5-羟色胺2A受体阻断剂对大鼠肝脏再生的影响

目的观察5-羟色胺2A受体阻断剂酮色林对大鼠肝部分切除后肝再生的影响,了解5-羟色胺及其受体在肝脏再生中的作用。方法 80只雄性Wistar大鼠随机分为实验组和对照组。采用肝大部分切除术建立肝再生模型,术后16 h分别给予腹腔内注射酮色林（实验组）和生理盐水（对照组）,采用免疫组化及流式细胞技术动态观察并比较两组大鼠术后24、36、48、72 h肝脏Ki67、增殖细胞核抗原的表达情况。结果大鼠肝大部切除术后24、36 h肝脏表达Ki67、增殖细胞核抗原最为活跃,而后表达逐渐下降。实验组大鼠肝脏表达Ki67、增殖细胞核抗原较对照组显著下降（P〈0.05）。结论 5-羟色胺2A受体阻断剂酮色林显著抑制大鼠肝大部切除术后的肝脏再生,说明5-羟色胺具有一定的促进肝再生的作用,2A受体是其重要的信号传导受体之一。     

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.     

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。     

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。     

骨髓间充质干细胞对大鼠减体积肝移植术后肝细胞再生与修复的作用

目的探讨骨髓间充质干细胞（BMSCs）对大鼠减体积肝移植术后肝脏再生与修复的影响。方法采用生化、免疫组化等方法检测术后不同时间的血清和肝脏标本。（1）取同周龄Wistar大鼠骨髓体外培养BMSCs,标记CSFE荧光后制备悬液;（2）建立大鼠50%减体积肝移植模型（供体鼠为SD大鼠,受体鼠为Wistar大鼠）,分为实验组（取BMSCs悬液经门静脉植入）和对照组（取等量生理盐水经门静脉注射）,观察大鼠生存状态,测量肝再生的相关指标。结果 （1）成功分离培养BMSCs;（2）术后2h,大鼠均自由活动饮水;（3）实验组肝组织中存在大量BMSCs,术后第2、3 d实验组肝体比显著高于对照组。病理显示：实验组肝细胞损伤较对照组轻微。结论植入BMSCs后,可以促进大鼠减体积肝移植术后肝细胞增殖和修复。     

氢质子波谱成像对热缺血再灌注损伤大鼠移植肝细胞再生能力的评价作用

目的 探讨磁共振氢质子波谱(1H-MRS)评价大鼠原位肝移植(OLT)热缺血模型肝细胞再生能力的意义,以及热缺血对于移植肝再生能力的影响. 方法 以实验组热缺血10min的大鼠肝移植模型和对照组无热缺血大鼠肝移植模型各30只为研究对象,移植术后分6个时间点(6 h、1 d、3 d、7 d、14 d、30 d)对每只大鼠行肝脏常规T1WI、T2WI成像及氢质子波谱扫描.扫描后取肝脏,测定肝细胞的细胞增殖核抗原(PCNA)表达,同时检测肝酶代谢水平. 结果 术后5个时间点实验组的肝细胞PCNA阳性率、胆碱峰与水峰的峰高比值均高于对照组,实验组和对照组胆碱峰/水峰峰高比值均与相应PCNA的阳性率呈显著正相关(r实验=0.819,P＜0.01;r对照=0.543,P＜0.01).实验组和对照组的血清ALT、AST术后明显升高,尤以术后6 h～3 d最为显著,实验组的ALT、AST明显高于对照组. 结论 热缺血再灌注损伤对于移植后肝细胞的再生能力有明显的影响,1H-MRS的胆碱峰可以无创伤地评价移植肝的肝细胞再生能力.     

术前不同引流方式减黄对梗阻性黄疸大鼠部分肝切除术后肝功能和肝再生的影响

目的 了解术前不同引流方式减黄对梗阻性黄疸(OJ) SD大鼠部分肝切除术(PH)术后肝功能和肝再生的影响.方法 建立OJ不同引流方式减黄70％部分肝切除SD大鼠动物模型.并在术后0、1、2、4、12、24、48和72h收集大鼠血液及肝脏组织标本,测定血清TBIL、ALB、ALT、AST水平,计算残肝重量、肝再生率,免疫组化法观察肝脏组织PCNA表达,ELISA法检测血清TNF-α水平.结果 PH术后各时段内引流(ID)组和外引流(ED)组TBIL、ALT、AST水平较OJ组均偏低.各时段ID组ALB水平较OJ组、ED组偏高.72 h肝再生率ID组高于ED组,ED组高于OJ组.3组PCNA水平均于12 h明显升高,ID组于24 h达高峰,OJ组、ED组高峰延迟至48 h且峰值偏低.PH术后各组血清TNF-α水平均呈上升趋势,ID组于12 h达高峰,OJ组、ED组均于24 h达高峰,各时段OJ组、ED组血清TNF-α水平较ID组均偏高.结论 内外引流术均可改善OJ所致的高胆红素血症和肝功能,并改善OJ大鼠残肝再生能力,但内引流效果更明显,且内引流术可以有效降低血清TNF-α水平.     

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．     

BMP2 mRNA在大鼠再生肝组织的表达与意义

目的:在基因水平上观察BMP2在肝组织的表达和肝再生过程中的动态变化特征,并结合NF-κB在肝再生过程中的表达变化特征,探讨BMP2对肝再生的影响.方法:健康成年♂Wistar大鼠54只,随机分为3组:正常对照(NC)组,即0 h组(n=6),假手术(SO)组(n=24),肝部分切除术(PH)组(n=24).采用原位杂交和免疫组化的方法分别测定大鼠再生肝脏组织中BMP2 mRNA和NF-κB的表达.结果:BMP2 mRNA表达在SO组与PH组6、12、24 h时间点组间存在统计学差异(灰度值:99.74 6.85 vs 114.41±5.12,130.59±6.74,113.74±7.32,均P<0.05).SO组与NC组无统计学意义.NC组肝组织内NF-κB阴性,SO组术后各时相点未见特异性染色.PH组NF-κB最初少量弱阳性表达,术后6、12、24 h,NF-κB表达逐步增强,灰度值降低(96.22±3.12,89.59±3.24,83.72±4.32),有统计学差异(P<0.05).结论:BMP2在正常大鼠肝组织中有表达且在肝再生过程中BMP2表达呈现先减弱后增强的趋势.NF-κB在正常大鼠肝脏组织中无表达,在肝再生过程中NFκB表达逐渐增强.BMP2可能抑制肝再生.     

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。     

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.     

Decreased expression of peroxisome proliferator-activated receptor alpha and liver fatty acid binding protein after partial hepatectomy of rats and mice.

BACKGROUND AIMS: Marked changes in metabolism, including liver steatosis and hypoglycemia, occur after partial hepatectomy. Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a nuclear hormone receptor that is activated by fatty acids and involved in hepatic fatty acid metabolism and regeneration. Liver fatty acid binding protein (LFABP) is an abundant protein in liver cytosol whose expression is regulated by PPAR alpha. It is involved in fatty acid uptake and diffusion and in PPAR alpha signaling. The aim of this study was to investigate the expression of PPAR alpha and LFABP during liver regeneration. METHODS: Male Sprague-Dawley rats and male C57 Bl/6 mice were subjected to 2/3 hepatectomy and LFABP and PPAR alpha mRNA and protein levels were measured at different time points after surgery. The effect of partial hepatectomy was followed during 48 h in rats and 72 h in mice. RESULTS: PPAR alpha mRNA and protein levels were decreased 26 h after hepatectomy of rats. The LFABP mRNA and protein levels paralleled those of PPAR alpha and were also decreased 26 h after hepatectomy. In mice, the mRNA level was decreased after 36 and 72 h after hepatectomy. In this case, LFABP mRNA levels decreased more slowly after partial hepatectomy than in rats. CONCLUSIONS: A marked decrease in PPAR alpha expression may be important for changed gene expression, e.g. LFABP, and metabolic changes, such as hypoglycemia, during liver regeneration.     

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.     

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.     

Role of angiotensin‐1 receptor blockade in cirrhotic liver resection

Background: The regeneration capacity of cirrhotic livers might be affected by angiotensin‐1 (AT1) receptors located on hepatic stellate cells (HSC). The effect of AT1 receptor blockade on microcirculation, fibrosis and liver regeneration was investigated. Materials and methods: In 112 Lewis rats, cirrhosis was induced by repetitive intraperitoneal injections of CCl₄. Six hours, 3, 7 and 14 days after partial hepatectomy or sham operation, rats were sacrificed for analysis. Animals were treated with either vehicle or 5 mg/kg body weight losartan pre‐operatively and once daily after surgery by gavage. Microcirculation and portal vein flow were investigated at 6 h. The degree of cirrhosis was assessed by Azan Heidenhein staining, activation of HSC by desmin staining, apoptosis by ssDNA detection and liver regeneration by Ki‐67 staining. Changes in expression of various genes important for liver regeneration and fibrosis were analysed at 6 h and 3 days. Haemodynamic parameters and liver enzymes were monitored. Results: Losartan treatment increased sinusoidal diameter, sinusoidal blood flow and portal vein flow after partial hepatectomy (P<0.05), but not after sham operation. AT1 receptor blockade resulted in increased apoptosis early after resection. HSC activation was reduced and after 7 days, a significantly lower degree of cirrhosis in resected animals was observed. Losartan increased the proliferation of hepatocytes at late time‐points and of non‐parenchymal cells early after partial hepatectomy (P<0.05). Tumour necrosis factor (TNF)‐α was significantly upregulated at 6 h and stem cell growth factor (SCF) was downregulated at 3 days (P<0.05). Conclusion: Losartan increased hepatic blood flow, reduced HSC activation and liver fibrosis, but interfered with hepatocyte proliferation after partial hepatectomy in cirrhotic livers.     

Shedding of TNFR1 in regenerative liver can be induced with TNFα and PMA

AIM: Liver regeneration is associated with apoptosis of hepatocytes, which is mediated via tumor necrosis factor receptor 1(TNFR1). The shedding of TNFR1 in liver regeneration and its mechanism to regulate this shedding were investigated.METHODS: The shedding of TNFR1 in liver regeneration and changes of TNF-α, PMA and plasma membrane purified from hepatocytes on this shedding process were measured with Western blot. Then, the relationship between TNFR1 shedding and apoptosis of hepatocytes induced by TNFα was studied by detecting apoptotic index.RESULTS: The shedding of TNFR1 began at 4 hours and terminated before 2 months after partial hepatectomy. In culture system, serum from rats at 36 h after partial hepatectomy could also promote this shedding process. With the stimulation of TNF α, PMA or purified plasma membrane from hepatocytes at 36 h after partial hepatectomy or from hepatocytes treated with TNF α for 2 h, membranous TNFR1 was also shed. With the stimulation of both TNF α and plasma membrane from hepatocytes affected with TNF α for 2 hor from hepatocytes at 36 h after partial hepatectomy, apoptotic index of hepatocytes decreased from 21% to 7.52 % and 8.45 %, respectively. PMA could also reduce apoptotic index to 13.67 %. This descent occurred in hepatocytes cultured in serum from rats at 36 h after partial hepatectomy too,but not in serum from rats at 2 months after partial hepatectomy and sham-operated rats.CONCLUSION: Shedding of TNFR1 may help reduce apoptosis of hepatocytes induced by TNFα. Membraneanchored metalloprotases could play a role in shedding membranous TNFR1. At the same time, PKC may take part in regulation of this shedding process.     

The effect of ursodeoxycholic acid on liver regeneration after partial hepatectomy in rats with non-alcoholic fatty liver disease

Aim:  To investigate the effect of ursodeoxycholic acid (UDCA) on liver regeneration following partial hepatectomy in rats with non-alcoholic fatty liver disease (NAFLD).
Methods:  UDCA was administered to seven rats (group 1) and physiological saline was administered both to seven rats (group 2) with NAFLD and to seven rats with normal livers (group 3). All rats underwent two-thirds hepatectomy and the remnant liver tissues were removed 48 h later. Mitotic index (MI) and levels of proliferating cell nuclear antigen (PCNA), glutathione (GSH) and malondialdehyde (MDA) were assayed.
Results:  MI and PCNA levels in group 2 were significantly lower than in groups 1 and 3, but the values in groups 1 and 3 were similar. The GSH levels of group 2 were significantly lower than those of group 3 in the hepatectomy tissues, and lower than those of groups 1 and 3 in the remnant tissues. The differences between GSH levels in groups 1 and 3 were not significant. MDA levels in hepatectomy and remnant tissues were significantly higher in group 2 compared to groups 1 and 3; values in groups 1 and 3 were similar.
Conclusion:  UDCA increases regeneration after partial hepatectomy in rats with NAFLD, possibly due to an attenuating effect on oxidative stress.