L-谷氨酰胺与肝脏大部分切除后的再生

背景：L-谷氨酰胺作为DNA和谷胱甘肽等合成的氮前体,在肝组织再生,肝细胞增殖的过程中扮演着极其重要的角色。 目的：观察经饮食由来补充L-谷氨酰胺对大鼠肝脏大部切除后肝再生能力的影响。 方法：Wistar大鼠随机分组3组,L-谷氨酰胺组和L-丙氨酸组大鼠肝切除前分别灌服10% L-谷氨酰胺或10%L-丙氨酸,肝切除后继续加入饮用水中饮用,对照组肝切除前后均使用饮用水。 结果与结论：大鼠肝切除后72 h L-谷氨酰胺组肝再生率明显高于对照组及L-丙氨酸组(P < 0.05)。肝切除后24 h和72 h L-谷氨酰胺组肝细胞增殖均明显高于对照组和L-丙氨酸组(P < 0.01;P < 0.05)。肝切除后24 h和72 h总RNA水平在两种氨基酸与对照组之间差异无显著性意义。肝切除后72 h基因组DNA的含量L-谷氨酰胺组显著高于对照组和L-丙氨酸组 (P < 0.05)。提示肝损伤围手术期投用高浓度L-谷氨酰胺对大鼠肝再生有促进作用,而投用L-丙氨酸则没有此作用。     

Facilitation of liver regeneration after partial hepatectomy by ventromedial hypothalamic lesions in rats

Whether or not the hypothalamus is involved in initiating hepatic DNA synthesis after partial hepatectomy is unclear. To determine the role of the ventromedial hypothalamic nuclei in liver regeneration after partial hepatectomy, we studied hepatic DNA synthesis during liver regeneration in rats with bilateral lesions of these nuclei. Lesions of the ventromedial hypothalamus accelerated the increase in hepatic DNA synthesis and raised the peak level of thymidine incorporation after partial hepatectomy. These effects of hypothalamic lesions were completely inhibited by hepatic vagotomy. Thus, lesions of the ventromedial hypothalamus appear to promote hepatic regeneration by increasing vagal stimulation of the liver.     

A study of endotoxin-associated hepatotoxicity on proliferating hepatocytes.

It is thought that regeneration of the liver provides a state of preparedness for the Shwartzman reaction and contributes to the development of endotoxin-associated massive hepatic necrosis following partial hepatectomy. Therefore we examined endotoxin hepatotoxicity in rats with hepatic regeneration after 35% hepatectomy and in rats with liver cell proliferation induced by lead nitrate. Biochemical and histopathological studies showed no enhanced endotoxin hepatotoxicity in either partially hepatectomized rats or in rats with lead nitrate-induced liver cell proliferation. These results indicate that the development of endotoxin-associated hepatic damage after partial hepatectomy may not relate to regeneration and proliferation of the liver.     

CK19和TGF-α在大鼠肝纤维化部分肝切除后残肝中的表达

目的　检测肝纤维化大鼠部分肝切除后不同时间点CK19及TGF-α的表达,了解其胆管再生情况。 方法　雄性SD大鼠,对照组和实验组各35只。实验组腹腔注射CCl4制备肝纤维化模型,两组均进行肝部分切除术。进行免疫组织化学、HE染色,图像分析和数据统计。 结果　验组和对照组术后随时间延长CK19表达均呈增强趋势; 实验组术后各时间点CK19的表达均高于对照组同时间点。实验组和对照组术后随时间延长TGF-α表达均呈上升趋势,但实验组上升速度明显较对照组缓慢; 实验组术后　0d、1d、3d的TGF-α表达高于对照组。 结论 (1) 肝纤维化大鼠肝部分切除后CK19呈现高表达,提示肝纤维化部分切除可以促进肝卵圆细胞的增殖和分化。(2) TGF-α对肝纤维化大鼠肝部分切除后肝卵圆细胞增殖及胆管再生的促进作用不明显。     

大鼠肝再生过程中线粒体外周型苯二氮卓类受体的研究

目的：研究肝细胞线粒体通透性转换（PT）的主要调节蛋白外周型苯二氮卓类受体(PBR)在肝再生过程中的表达及其与专一性配体结合动力学变化，探讨与线粒体PT的关系。〖HTH〗方法：健康成年雄性SD大鼠，随机分为3组：肝部分切除（PH）组，切除肝左叶和中叶约全肝的70%；假处理组，同样麻醉和开腹，但不切肝；正常组。手术后3 h、6 h、12 h、 24 h、 48 h、 72 h、 120 h 和168 h 分别以半定量RT-PCR法检测PBR mRNA表达的动态变化。利用PBR专一的配体［3H］PK11195测定肝再生时线粒体膜上PBR的含量以及受体与配体亲和力的变化。〖HTH〗结果：在肝再生过程中PBR基因表达与假处理组无显著差异；［3H］PK11195与PBR最大结合量(Bmax)显著低于对照组（P <0.05），其中PH后3 h 和120 h非常显著（P<0.01），168 h接近正常水平； 平衡解离常数（Kd）在PH后72 h和168 h明显低于假处理照组（P<0.01）。假处理组之间Bmax和Kd无明显差异。〖HTH〗结论：肝再生过程中肝线粒体PBR mRNA水平无明显变化，而PBR与配体结合动力学明显改变提示PBR与线粒体PT变化有关。     

Effects of the liver homogenate obtained following partial hepatectomy on cirrhosis of the liver.

The authors studied the influence of liver homogenates as a whole and collected at various intervals after partial hepatectomy on cirrhosis of the liver. The lyophilized homogenates were administered over a period of 6 weeks to various groups of albino rats pretreated with CCl4 for a period of 6 months. The normal liver homogenate did not influence the histological and biochemical picture of the hepatocirrhosis. The material collected 48 hours after partial hepatectomy causes a moderate stimulation of the mechanisms of parenchymatous regeneration. 7 days after partial hepatectomy (in the postmitotic period) the hepatic regenerate shows a biological effect with lysis of collagen fibres and protection of parenchymatous cells.     

大鼠部分肝切除术后Hedgehog信号分子的表达

目的: 探讨部分肝切除术后Hedgehog(Hh)信号分子的表达及其意义。方法: 切除肝脏左叶和中叶以建立SD大鼠部分肝切除术模型。18只雄性SD大鼠随机均分为3组:假手术组(A)、部分肝切除术组1(B)、部分肝切除术组2(C)。A、B组于术后24 h、C组于术后48 h取肝组织,用免疫组化染色法和Western blotting检测肝组织Ki-67、Sonic Hedgehog(Shh)、Indian Hedgehog(Ihh)和Glioblastoma-2(Gli-2)的表达。结果: HE染色显示B、C组可见肝细胞水肿,点状坏死。C组可见处于不同分裂期的细胞。B、C组Ki-67、Shh、Ihh和Gli-2的表达明显高于A组(P<0.01),C组Ki-67、Shh、Ihh及Gli-2的表达高于B组(P<0.01)。结论: 部分肝切除术后Hh信号通路被激活,可能参与促进肝脏修复再生。     

肝再生对大鼠胎肝细胞脾内移植后增殖的影响

目的:研究肝再生状态对大鼠胎肝细胞脾内移植后增殖影响。方法:分离孕3周SD大鼠胚胎肝细胞,将其移植入70%肝切除肝再生模型大鼠脾内,分别于移植后7 d和30 d应用流式细胞仪检测肝切除大鼠残肝细胞的细胞周期,用图像分析法检测脾内移植胎肝细胞面积密度。结果:移植后7 d,肝切除鼠残肝细胞S和G₂/M期细胞比例都明显少于对照组(P＜0.05),而其脾内移植胎肝细胞面积密度则显著高于对照组(P＜0.05);30 d后,各组间残肝细胞再生状态与移植胎肝细胞的面积密度均无明显差异。 结论:肝再生状态有利于大鼠胎肝细胞脾内移植后的增殖。     

In-vitro immune response of splenic lymphocytes to portal serum agents from rats undergoing hepatic regeneration or hepatic carcinogenesis.

To clarify the physiologic response of splenic lymphocytes to liver damage and the role of this response in regeneration versus malignant transformation, we cultured rat spleen lymphocytes with portal sera from rats subjected either to partial (70%) hepatectomy or to long-term oral administration of the hepatic carcinogen 3'-methyl-4-dimethylaminoazobenzene. Sera taken within 24h after partial hepatectomy contained a previously described signal protein which serves as a marker of liver damage. The MW 5,000-10,000 serum fraction also contained a factor that promoted cell growth, DNA synthesis, glucose utilization, and the production of anti-sheep erythrocyte plaque-forming cells in cultures of rat splenic lymphocytes. In contrast, the sera of rats subjected to liver damage by the carcinogen had no more effect on the cultured lymphocytes than sera from sham-operated or untreated controls. The signal protein was present initially in portal sera from carcinogen-treated rats, but decreased as hepatitis gave way to cirrhosis. Subsequent malignant transformation was marked by the appearance of serum alpha-fetoprotein. Our results suggest that activation of splenic lymphocytes by serum factor(s) is involved in hepatic regeneration and that this process is deranged in carcinogenesis.     

The extracellular matrix in hepatic regeneration. Localization of collagen types I, III, IV, laminin, and fibronectin

After partial hepatectomy, the liver is capable of complete regeneration, restoring normal hepatic size, architecture, and function. To study the role of the extracellular matrix in regeneration, the temporal and spatial sequence of deposition of several of its components, including collagen types I, III, and IV, laminin, and fibronectin, in rat liver, after an 80% hepatectomy, was characterized by light microscopy immunohistochemistry. A minimum of five animals were studied for each date. In agreement with previous reports, subsequent to 80% hepatectomy, there was a brisk mitosis of hepatocytes. The mitotic activity was maximal at 48 hours, primarily in the periportal and centrilobular zones, and resulted in the formation of hepatocyte clusters and widening of the hepatic plates. Of the extracellular matrix components studied, laminin was the one demonstrating the most dramatic changes. By 24 hours, laminin appeared in the hepatic sinusoids reaching a maximum staining intensity at 48 hours. Intracellular laminin was prominent in numerous non-parenchymal cells, with many having the morphology, location, and desmin content characteristic of Ito cells. Laminin staining decreased in the sinusoids at 4 days; however, some intracellular staining of Ito cells was present even at 8 days after hepatectomy. At the completion of regeneration, there was no evidence of any substantial change in the ratio: extracellular matrix/cell mass. The results indicate that: (a) hepatocytes can divide without prior removal of the subsinusoidal extracellular matrix; (b) during regeneration, hepatocyte division precedes sinusoidal formation; (c) during hepatic regeneration, and in spite of the presence of laminin in Ito cells, no basement membranes are formed; (d) the prominent expression of laminin and its proposed functions in morphogenesis suggest a critical role for this matrix component in the formation and reorganization of the regenerating liver.     

异种肝前体细胞移植治疗急性肝损伤大鼠

背景：成体肝前体细胞可在受体肝脏内定植并分化为肝细胞。不过,异种肝前体细胞移植能否促进急性肝损伤的恢复,脾脏微环境能否促进移植物的存活和向肝细胞分化,尚没有研究。 目的：评价异种肝前体细胞移植治疗急性肝损伤的作用;监测移植肝前体细胞在大鼠脾脏实质内的定植及向肝细胞的分化。 方法：体外培养雄性小鼠来源的肝前体细胞系肝上皮样前体细胞。通过CCl4腹腔注射联合2/3肝切除构建急性肝损伤大鼠模型,进行肝上皮样前体细胞脾脏移植。在肝切除后1,5,14和21 d,苏木精-伊红染色观察肝脏病理改变,全自动生化分析仪监测血清转氨酶变化,PCR反应检测脾脏组织Y染色体特异性序列Sry,脾脏CK-19和Alb免疫组织化学追踪移植肝上皮样前体细胞的植入和肝细胞分化。 结果与结论：肝上皮样前体细胞可在体外长期培养,保持增殖能力和双向分化潜能。肝上皮样前体细胞脾脏移植后,肝损伤大鼠肝细胞肿胀明显减轻,丙氨酸转氨酶和天门冬氨酸转氨酶下降更明显。移植后1,5,14和21 d,脾脏DNA中均能检测到Sry序列。在整个实验期间CK-19阳性细胞在大鼠脾脏实质内始终存在。Alb阳性细胞在移植后5 d在脾脏实质中出现,随后阳性细胞数逐渐增多。实验表明,移植肝前体细胞能在大鼠脾脏实质中植入,并分化为肝细胞,能有效促进CCl4腹腔注射联合2/3肝切除诱导的大鼠急性肝损伤的修复过程。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

Lateral hypothalamic lesions facilitate hepatic regeneration after partial hepatectomy in rats

It has been reported that ventromedial hypothalamic lesions facilitate hepatic regeneration through the hepatic vagal nerve after partial hepatectomy. However, whether the lateral area of the hypothalamus is involved in liver regeneration after partial hepatectomy is unknown. To determine the role of the lateral hypothalamic area in this phenomenon, we studied hepatic DNA synthesis during liver regeneration after partial hepatectomy with bilateral lesions of the area. Lesioning of the lateral hypothalamus accelerated the increase in hepatic DNA synthesis and raised the peak level of [methyl-³H]thymidine incorporation after partial hepatectomy. These effects of hypothalamic lesioning were inhibited by combined hepatic vagotomy and sympathectomy. Our results demonstrate that lesioning of the lateral hypothalamus promotes hepatic regeneration through the autonomic nervous system after partial hepatectomy and suggest that the lateral hypothalamic area is involved in liver regeneration through neural mediation.     

Differentially expressed genes in a porcine adult hepatic stem-like cell line and their expression in developing and regenerating liver

To identify differentially expressed genes in adult hepatic stem cells, we performed suppression-subtractive hybridization (SSH) between adult porcine hepatic stem-like cells (HSLCs) and hepatocytes, and the expression of selected genes was assessed in porcine fetal livers and regenerating liver in an 80% hepatectomy model. SSH and subsequent differential screening selected 39 clones that were expressed differentially in HSLCs, including six known genes, 10 unknown genes, one unidentified gene and some chimeric fragments. Four of these genes showed significantly higher expression in HSLCs than in mature hepatocytes: anti-leukoproteinase, matrix Gla protein, amyloid-beta precursor protein (APP) and dickkopf-3 (DKK-3). Among them, the mRNA expression of APP and DKK-3 was significantly higher in fifth GW fetal liver than in seventh and thirteenth GW fetal and adult livers, unlike the expression patterns of alpha-fetoprotein (AFP) or albumin. These mRNAs were detected in the parenchyma of fifth GW fetal liver, whereas in normal adult liver possible expression was limited to the periportal area. On the other hand, immunohistochemistry, Masson's trichrome staining and silver impregnation demonstrated APP and DKK-3 proteins in fifth GW fetal liver in which intralobular bile ducts and hepatic plates had not completely developed. DKK-3 and AFP mRNAs were upregulated on the seventh day (7D) after 80% hepatectomy. In the liver tissue, DKK-3 and AFP proteins were detected in mesenchymal cells in the periportal area and parenchyma, respectively. These data for DKK-3 expression in adult livers suggest the possible presence of adult HSLCs in the periportal area. The pattern of histological staining suggested that 7D liver was in the process of regeneration, showing a character similar to the fifth GW fetal liver. It is speculated that DKK-3 is upregulated in immature and developing livers, and has possible involvement in hepatic differentiation and liver regeneration.     

CK19和PCNA在肝纤维化大鼠部分肝切除后再生肝中的表达

目的　检测肝纤维化大鼠部分肝切除后不同时点CK19及PCNA的表达,了解胆管再生情况。 方法　雄性SD大鼠,对照组和实验组各42只,实验组腹腔注射CCl4制备肝纤维化模型,两组均进行部分肝切除术,在不同时点取材后利用HE、免疫组化及免疫荧光双标染色等方法检测CK19和PCNA的表达情况。 结果　实验组和对照组术后随时间延长CK19表达均呈增强趋势,且实验组术后各时间点CK19的表达均高于对照组同时间点。两组PCNA的表达量都随时间推移逐步升高,但实验组大鼠明显上升缓慢,持续时间延长,表达高峰晚于对照组出现。 结论　 （1）肝纤维化大鼠部分肝切除能刺激肝卵圆细胞增殖和向胆管细胞的分化,而正常大鼠部分肝切除后再生肝中的胆管上皮细胞主要来源于原有细胞的代偿性增生。（2）术前肝纤维化大鼠的肝脏细胞就出现了增殖修复,术后由于肝纤维化大鼠本身肝脏受到伤害,因此肝脏的有效再生细胞数低于正常肝脏。     

Effect of toluene exposure on the liver under different experimental conditions

The effect of exposure to toluene inhalation was studied in adult mice, rats, and rabbits, and, further, in rats of different age groups and rats subjected to partial hepatectomy or ligation of the common bile duct. It was found that (1) toluene has no substantial liver damaging effect on the mouse, rat, or rabbit; (2) induction of microsomal monooxygenase enzymes by toluene is almost completely independent of physiological changes affecting the functional-anatomical state of the liver (aging), of restriction of the functional capacity of the organ (partial hepatectomy), and of irreversible hepatic damage (bile duct ligation); (3) toluene does not in any way interfere with surgically induced hepatic damage. The consequences of surgical interventions, in turn, just like the age-dependent changes, do not modify the minimal ultrastructural damaging effect of toluene.     

Transforming Growth Factor α Levels in Liver and Blood Correlate Better than Hepatocyte Growth Factor with Hepatocyte Proliferation during Liver Regeneration

Transforming growth factor α (TGFα) and hepatocyte growth factor (HGF) are mitogens for hepatocytes in vitro and in vivo, produced by hepatocytes or nonparenchymal cells such as stellate cells in the liver. It is still uncertain whether TGFα and HGF are essential for liver regeneration. To assess the role of these growth factors in liver regeneration, their circulating and hepatic levels were studied in various rat models of liver regeneration. Hepatic and plasma HGF levels were increased with increased number of mitotic hepatocytes in rats after partial hepatectomy or carbon tetrachloride intoxication. However, hepatic HGF levels were decreased despite an increased number of mitotic hepatocytes and increased or unchanged plasma HGF levels in rats given phenobarbital and in rats after dimethylnitrosamine intoxication, which can induce hepatic necrosis after apoptosis of hepatic stellate cells. In contrast, hepatic and serum TGFα levels were increased in all of the models. In sham-operated rats with no increased number of mitotic hepatocytes, hepatic and circulating levels of HGF were increased, whereas those levels of TGFα were unchanged. The results indicate that TGFα levels in liver and blood more closely correlate with hepatocyte mitogenesis than HGF levels.     

肝细胞生长因子和C-met在肝纤维化肝部分切除后残余肝组织中的表达

目的 探讨肝细胞生长因子（HGF）和C-met在纤维化肝部分切除后残余肝组织中的表达变化。方法 雄性SD大鼠130只,随机分为正常组（n=7）、正常肝部分切除组（n=50）、肝纤维化组（n=7）和纤维化肝部分切除组（n=66）。正常肝部分切除组和纤维化肝部分切除组分别在术后12 h、1 d、3 d、5 d、7 d和14 d 取材,运用免疫组织化学和Western blotting方法,检测残肝组织中HGF、C-met的表达。结果 免疫组织化学显示,正常肝部分切除组,HGF表达于术后12 h达高峰,并维持高峰平台,7 d后逐渐降低,于14 d接近术前水平;C-met术后迅速上升,3 d达到高峰,而后逐步下降,14 d降回术前水平。纤维化肝部分切除组,HGF表达于术后迅速下降,12 h又迅速上升,于1 d时达高峰,然后逐步下降,14 d降至最低点;C-met术后迅速下降,12 h开始缓慢下降,于术后3 d达最低点,后缓慢上升,7 d又迅速上升,14 d达最高点。Western blotting显示,HGF、C-met蛋白条带变化规律与免疫组织化学结果吻合。结论 HGF和C-met同时高水平表达有利于肝细胞的分裂增殖,提示HGF和C-met表达不同步,可能是纤维化肝术后再生困难的重要原因。     

Limited therapeutic efficacy of thrombopoietin on the regeneration of steatotic livers

Liver regeneration after partial hepatectomy is impaired in steatotic livers of leptin-deficient ob/ob mice. Previous studies have shown that thrombopoietin (TPO) promotes liver regeneration and improves liver cirrhosis by an increase of platelet counts and the expansion of hepatic progenitor cells. Herein we studied whether TPO exerts pro-proliferative and hepatoprotective effects and thereby improves the regenerative capacity of steatotic livers. For this purpose, we studied hepatic regeneration at day 2, 3, 7 and 10 in a model of 55% hepatectomy in obese (ob/ob) and non-obese (C57BL/6J) mice. Liver function and injury, platelet counts, weight of the regenerated liver, proliferating liver cells as well as the number of hepatic (CK19-positive) oval cells were quantified by biochemical and immunohistochemical analysis. As expected, obese mice had a markedly decreased regenerative capacity of livers compared with lean animals. Pretreatment of mice with recombinant TPO (12.5 μg/kg) had no evident effect on regeneration of fatty livers, but ameliorated acute liver damage in obese mice, as indicated by decreased liver enzyme release early after resection. TPO was unable to enhance hepatocyte proliferation, but increased proliferation of non-parenchymal cells, including CK19-positive oval cells, at later observation time points after resection. Interestingly, TPO completely inhibited the resection-induced increase of plasma triglycerides immediately after resection in non-obese mice. In conclusion, TPO slightly prevents acute liver damage after resection in obese mice, but fails to significantly enhance regeneration of fatty livers.     

Evidence for non-traditional activation of complement factor C3 during murine liver regeneration

Complement signaling has been implicated as important for normal hepatic regeneration. However, the specific mechanism by which complement is activated during liver regeneration remains undefined. To address this question, we investigated the hepatic regenerative response to partial hepatectomy in wildtype mice, C3-, C4-, and factor B-null mice, and C4-null mice treated with a factor B neutralizing antibody (mAb 1379). The results showed that following partial hepatectomy, C3-null mice exhibit reduced hepatic regeneration compared to wildtype mice as assessed by quantification of hepatic cyclin D1 expression and hepatocellular DNA synthesis and mitosis. In contrast, C4-null mice and factor B-null mice demonstrated normal liver regeneration. Moreover, animals in which all of the traditional upstream C3 activation pathways were disrupted, i.e. C4-null mice treated with mAb 1379, exhibited normal C3 activation and hepatocellular proliferation following partial hepatectomy. In order to define candidate non-traditional mechanisms of C3 activation during liver regeneration, plasmin and thrombin were investigated for their abilities to activate C3 in mouse plasma in vitro. The results showed that both proteases are capable of initiating C3 activation, and that plasmin can do so independent of the classical and alternative pathways. Conclusions: These results show that C3 is required for a normal hepatic regenerative response, but that disruption of the classical- or lectin-dependent pathways (C4-dependent), the alternative pathway (factor B-dependent), or all of these pathways does not impair the hepatic regenerative response, and indicate that non-traditional mechanisms by which C3 is activated during hepatic regeneration must exist. In vitro analysis raises the possibility that plasmin may contribute to non-traditional complement activation during liver regeneration in vivo.