖套法建立大鼠部分肝移植模型, 将24只受体大鼠随机分为nodosin组和对照组。nodosin组于肝移植术后经尾静脉注射nodosin 100μg/ml。在术后3 d和7 d分别处死大鼠取外周血浆及肝脏组织标本。分光光度法检测外周血浆中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)和白蛋白(ALB)含量; 光学显微镜下观察肝组织形态学改变; 采用免疫组织化学法检测肝组织增殖细胞核抗原(PCNA)抗体的表达水平; 采用蛋白印迹法(Western blot)检测肝组织中磷酸化蛋白激酶(p-AKT)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、细胞周期素D1(cyclin D1)、血红素加氧酶(HO)-1蛋白的表达水平; 采用磷脂酰丝氨酸外翻分析(Annexin V法)和dUTP缺口末端标记(TUNEL)法检测肝细胞的凋亡情况。结果  术后3 d和7 d, 与对照组比较, nodosin组大鼠血清中ALT、AST含量较低, ALB含量较高(均为P < 0.05), 同时nodosin能够减轻大鼠肝移植术后肝组织病理损伤。nodosin组的PCNA阳性表达细胞数明显多于对照组(P < 0.05)。nodosin组增殖相关蛋白p-AKT、p-mTOR、cyclin D1和HO-1蛋白表达水平均明显高于对照组(均为P < 0.05)。nodosin组发生凋亡的肝细胞数量和比例明显低于对照组(P < 0.05)。结论  大鼠肝移植术后应用nodosin具有减少肝细胞凋亡数量, 促进肝细胞增殖的作用。     

急性肝功能衰竭肝细胞移植后的肝再生研究进展

急性肝功能衰竭（acute liver failure，ALF）的治疗十分棘手。自1985年开始采用急诊行原位肝移植治疗以来，术后一年生存率已提高至83％。但是急诊行原位肝移植存在供体短缺等缺点，其应用受到一定限制。近年肝细胞移植的研究取得很大进展。有望成为治疗ALF的另一种有效手段。由于肝脏的再生潜力很大．移植肝细胞所提供的暂时、有效的肝功能支持．为受体肝脏的再生恢复赢得了时间，从而可使ALF病人有时间等待肝移植，甚至有可能避免肝移植。研究ALF肝细胞移植后的肝再生及其机制．探讨其影响因素．对肝细胞移植的临床应用具有重要的意义。本文就有关ALF肝细胞移植后肝再生的研究进展综述如下。     

Deletion variant of hepatocyte growth factor prolongs allograft survival after liver transplantation in rats.

BACKGROUND: Despite continued progress in the development of immunosuppressive agents, allograft rejection remains an important cause of morbidity and mortality after liver transplantation. We examined the effect of the deletion variant of hepatocyte growth factor (dHGF) on allograft rejection after liver transplantation. METHODS: Male Dark Agouti rats (RT1a) were selected as donors and male Lewis rats (RT1l) as recipients for a rejection model. The recipients were divided into 2 groups after orthotopic liver transplantation (OLTx): in the dHGF group dHGF was given intravenously twice a day (1 mg/kg/day) after OLTx, whereas in the control group vehicle buffer was given intravenously daily twice after OLTx. The survival period, serum chemistry studies, and histopathologic findings were then compared between the 2 groups. RESULTS: The mean survival period after OLTx in the dHGF group was significantly longer than that in the control group (21.4 +/- 1.3 days vs 11.8 +/- 0.4 days, P < .001). On the 10th posttransplant day the serum albumin level significantly improved in the dHGF group (P < .01), and the serum total bilirubin and aspartate aminotransferase levels were significantly lower in the dHGF group (P < .01 and P < .05, respectively). On the 10th posttransplant day a histologic examination revealed no apparent difference in the severity of rejection between the 2 groups. The number of proliferating cell nuclear antigen-positive hepatocytes in the dHGF group significantly increased (P < .01), whereas the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling-positive hepatocytes were significantly reduced in the dHGF group (P < .01) in comparison with those in the control group. CONCLUSION: dHGF has an antiapoptotic property as well as a proliferative and protective effect on hepatocytes under allograft rejection. dHGF might serve as a novel agent for reducing the harmful effects of hepatic allograft rejection in rats.     

大鼠小体积肝移植后肝细胞再生的研究

目的 探讨小体积肝移植术后肝再生的情况。方法 建立大鼠30％原位肝移植模型,实验分为肝切除（PH）组、全肝移植（0LT）组和30％小体积肝移植（30％POLT）组,观察1w生存率,并于术后1、2、3、7d检测肝细胞增殖活性、肝功能和肝组织学变化。结果 各组1w生存率均为100％;30％POLT组术后2d达到增殖高峰,峰值与PH组无差异（P〉0．05）;其肝功能酶学指标在术后1、3d明显升高,组织学见肝细胞核分裂极其活跃。结论 冷缺血1h的30％供肝和肝切除后肝脏具有同样的增殖活性,仅增殖高峰稍晚;较短的冷缺血时间以及熟练的手术技术可能对小体积供肝的再生起重要作用。     

Acute impairment of hepatic microcirculation and recruitment of nonparenchymal cells by intrasplenic hepatocyte transplantation

Background/Purpose

Over the last 20 years, hepatocyte transplantation (HcTx) has advanced from the experimental to the clinical stage. To date, HcTx has been performed in 30 patients in the United States. Regardless whether hepatocytes are transplanted into the spleen and migrate to the liver or are injected directly into the portal vein, transplanted liver cells will, to some extent, congest the recipient liver microcirculation. The potential negative consequences of intrasplenic HcTx were the subject of this study.

Methods

By using intravital microscopy, the authors investigated whether intrasplenic HcTx of 20 × 10⁶ allogenic hepatocytes would influence liver perfusion, excretory liver function, and nonparenchymal cells (Kupffer and Ito cells) in vivo.

Results

The sinusoidal perfusion rate declined significantly from 94% (control) to 84% on day 1 and 76% on day 7. Bile acid excretion decreased in a similar fashion from 0.924 mg/h (control) to 0.669 mg/h on day 7. The authors observed a significant increase of Ito cells from 81.1 cells per microscopic field (control) to 97.1 (day 1) and an increase of Kupffer cells (KC; 6.1 cells per microscopic field on day 1 v 3.8 on control).

Conclusions

This study shows an acute impairment of hepatic microcirculation and hepatucellular function along with an recruitment and activation of nonparenchymal cells in the early posttransplantation period after intrasplenic HcTx. Kupffer cell recruitment indicates an activation of local host defense, and Ito cell activation implies the initiation of liver repair mechanisms owing to ischemia-related cell damage.     

肝卵圆细胞在肝硬化组织中介导肝脏再生的研究进展

肝脏依靠肝细胞的自我更新和肝卵圆细胞(HOC)的增殖分化两大途径参与肝损伤的修复。HOC是一类具有多向分化潜能的肝干细胞,在肝脏再生中扮演重要角色。肝硬化肝细胞再生能力低下,HOC参与肝脏损伤的修复和重建,其活化,增殖及分化等与肝硬化微环境密切相关。因此,深入研究HOC在肝硬化中介导肝脏再生的机制及细胞移植的优势,将为治疗终末期肝硬化提供新策略。笔者就HOC的特征、其在肝硬化微环境下的作用、以及肝硬化微环境对HOC介导肝脏再生的调控等研究进展进行综述。     

Treatment of carbon tetrachloride and phenobarbital-induced chronic liver failure with intrasplenic hepatocyte transplantation

Hepatocyte transplantation (HTx) has been shown to improve the survival of laboratory animals with experimentally induced acute liver failure and to ameliorate the physiologic abnormalities associated with liver-based metabolic deficiencies. However, the role of HTx in the treatment of liver cirrhosis (LC) has not been adequately studied. In order to address this issue, HTx was performed in rats following induction of stable LC using phenobarbital (PhB) and carbon tetrachloride (CCl4). Intrasplenic transplantation of 50 x 10(6) primary hepatocytes could significantly improve liver functions and prolong the survival of rats with irreversible, decompensated LC.     

异甘草酸镁对肝硬化大鼠肝部分切除术后肝功能及肝再生的影响

目的 研究异甘草酸镁对肝硬化大鼠部分肝切除术后肝功能和肝再生的影响.方法 45只肝硬化Wistar大鼠行2/3肝部分切除,并随机分为对照组(A组)、治疗组(B组)、术前3 d治疗组(C组).手术当天起,B组给予腹腔注射异甘草酸镁(60mg·kg-1·d-1),A组给予同等剂量生理盐水,C组在手术前3 d即给药(60 mg·kg-1·d-1).各组大鼠分别于术后1、2、7 d处死,检测肝功、血清肝细胞生长因子(HGF)及磷脂酶A2(PLA2)、5-溴脱氧尿苷(BrdU)标记指数、肝再生率.结果 A组在术后第1天,BrdU标记指数及HGF均低于C组(分别t=2.831,3.427,均P＜0.05),而PLA2高于B、C组(分别t=2.794,2.902,均P＜0.05);在术后第2天A组BrdU标记指数均低于B、C组(分别t=2.736,3.083,均P＜0.05),HGF水平与其他两组比较,差异均无统计学意义,PLA2仍高于B、C组(分别t=2.794,2.902,均P＜0.05);A组术后第1、2天ALT、AST、TP水平及肝再生率与B、C组比较,差异均无统计学意义;术后第7天A组AST高于其他两组(A组与B组比较t=4.508,P＜0.05;A组与C组比较t=2.967,P＜0.05),TP水平及肝再生率则均低于B、C组(TP:A组与B组比较t=2.838,P＜0.05;A组与C组比较t=2.743,P＜0.05);肝再生率:(A组与B组比较t=3.316,P＜0.05;A组与C组比较t=4.093,P＜0.05),而BrdU标记指数、HGF、PLA2三组间比较,差异均无统计学意义.B组术后第1天BrdU标记指数及HGF均低于C组(t=2.831,P＜0.05;t=2.836,P＜0.05).结论 异甘草酸镁可降低肝硬化大鼠部分肝切除术后转氨酶水平,改善肝脏功能,促进肝细胞增生.     

奥美拉唑对大鼠减体积肝移植后肝再生的影响

目的观察奥美拉唑对大鼠减体积肝移植肝细胞再生的影响。方法建立大鼠减体积肝移植模型，实验组移植后即时给予奥美拉唑，对照组予生理盐水。两组分别于肝移植术后分为5组（n=8），观察术后3、5、7、10、14d血清丙氨酸转氨酶、天冬氨酸转氨酶值、移植肝重／供肝减体积前全肝重比值、移植肝细胞有丝分裂指数（MI）、增殖细胞核抗原（PCNA）表达指数、溴脱氧尿嘧啶核苷（BrdU）掺入指数及血清胃泌素值。结果大鼠减体积肝移植术后5d肝细胞再生达高峰，实验组的再生活性显著高于对照组，MI为（2．54±0．24）％和（1．71±0．16）％（P〈0．01）、PC—NA指数为（26．96±2．09）％和（18．73±1．94）％（P〈0．01）、BrdU指数为（10．24±1．11）％和（5．75±0．88）％（P〈0．01）。术后7d，实验组和对照组移植肝重／全肝重比值分别为（76．3±1．6）％和（71．2±1．0）％（P〈0．05），血清胃泌素水平分别为（441．9±25．9）ng／L和（292．9±14．2）ng／L（P〈0．05）。术后14d，实验组和对照组移植肝重／全肝重比值分别为（94．5±1．7）％和（86．9±1．5）％（P〈0．01），血清胃泌素水平分别为（487．8±29．4）ng／L和（291．7±21．6）ng／L（P〈0．01）。实验组和对照组血清ALT、AST值差异无统计学意义。结论奥美拉唑能促进减体积肝移植术后的肝细胞再生，其作用可能与胃泌素分泌增高有关。     

DiI荧光示踪剂在大鼠脾内肝细胞移植中的研究

目的 探讨大鼠肝细胞在脾内移植后的生存、迁移及其并发症情况。方法 先用ＤｉＩ荧光示踪剂标记肝细胞再行脾内肝细胞移植。在不同时间段取肝、脾、肺、心、肾 、胸腺组织冷冻切片,在荧光显微镜下用荧光和普通光示踪观察。结果 ＤｉＩ荧光示踪剂能很好地标记肝细胞,６０ｄ实验期内,移植细胞能在所取各器官中生存并保持良好形态,未发现有区域梗塞灶、同时还反映了其在肝实质中的分布。结论 ＤｉＩ是一种新颖、使用简便的荧光     

Effects of intrasplenic injection of hepatocytes, hepatocyte fragments and hepatocyte culture supernatants on D-galactosamine-induced liver failure in rats

Intraperitoneal injection of 0.5 g/kg D-galactosamine results in 95% lethal acute acute liver failure in male Fisher 344 rats. Intrasplenic injection of viable syngeneic hepatocytes 20-28 h after poisoning improves survival in a dose-dependent fashion, 10(7) cells being the optimal dose with a survival rate of 47.1%. While nonviable cells and hepatocyte fragments are totally ineffective, 42.9% of rats survive after injection of 28-hour liver cell culture supernatant. It is concluded that soluble factors generated by cultured cells in vitro or intrasplenically transplanted cells improve survival either by a direct hepatotrophic effect, by stimulation of the reticulo-endothelial system or by an unspecific humoral mechanism.     

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。     

肝硬化鼠肝移植后早期全身和内脏血流动力学的变化

目的 观察正常鼠肝移植及及肝硬化鼠肝移植术早期全身和内脏血流动力学的变化。方法 实验动物随机分为正常鼠（NL,10只）、肝硬化鼠（IHPH,10只）、正常鼠肝移植（NL－OLT,9只）、肝硬化鼠肝移植（IHPH－OLT,16只）组。分别采用放射性微球法行血流动力学研究。结果 NLOLT鼠绝大多数血流动力学参数与NL鼠比较差异无显著意义。IHPH及IHPH－OLT 3d,7d组心输量和内脏血流量和内脏血流量增加,平均动脉压、周围血管总阻力和内脏血管阻力降低。内脏血流动力学紊乱较全身明显。结论 肝硬化鼠肝移植后的血流动力学紊乱可能与移植前已存在的病理生理因素有关。     

Changes after major resection of experimental cirrhotic liver

In an investigation of resectability and changes in the remnant liver after major resection of the cirrhotic liver, liver cirrhosis was induced experimentally in dogs—hemodynamically, by constriction of the hepatic vein and pharmacologically, by the administration of DMNA—after which approximately 45 per cent of the liver was resected.     

药物性肝衰大鼠脾内移植异种肝细胞治疗前后的CD4，CD8的变化及意义

为探讨异种肝细胞脾内移植治疗大鼠药物性肝衰的意义，观察大鼠肝功能、存活率及ＣＤ４，ＣＤ８在免疫排斥反应中的作用。笔者以豚鼠肝细胞为供体，ＳＤ大鼠为受体，Ｄ-氨基半乳糖(１９.５ml/㎏)腹腔内一次性注射，制作肝衰模型。４８h后将微囊化的游离豚鼠肝细胞移植于大鼠脾脏内。以豚鼠裸肝细胞移植及生理盐水脾脏注射为对照。移植后１４d测定存活率和肝功能，检查病理切片及ＣＤ４，ＣＤ８的变化。结果示微囊化肝细胞移植组存活率８０.0％，明显高于生理盐水组（２５.0％）和裸肝细胞移植组（７０.0％）（分别为P<０.０１和P<０.０５）。微囊肝细胞移植组１４ｄ总胆红素和ＡＬＴ，明显低于生理盐水组和裸肝细胞组。移植后７２ｈ和１４ｄ分别测定受体脾脏ＣＤ４，ＣＤ８淋巴细胞，移植后７２ｈ，１４ｄ微囊化肝细胞组、裸肝细胞组CD4，CD8均呈阳性。提示：大鼠药物性肝衰微囊化肝细胞脾内移植后可维持受体存活１４ｄ，体液和细胞免疫均参与排斥反应。肝细胞微囊化处理可延迟细胞免疫的发生时间。