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。     

丹参对暴发性肝衰竭大鼠肝损伤及肝再生的影响

目的：探讨丹参对暴发性肝衰竭（FHF）大鼠肝损伤及肝再生的影响。方法：Wistar大鼠随机分为4组：正常对照组、FHF组、丹参组、促肝细胞生长素（PHGF）组。FHF、丹参组和PHGF组动物模型采用TAA皮下注射,剂量按每kg体重600mg,2次,每次间隔24h,复制FHF动物模型。丹参组和PHGF组动物除皮下注射TAA外,于实验前3天至实验结束分别于皮下注射丹参注射液1ml/100g和PHGF1ml/100g,正常对照组和FHF组大鼠同时皮下注射生理盐水1ml/100g。FHF组、丹参组和PHGF组,于第2次注射TAA后24h,随机取大鼠各8只,腹主动脉取血测肝功能。迅速取肝组织,用10%甲醛液固定,制成石蜡切片,检测肝细胞有丝分裂指数（MI）和增殖细胞核抗原（PCNA）。结果：丹参具有降低FHF大鼠ALT、AST及TBil,显著提高肝细胞MI和PCNA的作用（P〈0.05,P〈0.01）。结论：丹参具有改善FHF大鼠肝功能和促进肝细胞增殖及再生的作用。     

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。     

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     

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。     

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.     

肝细胞生成素及肝部分切除诱导肝再生基因PC3的表达

目的 观察重组人肝细胞生成素（rhHPO)和肝部分切除迅速诱导瞬时早期反应基因表达情况。方法 利用表达性差异显示分析技术和序列分析研究2／3肝部分切除后1h及原代培养大鼠肝细胞体系的选择性基因表达。结果 揭示EST集中的大部分为瞬时早期反应基因，发现一种可能与肝再生调控相关的新基因PC3，Northern杂交证实2／3肝部分切除可迅速诱导PC3基因的表达，其表达高峰为术后1－2h。HPO在原代培养大鼠肝细胞体系中可迅速诱导瞬时早期反应基因（c-fos,LRF-1和PC3等）表达。结论 HPO和肝部分切除可迅速诱导瞬时早期反应基因表达，PC3基因是一种与肝再生调控密切相关的早期反应基因。     

肝细胞生长因子与肝再生增强因子重组表达质粒对大鼠肝纤维化的影响

目的 研究肝细胞生长因子(HGF)与肝再生增强因子(ALR)重组表达质粒对大鼠实验性肝纤维化的治疗作用.方法 建立二甲基亚硝胺肝纤维化模型后的90只S-D大鼠分为空白组、pcDNA3.1治疗组、pcDNA3.1-HGF治疗组、pcDNA3.1-ALR治疗组、pcDNA3.1-HGF与pcDNA3.1-ALR共治疗组、pcDNA3.1-HGF-ALR治疗组,每组15只.各治疗组大鼠每24小时经尾静脉注射1次相应质粒0.1 μmol,连续3次,空白组不接受任何治疗,另取同批次S-D大鼠10只作为对照组.治疗结束后4 d处死大鼠,留取肝组织采用HE染色观察肝脏的病理形态,采用免疫组织化学染色检测增殖细胞核抗原(PCNA)和c-jun的表达.计量资料采用单因素方差分析,两两比较采用SNK检验,计数资料采用Fisher确切概率法分析.结果 空白组和pcDNA3.1治疗组大鼠肝组织有明显的条索状纤维间隔形成,可见假小叶,两组肝纤维化程度差异无统计学意义(x2=0.317,P=1. 000);其他治疗组肝纤维化均有不同程度改善,以pcDNA3.1-HGF-ALR治疗组改善最明显.对照组肝组织PCNA和c-jun表达均较低,其吸光度值分别为8.6±1.9和3.2±1.2;空白组与pcDNA3.1治疗组肝组织PCNA和c-jun表达均增加,其吸光度值分别为24.1±3.0.24.5±4.3与23.8±3.1、24.9±4.2,与对照组比较,差异有统计学意义(均P＜0.01);其他治疗组的PCNA表达显著增加,c-jun表达显著降低,均以pcDNA3.1-HGF-ALR治疗组的变化最明显.结论 重组表达质粒pcDNA3.1-HGF-ALR能较好地改善大鼠实验性肝纤维化,并可能通过促进肝细胞增殖和抑制原癌基因c-jun的表达来发挥其抗肝纤维化作用.     

白藜芦醇对部分肝切除术大鼠肝再生和肝组织NK细胞活性的影响*

目的 探讨白藜芦醇(RES)对肝部分切除大鼠肝组织自然杀伤(NK)细胞活性和肝再生的影响。方法 随机将90只SD大鼠分为对照组、小剂量白藜芦醇和大剂量白藜芦醇处理组,各组30只,分别经尾静脉注射生理盐水、白藜芦醇10 g.kg^-1·d^-1和20 g.kg^-1·d^-1,连续5 d,然后行70%肝脏切除术。检测肝脏再生指数,使用流式细胞术测定肝组织NK细胞百分率,采用⁵¹Cr释放法测定NK细胞杀伤活性,采用蛋白印迹法检测肝组织增殖细胞核抗原(PCNA)和CyclinD1蛋白表达。结果 在24 h和48 h,大剂量白藜芦醇处理组大鼠肝脏再生指数分别为(1.89±0.04)和(2.45±0.07),显著大于小剂量白藜芦醇处理组【(1.59±0.06)和(2.12±0.05),P<0.05】或对照组【(1.43±0.03)和(1.92±0.05),P<0.05】;大剂量白藜芦醇处理组大鼠NK细胞百分率分别为(24.62±1.36)%和(25.47±1.19)%,显著高于小剂量白藜芦醇处理组【(22.21±1.98)%和(22.36±1.78)%,P<0.05】或对照组【(17.36±1.78)%和(18.65±1.69)%,P<0.05】;大剂量白藜芦醇处理组大鼠NK细胞杀伤活力分别为(48.48±2.69)%和(49.01±2.78)%,显著高于小剂量白藜芦醇处理组【(41.88±2.65)%和(42.32±2.58)%,P<0.05】或对照组【(28.32±2.36)%和(30.12±2.36)%,P<0.05】;大剂量白藜芦醇处理组大鼠肝组织PCNA和CyclinD1蛋白相对表达量显著强于小剂量白藜芦醇处理组和对照组,差异有统计学意义(P<0.05)。结论 RES可以促进肝部分切除术大鼠肝再生,可能与增强了NK细胞活性和相关蛋白表达有关。     

肝再生大鼠血清诱导骨髓干细胞向肝细胞分化的实验研究

目的观察肝大部切除大鼠再生血清和肝细胞生长因子(HGF)诱导骨髓干细胞向肝实质细胞分化的作用；探讨骨髓干细胞向肝细胞分化和增殖的体外培养条件和方法，为患者应用自身骨髓细胞修复损伤肝脏提供实验依据。方法制作肝大部切除大鼠肝再生动物模型，收集术后24h血清；分别应用鼠肝再生血清和HGF对大鼠骨髓细胞进行定向诱导分化培养；以免疫组化、RTPCR和western blot等方法对分化不同阶段细胞进行检测。将分化细胞经尾静脉输入同系大鼠，观察输注细胞在肝、脾内的生长情况。结果活体移植分化细胞能定向迁移至肝和脾；体外及体内分化细胞在mRNA水平和蛋白质水平均表达白蛋白，并在一定时期内表达甲胎蛋白。结论大鼠骨髓干细胞在肝大部切除再生血清或HGF的诱导下能横向分化为肝实质样细胞。     

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.     

Appearance of an inhibitory cell nuclear antigen in rat and human serum during variable degrees of hepatic regenerative activity

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丹黄方对部分肝叶切除大鼠肝组织中肝细胞生长因子的影响

[目的]探讨丹黄方对部分肝叶切除大鼠肝组织中肝细胞生长因子（hepatocyte growth factor，HGF）mRNA表达的影响。[方法]采用部分肝叶切除肝再生模型。24只大鼠随机分为假手术组、手术组、丹黄方组和促肝细胞生长素（pHGF）组。除假手术组外，其余3组接受部分肝叶切除手术。从手术前3d至手术后48h，丹黄方组给予丹黄方（10g／kg）灌胃及0．85％氯化钠（4．0ml／kg）腹腔注射，每天1次；pHGF组给予0．85％氯化钠（4．0ml／kg）灌胃和pHGF（1ml／100g）腹腔注射，每天1次；手术组及假手术组仅给予0．85％氯化钠（4．0ml／kg）灌胃和腹腔注射。采用RT-PCR方法检测HGFmRNA的表达。[结果]丹黄方组肝组织中HGFmRNA的表达显著高于假手术组和手术组（P〈0．01），与pHGF组比较差异无统计学意义（P〉0．05）。[结论]丹黄方具有促进部分肝叶切除大鼠肝组织中HGFmRNA的表达作用。     

Effects of emodin and double blood supplies on liver regeneration of reduced size graft liver in rat model

AIM: To study the influences of emodin and reconstruction of double blood supplies on liver regeneration of reduced size graft liver in rat model. METHODS: A total of 45 SD-SD rat reduced size liver transplantation models were randomly divided into three groups (A-C). The conventional reduced size liver transplantation was performed on rats in group A, while the hepatic artery blood supply was restored in groups B and C. The emodin (1.5 mg/kg/d) was given by intraperitoneal route in group C only. The recipients were killed on the seventh day after the operation. The proliferative cell nuclear antigen (PCNA), TBil and ALT of serum were detected, and the pathological changes of liver cell were observed. RESULTS: The numbers of the rats that survived in A, B, and C group on the seventh day after operation were 14, 13, 13, respectively. The levels of TBil (31.5±5.2 μmol/L, 23.2±3.1 μmol/L vs 38.6±6.8 μmol/L), and ALT (5 351±1 050 nKat, 1300±900 nKat vs 5779±1202 nKat) in serum in groups B and C were lower than those in group A (P<0.05), while the expression of PCNA in groups B or C was higher than that in group A (22.0±3.5%, 28.2±4.2% vs 18.6±3.2%, P<0.05). The deeper staining nuclei, double nuclei, multi-nuclei and much glycogen were observed in liver cells of groups B and C, especially in group C, while fewer were found in liver cells of group A. CONCLUSION: The reconstruction of arterial blood supply is very important for rat liver regeneration after reduced size liver transplantation. Emodin has the effect of promoting liver regeneration and improving liver function in rats after reduced size transplantation. The possible mechanism is improving proliferation of liver cell and protecting liver cells from injury.     

Regenerative activity and liver function following partial hepatectomy in the rat using (31)P-MR spectroscopy

The aim of the present study was to determine whether alterations in hepatic energy expenditure following partial hepatectomy (PHx), as documented by in vivo hepatic (31)P-MRS, correlate with standard parameters of hepatic regeneration and/or liver function. In addition, we sought to determine whether changes in hepatic energy levels are proportional to the extent of hepatic resection. Adult male Sprague-Dawley rats (4-7 per group) underwent a 40%, 70%, or 90% PHx or sham surgeries. Magnetic resonance spectroscopy (MRS) examinations were performed on each animal 24 or 48 hours thereafter. After MRS examinations, [(3)H]thymidine incorporation into hepatic DNA, proliferating cell nuclear antigen (PCNA) protein expression, and serum bilirubin determinations were performed on each rat. Twenty-four hours following surgery, rats that had undergone 70% PHx had unchanged adenosine triphosphate (ATP) levels but significantly lower ATP/inorganic phosphate (Pi) ratios (P <.05), whereas, at 48 hours post-PHx, both ATP and ATP/Pi levels were lower than in sham- and nonoperated controls (P <.05). Hepatic regeneration and liver dysfunction mirrored these changes; correlations existed between ATP/Pi ratios and [(3)H]thymidine incorporation (r = -0.61, P <.005), PCNA protein expression (r = -0.62, P <.005), and serum bilirubin (r = -0.49, P <.05). For rats that had undergone graded resections, depleted energy levels 48 hours post-PHx were proportional to the extent of resection, degree of enhanced regenerative activity, and liver dysfunction. In conclusion, (31)P-MRS-generated ATP/Pi index is a noninvasive, robust determination that correlates with standard parameters of hepatic regeneration and function.     

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.     

Effect of antihypertensive agents on stellate cells during liver regeneration in rats

BACKGROUND: Although most studies have focused on the hepatocytes, all the hepatic cells participate in the regenerative process, among them the stellate cells. The stellate cells are mesenchymal cells involved in local neurotransmission and paracrine regulation of several liver functions. Acute hepatic tissue loss promotes the proliferation and activation of stellate cells from a quiescent state to myofibroblast-like cells. AIM: Investigate the effects of antihypertensive agents on the stellate cell population during the liver regenerative phenomenon in rats. METHODS: Adult male Wistar rats received lisinopril, losartan, bradykinin, or saline solution in a proportional volume, intraperitoneally, before and after 70% partial hepatectomy. Animals from the experimental and saline groups were sacrificed at 36 hours after partial hepatectomy. The alpha-smooth muscle actin labelled stellate cells population was counted in the periportal and pericentral zones of the liver specimen. RESULTS: The labelled stellate cells were more numerous in the control group both in the periportal and pericentral zones at 36 hours after partial hepatectomy than at the other times. The population of stellate cells was significantly lower in the losartan group and higher in the bradykinin and lisinopril groups than in the control group. CONCLUSIONS: These results suggest that losartan can inhibit and bradykinin and lisinopril can stimulate the stellate cell population during liver regeneration in rats. These cells synthesize several substances to stimulate liver regeneration.     

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.