5-HT及其受体在大鼠肝再生过程中的作用

目的分析5-羟色胺（5-HT）及其受体在大鼠肝脏再生过程中的作用。方法将50只雄性Wistar大鼠随机分成实验组和对照组。实验组大鼠于肝大部分切除术后24、36、48和72h处死,对照组行假手术。采用流式细胞技术检测大鼠肝脏增殖细胞核抗原（PCNA）表达、免疫组化法检测Ki67及5-HT表达、实时荧光定量PCR检测5-HT2A、2B受体亚型的表达。结果肝大部切除术后大鼠肝脏重量逐渐增加,PCNA和Ki67表达于术后24和36h达高峰;在肝切除术后各个时间点5-HT2A、2B受体在肝脏中的表达均显著升高,以术后36h最高;术后36h空肠嗜铬细胞5-HT含量高于24h,且都高于正常大鼠。结论肝脏再生过程中5-HT合成及其受体表达均显著上调,可能与肝脏的再生有关。     

靶向SMAD3基因的shRNA重组慢病毒对大鼠肝再生的作用

目的:应用大鼠肝大部切除肝再生模型,将前期构建好的靶向SMAD3基因的shRNA重组慢病毒注射入大鼠体内,观察SMAD3 shRNA对大鼠肝再生的影响.方法:将60只♂Wistar大鼠随机分为3组:SMAD3 shRNA组(20只)、shRNA对照组(20只)、生理盐水对照组(20只),通过脾脏注射方法给药,慢病毒给药剂量为1.0×108TU/只,生理盐水对照组给予等体积500L生理盐水.给药96h后行2/3肝大部切除,构建大鼠肝再生模型;肝大部切除术后96h各组分别除死大鼠7只,剩余大鼠于144h处死,收集肝脏标本,Real-Time PCR、免疫组织化学检测肝组织SMAD3表达,免疫组织化学检测肝组织Ki67表达,测定大鼠肝质量/体质量,观察SMAD3 shRNA对肝再生的影响.结果:Real-Time PCR检测显示,通过脾脏注射慢病毒,在96、144h处死时间点,SMAD3 shRNA组SMAD3 mRNA分别较shRNA对照组平均下降73%、63%.免疫组织化学检测显示SMAD3蛋白表达明显下降.Ki67免疫组织化学结果显示,肝大部切除术后96、144h,SMAD3 shRNA组Ki67表达阳性细胞数均明显多于生理盐水对照组及shRNA对照组,表明抑制SMAD3表达后肝细胞增殖活跃.大鼠肝质量与体质量比值显示,SMAD3 shRNA组分别较生理盐水对照组及shRNA对照组有增加趋势(96h:4.50±0.43vs3.97±0.55vs3.98±0.40,144h:4.66±0.54vs4.15±0.51vs4.20±0.34),但没有统计学意义(P>0.05).结论:SMAD3 shRNA在大鼠体内可一定程度上促进肝细胞增殖,但对肝再生的促进作用尚弱.     

非酒精性脂肪肝大鼠部分肝切除术后肝再生功能的变化

目的 探讨大鼠非酒精性脂肪肝（NAFLD）部分肝脏切除术后肝再生功能的变化。方法 80只Wistar大鼠，随机分为正常对照组（C组，35只）与NAFLD组（F组，45只），C组给予正常饮食喂养，F组给予高脂饲料喂养。在喂养至第12周时行70％肝切除术，两组动物分别于术后0、1、12、24、36h处死，取出残肝，计算再生肝重比；光镜下计数核分裂肝细胞；透射电镜观察术后肝细胞超微结构的变化；免疫组织化学染色法检测增殖细胞核抗原阳性表达率；半定量逆转录聚合酶链反应检测细胞周期蛋白D1的表达变化。结果 光镜和电镜观察显示F组肝窦狭窄迂曲，细胞质内大量脂滴沉积，细胞核小，细胞器少，能量代谢及细胞增殖均不活跃。F组术后12、24、36h核分裂相计数明显低于C组同时相点（P〈0．01）；F组术后再生肝重比、S期细胞分数及增殖指数也较C组下降，差异有统计学意义（P〈0．01）；F组增殖细胞核抗原阳性率、细胞周期蛋白D1的mRNA表达在术后12、24、36h均明显低于C组同时相点（P〈0．01）。结论 中至重度NAFLD大鼠部分肝切除术后DNA合成高峰滞后，肝再生延迟，再生进程主要被阻滞在细胞周期的G1／S期调控点。     

重组人肝再生增强因子对大鼠肝部分切除后肝再生的影响

目的观察原核表达的重组人肝再生增强因子（rhALR）对大鼠肝再生的影响。方法按Higgins方法进行大鼠34％肝切除。术后4-6h各实验组大鼠经腹腔注射rhALR剂量分别为50μg、100μg、200μg、400μg、800μg,对照组给生理盐水。术后30h杀鼠取肝,增殖核抗原（PCNA）免疫组化染色和HE染色,进行肝细胞核PCNA阳性细胞计数和有丝分裂计数。结果rhALR能促进肝部分切除后大鼠肝细胞的有丝分裂和细胞核PCNA的表达,并呈现一定的剂量依赖关系。结论rhALR能促进在鼠肝再生和肝细胞增殖。     

白藜芦醇促进肝部分切除术后肝再生的实验研究

目的研究白藜芦醇对小鼠70%肝切除后残余肝的再生是否有促进作用。方法实验动物为雄性C57BL/6小鼠。将100只小鼠随机分为实验组(白藜芦醇预处理组)和对照组(生理盐水预处理组)。采用肝大部分切除术建立肝再生模型,术前连续5 d分别给予小鼠腹腔内注射白藜芦醇12 g/kg(实验组)和生理盐水(对照组),第5天注射完白藜芦醇和生理盐水2 h后给两组小鼠分别进行70%的肝切除手术(pH)。用肝重/体重比,实时定量聚合酶链式反应及免疫组化等方法来评估白藜芦醇对小鼠肝再生的促进作用。结果 pH术后36 h、48 h实验组与对照组相比,肝重/体重比增高(4.56±0.07对3.93±0.07;5.36±0.07对4.6±0.09)。肝脏ki-67术后36 h表达最为活跃,48 h后下降,实验组与对照组相比ki-67表达明显增高。实验组中组织肝细胞生长因子(HGF)及肿瘤坏死因子(TNF-α)水平明显比对照组增强。结论白藜芦醇能明显促进小鼠部分肝切除后的肝再生。     

粒细胞集落刺激因子促进大鼠移植肝的再生作用

目的:探讨部分肝移植后,粒细胞集落刺激因子(granulocyte colony-stimulating factor, G-CSF)对部分肝移植物再生的促进作用．方法:采用改良“二袖套法”建立SD大鼠 50％部分肝移植模型,受体鼠随机分为实验组和对照组,术后分别注射(sc)G-CSF和相同体积的生理盐水5 d．观察大鼠移植肝存活时间．术后1,3,5,7和14 d取血清和移植肝脏,测移植物与受体质量比(graft-recipient weight ratio,GRWR),检测血清生化指标,并采用免疫组织化学方法观察肝内的增殖细胞核抗原 (proliferating cell nuclear antigen,PCNA)的表达．结果:实验组移植肝存活率较对照组高(90％ vs 60％,X2=5．03,P<0．05),术后第3-5天,实验组GRWR较对照组明显增加(P<0．05)．两组肝再生均于移植后3 d达高峰．与对照组相比, 实验组肝坏死灶少,AST,ALT水平低(3 d:t= 17．61,P<0．05;t=20．16,P<0．05;5 d:t=15．64, P<0．05;t=23．08,P<0．05),白蛋白水平高(3 d: 36．2±4．7 vs 29．5±3．4,P<0．05;5 d:43．2± 4．1 vs 33．8±3．9,P<0．05),PCNA表达升高(t= 23．08,P<0．05)．结论:大鼠部分肝移植后G-CSF可以促进肝细胞再生,减轻肝损伤．     

生长激素对鼠部分肝切除术后肝再生影响

目的　探讨生长激素对 70 %肝切除后肝再生的影响。方法　 60只SD大鼠随机分为对照组及生长激素组 ,按Higgins方法行 70 %肝切除术 ,术后给药并分批于术后 6、2 4、48、72、96h处死 ,作如下比较 :①残肝肝重 ;②增殖细胞核抗原 (PCNA)标记指数 ;③图像定量分析法测量PCNA阳性产物面积及灰度值。结果　与对照组比较 ,生长激素组残肝肝重、PCNA标记指数、PCNA阳性产物面积在术后均显著增高 (P <0 .0 5 ) ,而灰度值则显著降低 (P <0 .0 5 )。结论　生长激素具有强烈促进肝细胞增殖和刺激肝再生的作用     

粒细胞集落刺激因子促进小鼠肝再生的作用

目的:探讨粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)促进肝大部切除小鼠肝再生的作用.方法:建立小鼠肝大部分切除(约70%)模型,将肝切除小鼠随机分为3组.单纯肝切除组:肝切除后24 h,腹腔注射生理盐水5 d:G-CSF+肝切除组:rhG-CSF 150 μg/(kg·d)腹腔注射5 d,24 h后进行肝切除:肝切除+G-CSF组:肝切除术后24 h,rhG-CSF 150 μg/(kg·d)腹腔注射5 d.于术后7 d取血清和肝组织,用全自动生化分析仪检测血清肝功能指标,并采用免疫组织化学方法观察肝内的增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)的表达及BrdU阳性细胞.结果:肝切除+G-CSF组和G-CSF+肝切除组Brdu及PCNA表达及BrdU阳性细胞明显升高,与对照组比较差异均具有统计学意义(PCNA:74.08%±8.86%,68.91%±9.64% vs 57.36%±13.37%,均P<0.05);肝组织病理检查发现G-CSF+肝切除组27%(3/11)小鼠肝组织出现了不同程度的炎症反应,肝功生化学也显示血清ALT及AST升高,且其差异有统计学意义(均P<0.05).结论:G-CSF具有明显促进肝大部切除小鼠肝再生的作用,但也容易诱发肝脏炎症反应.     

过表达肝细胞核因子4α脐带间充质干细胞促进小鼠大部肝切后肝再生

目的：探讨人源性脐带间充质干细胞（UC-MSC）以及过表达肝细胞核因子4α（HNF4α）的UC-MSC能否促进小鼠大部肝切后肝再生。方法体外分离、培养、鉴定人UC-MSC,慢病毒转染过表达HNF4α。体外收集细胞培养上清液,将L02与上清液共培养,通过细胞增殖实验试剂盒（CCK8）检测细胞增殖活性。体内实验建立肝大部切除模型（约70%）,分别经尾静脉向肝切除小鼠移植0.9%生理盐水（NS）、MSC、MSC-HNF4α。48h后比较3组肝切后肝再生的变化,通过免疫组化来观察肝标本Ki67的表达。结果成功分离鉴定UC-MSC,并且成功建立稳定过表达HNF4α的MSC。体外实验MSC组和MSC-HNF4α组中L02的增殖能力都明显高于NS组（P＜0.01）,MSC组高于MSC-HNF4α组（P＜0.05）。同样体内实验相对于NS组,经MSC或MSC-HNF4α细胞处理的肝脏,其Ki67的表达明显高于NS组（ P＜0.01）,同样MSC组高于MSC-HNF4α组（P＜0.05）。结论 UC-MSC和过表达HNF4α的MSC都分泌一系列因子促进肝再生。     

Notch/Jagged信号在肝部分切除后肝再生中的表达

目的研究Notch/Jagged信号传导通路在大鼠肝部分切除术后肝再生中所起的作用。方法取雌性wister大鼠行肝部分切除术,术后0 min、5 min、15 min、30 min、1 h、3 h、6 h、12 h、1 d、2 d、4 d、7 d留取再生肝组织,观察大体组织变化,检测Notch-1和Jagged-1蛋白的表达,并通过RT-PCR检测Notch-1和Jagged-1的mRNA的表达。结果再生肝Notch-1蛋白第2 d在门脉周围细胞表达增强,第4 d在肝血窦内皮细胞表达增强,Jagged-1在正常肝脏标本中在胆管分布,在肝细胞也有少量表达。在再生的肝上,第2 d在门脉周围的肝细胞上较强地表达,第4 d在胆管内皮细胞表达增强。Notch-1的mRNA表达量在6 h-2 d下调,Jagged-1的mRNA表达量在3-6 h上调,12 h-2 d下调,4 d恢复。统计学处理采用方差分析方法、t检验及直线相关方法（P〈0.05）。结论Notch/Jagged信号通路的激活在肝再生中扮演着重要的角色。它可以促进胆管的形成和结构维持,有助于新生血管的形成及肝细胞的增殖。     

Effect of serotonin receptor 2 blockage on liver regeneration after partial hepatectomy in the rat liver.

The effect of serotonin receptor 2 blockade (5-HT(2)) on liver regeneration after 30-34% and 60-70% partial hepatectomy in the rat liver was investigated. Materials and methods: Male Wistar rats were subjected to 60-70% (group I) and 30-34% (group II) partial hepatectomy. Serotonin receptor 2 blockade was exerted by intraperitoneal administration of ketanserin at different doses and time points after partial hepatectomy. The rats of all groups were killed at different time points until 96 h after partial hepatectomy. The rate of liver regeneration was evaluated by the mitotic index in hematoxylin and eosin sections, the immunochemical detection of Ki67 and proliferating cell nuclear antigens, the rate of [(3)H]-thymidine incorporation into hepatic DNA and liver thymidine kinase enzymatic activity. Results: Liver regeneration peaked at 24 and 32 h after partial hepatectomy in 60-70% hepatectomized rats. In 30-34% hepatectomized rats liver regeneration peaked at 60 h, whereas low rates of regenerative activity were observed between 24 and 72 h after partial hepatectomy. Ketanserin administration arrested liver regeneration only when administered at 16 h after 60-70% partial hepatectomy. Ketanserin also abrogated the observed peak of regenerative activity at 60 h in 30-34% hepatectomized rats when administered at 52 h after partial hepatectomy. All indices of liver regeneration were affected by ketanserin administration. Conclusions: Serotonin receptor 2 blockade can arrest liver regeneration only when administered close to G1/S transition point, and that while serotonin may be a cofactor for DNA synthesis, it does not play a role in initiation of liver regeneration.     

Liver regeneration is promoted by increasing serotonin content in rat liver with secondary biliary cirrhosis

Aim: Liver cirrhosis clinically shows thrombocytopenia and hypersplenism. Although splenectomy is performed to achieve higher platelet count and better hemostasis, the effect of splenectomy for liver cirrhosis remains unclear. The aim of the present study that was focused on serotonin was to investigate the relationship between splenectomy and liver regeneration in rats with secondary biliary cirrhosis. Methods: Liver cirrhosis was induced in Sprague–Dawley rats by bile duct ligation (BDL). In addition, splenectomy and administration of ketanserin, which selectively antagonizes 5‐HT2A and 2B serotonin receptors, were performed. Three weeks after the interventions, whole blood, plasma, serum, and liver specimens were obtained for the following studies: peripheral platelet counts, hemodynamics of serotonin, histopathological examination, immunostaining, and quantification of mRNA expression. Results: Splenectomy induced thrombocytosis, and increased serotonin content in cirrhotic liver. Stimulation of liver regeneration was indicated by the following parameters: hepatocyte ratio to the entire liver area, Ki67‐positive hepatocyte count, and expression of phosphorylated extracellular signal‐regulated kinases. This enhancement of liver regeneration was negated by ketanserin. Conclusion: Our results showed that splenectomy promoted liver regeneration by increasing serotonin content in liver even under cirrhotic conditions.     

Effect of vascular endothelial growth factor on hepatic regenerative activity following partial hepatectomy in rats.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is an angiogenic factor with a growth-promoting effect that is thought to be restricted to vascular endothelial cells. Its essential role during liver regeneration has yet to be determined. The aim of this study was to document the effect of exogenous VEGF administration on liver regeneration in rats undergoing submaximal hepatic resections. METHODS: Adult male Sprague-Dawley rats (n = 4/group) undergoing 30% partial hepatectomy were administered 200 ng VEGF165 intravenously and were sacrificed at 24, 36, and 48 h postoperatively. Liver regeneration was monitored by measuring the restituted liver mass, proliferating cell nuclear antigen (PCNA) immunostaining, and hepatic PCNA protein by Western blot. RESULTS: Changes in restituted liver mass 48 h postsurgery were more prominent, but did not differ statistically between VEGF-treated and control rats (47% vs. 29%; p<0.06). Nevertheless, PCNA immunostaining showed increased labeling index of hepatocytes, apparent at 36 and 48 h after partial hepatectomy (38% vs. 18% [p<0.041 and 42% vs. 11% [p<0.021], respectively). Hepatic PCNA proteins measured by Western blot showed a 3-fold increase in VEGF-treated rats 48 h postsurgery compared with controls (p<0.01). CONCLUSION: Exogenous VEGF administration early after partial hepatectomy stimulates liver regeneration in rats. Whether or not VEGF165 is a direct mitogen for hepatocytes remains to be determined.     

Regeneration of liver with marked fatty change following partial hepatectomy in rats

Resection of liver for primary and metastatic tumors and living donor liver transplantation has become a common clinical practice. The success of recovery depends on the regeneration and functions of the remnant liver. However, information on the regenerative potential of liver with steatosis and steatohepatitis, a common clinical problem in this country, is incomplete. Therefore, we evaluated regeneration after two-thirds partial hepatectomy (PH) in male F-344 rats with marked steatosis and mild steatohepatitis induced by feeding choline-deficient diet. Choline-deficient rats and control rats were killed at 24, 36, 48, 72, and 96 h after PH. Liver regeneration was determined by measuring mitotic activity and bromodeoxyuridine (BrdU) labeling in hepatocytes. Livers of rats maintained on the choline-deficient diet showed marked steatosis and mild steatohepatitis. In these animals the levels of serum and liver triacylglycerols (TG) were low and very high, respectively, when compared to controls. In control rats mitotic and BrdU labeling indices were maximal at 24 h followed by a rapid decline, whereas in choline-deficient rats both these indices increased significantly at 36 h and decreased gradually over the next 60 h. By 96 h the size of livers in both groups was comparable. The results of this study indicate that regeneration in the liver of rats with marked steatosis is not impaired.     

Ischemic preconditioning improves liver regeneration by sustaining energy metabolism after partial hepatectomy under ischemia in rats.

BACKGROUND: The protective effect of ischemic preconditioning (IPC) has been reported on improvement of survival, reduction of liver necrosis and enhancement of the regenerative capacity of hepatocytes after partial hepatectomy. This study was undertaken to confirm that IPC has a significant impact on regeneration of hepatocytes after partial hepatectomy in ischemically damaged liver. In addition, we sought to examine the role of adenine nucleotides in this process. METHODS: Wistar rats were subjected to 60 min of total hepatic ischemia, followed by 70% hepatectomy. The animals were subdivided into an IPC (10/15 min) group and a non-IPC (control) group. Liver function tests and arginase activity were analyzed. Hepatic adenosine triphosphate (ATP), adenosine diphosphate and adenosine monophosphate were measured using gradient high-performance liquid chromatography. The liver regeneration was identified using relative liver weight and proliferating cell nuclear antigen (PCNA) labeling index. RESULTS: IPC treatment improved serum liver enzymes and tissue arginase activity (P<0.05) when compared with the control group. The preconditioned livers were associated with upregulation of ATP expression and also increased tissue energy charge. Regenerated liver weight in the IPC group was significantly higher than in the control group (P<0.05). The PCNA labeling index in the remnant livers in the IPC group was also significantly increased at 24 and 48 h after partial hepatectomy (P<0.05). CONCLUSION: These results suggest that IPC-augmented liver regeneration after hepatectomy, probably due to the stabilization of energy metabolism in rats.