肝再生过程中肝星状细胞及基质金属蛋白酶的动态变化

目的研究大鼠肝大部切除(PH)后再生过程中肝星状细胞(HSCs)的动态变化及肝MMP-2、MMP-9的活性变化,探讨肝星状细胞在肝再生中的作用。方法按Higgins等方法制备肝大部切除动物模型,于术后恢复不同时程取材,免疫组织化学法检测肝HSCs的动态变化,明胶酶谱电泳法检测肝中MMP-2、MMP-9的变化。结果(1)正常肝小叶内HSCs呈网架状分布,PH后Desmin阳性HSCs的数量递减;胶质纤维酸性蛋白(GFAP)阳性HSCs的数量也呈递减趋势。(2)PH后再生过程中,肝脏MMP-2、MMP-9的表达逐渐增多。结论肝再生过程中HSCs的增殖反应较慢且维持的时间短,这不同于肝纤维化等肝病过程中HSCs维持较长时间的激活状态。肝再生过程中基质金属蛋白酶-2,-9的表达发生显著改变,提示基质金属蛋白酶-2,-9参与了肝再生过程。     

大鼠肝大部切除后肝再生过程中肝小叶结构的重建

目的:观察正常大鼠肝大部切除后肝再生过程中肝小叶大小的动态变化,有助于探明大鼠肝再生过程中肝小叶结构的重建过程,并提供组织学依据.方法:正常雄性Wistar大鼠随机分为正常对照组、假手术组和部分肝切除组.采用Higgins和Anderson方法行大鼠70％肝切除,术中即刻称取切除肝叶的湿重.分别于术后12 h、24h、72 h、120 h、1周、2周处死大鼠,留取全部肝组织称湿重,统一留取肝右叶肝组织中性甲醛固定,石蜡包埋连续切片进行H-E染色,显微镜下观察再生肝肝小叶大小变化.结果:正常大鼠部分肝切除后,12h可观察到肝小叶面积开始增大,120 h达高峰,从1周开始可见汇管区门静脉终末支的增大并与邻近中央静脉相联系,肝小叶结构逐步重建,2周时肝小叶面积接近正常,小叶数目增加.正常对照组和假手术组大鼠未见上述动态变化.结论:正常大鼠部分肝切除后,肝再生通过早期肝小叶面积增大,后期肝小叶数目增多而恢复.     

大鼠肝再生过程中caspase-8的表达与时间相关性

目的:观察大鼠肝再生过程中caspase-8表达的动态变化,探讨大鼠肝再生过程中细胞凋亡的调控机制.方法:健康雄性SD大鼠75只,随机分为3组,手术组35只,10％水合氯醛麻醉后行70％肝大部切除;假手术组35只,正常对照组5只.手术组和假手术组各自分为7个亚组,即手术完成后分别饲养大鼠3h、6h、24 h、3d、7d、11d、14d后处死,切取残肝,称重;正常组直接切取肝,称重后取材进行组织学处理,石蜡包埋,切片,做免疫组织化学显色,检测不同时间点caspase-8的表达及分布.结果:caspase-8蛋白阳性产物呈棕黄色,主要分布于细胞核.手术组caspase-8表达趋势为术后3h阳性细胞表达率开始上升,术后7d达到峰值后开始下降,但14 d时仍明显高于假手术组和正常对照组.假手术组术后3hcaspase-8表达开始上升,到24 h时达峰值并开始下降,11d时恢复到正常水平.结论:肝大部切除后肝再生中,存在着细胞凋亡的分子调控机制,早期细胞凋亡活性的升高可能为手术应激反应所致,后期持续增高的细胞凋亡则可能与肝再生终止和肝组织结构重塑的调控有关.     

肝再生过程中肝和脑垂体纤维粘连蛋白表达的变化

目的；探讨大鼠肝大部分切除再生过程中肝和垂体内纤维粘连蛋白变化变化，方法：用免疫组织化学法观察鼠肝大部切除术０．５天，１天，１．５天，２天，３天，７天时，肝内和垂体远侧部滤泡状细胞细胞纤维粘连蛋白的变化，并用图像分析仪进行定量测定，结果：肝大部切除术后１．５天，肝内纤维粘连蛋白阳性染色增强，基平均光密度高于对照组（Ｐ〈０．０５）；术后２～３天，镜下可见纤维粘连蛋白染色呈强阳性，尤其在肝组织增生部位     

肝硬变大鼠肝大部分切除后肝再生的实验研究

本实验通过用四氯化碳(CCl_4)诱发大鼠肝硬变模型,进行肝大部分切除.观察残肝的再生动态变化.发现四氯化碳诱发的中轻度肝硬变大鼠可以耐受70%的肝切除,其残肝有再生能力,但较正常鼠肝缓慢.术后1个月左右才恢复术前大小.     

大鼠再生肝提取物对肝再生中肝细胞增殖的影响

大鼠肝大部(67%)切除24小时后取残肝匀浆,经加热,酒精沉淀、透析等步骤制备提取物,腹腔注射给正常大鼠和32%肝切术后的大鼠。光镜下观察肝组织切片,计算肝细胞有丝分裂百分率。鼠再生肝提取物可使32%肝切术后24-48小时再生肝细胞有丝分裂百分率明显增加,尤其在术后30小时可达对照组的3.9倍。结果表明,再生肝提取物中含有能够促进肝细胞增生的肝再生刺激因子(HSS),此因子对增殖前期(G_0/G_1早期)细胞作用不明显。     

肝纤维化大鼠部分肝切除后肝星状细胞活化和肝细胞生长因子的表达

目的：研究肝星状细胞在肝纤维化大鼠部分肝切除后的活化情况及其对肝细胞生长因子表达和肝再生的影响。方法：成年雄性SD大鼠，随机分成3组：正常组、肝纤维化组和肝纤维化大鼠部分肝切除组。其中肝纤维化大鼠部分肝切除组根据术后取材时间又分为6小组，分别于术后12h、1、3、5、7和14d取材。计算肝指数评价肝再生情况；用免疫组化、免疫荧光和免疫印迹方法检测各组大鼠肝组织中α平滑肌肌动蛋白和肝细胞生长因子的表达情况。结果：肝纤维化大鼠部分肝切除后肝指数逐渐增加，但递增速度缓慢；肝组织中α平滑肌肌动蛋白的表达呈现出先降低后升高的规律；肝细胞生长因子表达早期下降，而后升到一最高值后开始降低。结论：（1）肝纤维化大鼠部分肝切除后残肝可以再生；（2）活化肝星状细胞术后呈现出先减少后增多的规律性变化；（3）肝星状细胞可能是术后肝细胞生长因子表达和残肝再生的重要影响因素。     

库普弗细胞对原代培养贮脂细胞激活的调节作用

目的：观察库普弗细胞在贮脂细胞激活中的促进转化、促增殖及促进细胞外基质合成的效应；方法：采用库弗细胞和贮脂细胞分离培养，流式细胞仪测定ＤＮＡ含量，免疫组化和图像分析对贮脂细胞中Ⅰ、Ⅲ、Ⅳ型胶原和纤维粘连蛋白进行定量分析，液闪法检测贮脂细胞中＾３Ｈ－脯氨酸参入量；结果：（１）分离培养的贮脂细胞完全符合该细胞的各种特征；（２）贮脂细胞是肝纤维化中细胞外基质过度沉积的重要细胞来源之一；（３）库普弗细胞可     

大鼠肝大部切除与D-氨基半乳糖肝中毒后肝细胞增殖及甲胎蛋白和白蛋白免疫细胞化学研究

大鼠肝大部切除后和D-氨基半乳糖肝中毒后第1～7天,逐日取肝组织及血液样本,观察两种类型损伤后肝再生中肝细胞的增殖分化,以及血清甲胎蛋白(AFP)与白蛋白(ALB)浓度变化及其免疫细胞化学变化。结果表明:1.肝大部切除后肝细胞分裂高峰(1.5％)出现于术后第2天,半乳糖肝中毒组的高峰在第3天,且峰值仅为前者的一半(0.8％)。2.半乳糖肝中毒后,门管区和小叶周边带出现许多小型细胞,而大部切除后的肝内无此种小型细胞。3.AFP与ALB免疫细胞化学观察表明,两种损伤后肝再生中均有AFP阳性肝细胞,于第2～3天增多,第4天后渐少。部分小型细胞呈AFP阳性。电镜下见粗面内质网、滑面内质网、高尔基复合体及核周隙等处显示AFP阳性。ALB阳性细胞第1～3天较少,于第4天起渐增多。4.血清AFP含量于第1天开始升高,第4天达高峰,此后下降,ALB含量变化恰与AFP相反,第3天下降至最低点,此后回升。     

转化生长因子β1在大鼠肝内的原位表达

为探讨转化生长因子β１（ＴＧＦβ１）对肝细胞增殖的调节作用，用ＴＧＦβ１单克隆抗体和免疫组织化学方法研究ＴＧＦβ１在大鼠胎肝、正常成年肝、再生肝和癌变肝中的表达。结果表明：正常成年大鼠肝的大部分血管内皮细胞及胆管上皮细胞表达ＴＧＦβ１。大鼠胎肝的少量血窦内皮细胞表达ＴＧＦβ１，出现后表达逐渐增强，至生后３０ｄ的表达水平同成年肝。肝大部分切除１￣２ｄ，血窦内皮细胞ＴＧＦβ１表达增强，于大部切除后１０     

人肝贮脂细胞表达结蛋白的免疫细胞化学观察

用免疫细胞化学法观察了正常人肝贮脂细胞表达结蛋白中间丝。发现结蛋白阳性细胞位于血窦旁旁狄氏间隙内,呈星形或纺锤形,于肝小叶内大致均匀分布,有些细胞内含有空泡,每高倍(400X)视野细胞数为15～25个。汇管区结缔组织内及肝静脉周围散在少数与小叶内形态相似的结蛋白阳性细胞。结果表明正常人肝贮脂细胞表达结蛋白,可作为研究该细胞的良好标志,并支持了贮脂细胞的肌源性学说。[关键词]贮脂细胞,结     

A STUDY ON THE MECHANISM OF ACUTE HEPATIC NECROSIS FOLLOWING PARTIAL RESECTION OF THE LIVER

Endotoxin was injected intravenously into rats 48 hours after 70% he-patectomy. The remaining hepatic tissue demonstrated marked hemorrhagic necrosis in high frequency. However, hepatic lesions were slight in other groups such as the group in which heparin was administered simultaneously with injection of endotoxin 48 hours after 70% hepatectomy and the group in which endotoxin was injected 10 days after 70% hepatectomy. Difining of this marked necrosis to be a univisceral Shwartzman reaction in the liver may be justified from (i) conformity of experimental manipulations to those employed in the Shwartzman reaction, (ii) macroscoplcal as well as histological evidence, (iii) formation of microthrombl in hepatic lesions, (iv) inhibition of occurrence of the reaction by heparin, and (v) absence of any remarkable change in other organs. We concluded that the hepatic regenerative state might correspond theoretically to a state of preparedness for the Shwartzman reaction.     

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.     

血管内皮生长因子在大鼠肝再生过程中的表达变化

目的通过检测血管内皮生长因子（VEGF）在大鼠肝再生过程中表达量的变化,探讨VEGF在肝再生中的作用。方法300只SD大鼠随机分为正常对照组、假手术组和实验组。实验组大鼠切除2／3肝,分别于术后不同时段取肝组织或原位灌注分离肝实质细胞。用免疫组织化学和Western blotting技术检测肝组织和肝实质细胞中VEGF的表达变化。结果正常肝组织的VEGF阳性细胞很少;肝切除（PH）后24h阳性细胞数显著增加（P〈0．01）,主要分布于门管区周围;PH72h时阳性细胞数达到高峰,几乎遍及整个肝小叶;从PH120h起VEGF阳性细胞数逐渐减少至正常。Western blotting检测结果表明,肝实质细胞的VEGF表达量于PH0～12h较少,PH12h后增多,PH24—72h的表达量约为PH0～12h的2倍,PH72h后逐渐下降,至120h恢复正常;肝组织VEGF的表达量于PH0～12h较少,PH12h后逐渐增多,PH72h时出现高峰,约为PH0～12h的4倍;肝组织VEGF的表达量大于肝实质细胞的表达量。结论VEGF可能是参与肝脏组织结构重建的重要的生长因子：肝实质细胞是肝中VEGF的雷耍来源之一。     

Immunohistochemical detection of proliferating lipocytes in regenerating rat liver

For the detection of proliferating lipocytes in regenerating liver, partially hepatectomized rats were injected intraperitoneally with 5-bromo-2'-deoxyuridine (BrdUrd, 50 mg/kg body weight) and killed 1 h later. Acetone-fixed frozen liver sections were used for the simultaneous detection of cytoplasmic desmin and BrdUrd-labelled nuclei of lipocytes using double immunohistochemical procedures. The best results were obtained with the sequences: rabbit anti-desmin----biotinylated anti-rabbit IgG----avidin-biotin-peroxidase complex----3,3'-diaminobenzidine tetrahydrochloride, followed by DNA denaturation----mouse anti-BrdUrd----anti-mouse IgG----peroxidase-anti-peroxidase complex----4-chloro-1-naphthol. With this method, cytoplasmic desmin was stained a brown colour, which sharply contrasted with the blue-stained BrdUrd-labelled nuclei. Unlabelled nuclei appeared green after counterstaining with methyl green. No cross-reaction between immunoreagents of desmin and BrdUrd stainings was observed. The labelling index of lipocytes peaked (25.7 per cent) 48 h after partial hepatectomy, whereas it was 3.7 per cent in normal rat liver.     

The significance of lipid peroxidation (free radical) on hepatocellular dysfunction following hepatectomy

The possibility of extended hepatectomy was examined clinically and experimentally, with special reference to relation between remnant liver dysfunction and lipid peroxidation. Clinical study: When the functional reserve of the remnant liver prior to hepatectomy was below the critical level, both serum phospholipase A2 and plasma lipid peroxide increased and severe hepatic insufficiency occurred. Experimental study in dogs: The amount of lipid peroxide in the plasma and remnant liver increased more after 84% hepatectomy than after 70% hepatectomy, causing significant impairment of the remnant liver function. To prevent hepatic dysfunction after hepatectomy, Coenzyme Q10, an antioxidant and a membrane stabilizer, were administered. These substances reduced the increase of lipid in the plasma and remnant liver, and restored the liver function with subsequent good regeneration. The results suggest that the administration of Coenzyme Q10 may be useful for recovery of liver function and a more favorable prognosis following the extended hepatectomy.     

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.     

Experimental models of hepatectomy and liver regeneration using newborn and weaning rats

OBJECTIVES: Liver regeneration is a complex process that has not been completely elucidated. The model most frequently used to study this phenomenon is 70% hepatectomy in adult rats; however, no papers have examined this effect in developing animals. The aims of the present study were: 1) to standardize two models of partial hepatectomy and liver regeneration in newborn suckling and weaning rats, and 2) to study the evolution of remnant liver weight and histological changes of hepatic parenchyma on the days that follow partial hepatectomy. METHODS: Fifty newborn and forty-four weaning rats underwent 70% hepatectomy. After a midline incision, compression on both sides of the upper abdomen was performed to exteriorize the right medial, left medial and left lateral hepatic lobes, which were tied inferiorly and resected en bloc. The animals were sacrificed on days 0 (just after hepatectomy), 1, 2, 3, 4 and 7 after the operation. Body and liver weight were determined, and hepatic parenchyma was submitted to histological analysis. RESULTS: Mortality rates of the newborn and weaning groups were 30% and 0%, respectively. There was a significant decrease in liver mass soon after partial hepatectomy, which completely recovered on the seventh day in both groups. Newborn rat regenerating liver showed marked steatosis on the second day. In the weaning rat liver, mitotic figures were observed earlier, and their amount was greater than in the newborn. CONCLUSIONS: Suckling and weaning rat models of partial hepatectomy are feasible and can be used for studies of liver regeneration. Although similar, the process of hepatic regeneration in developing animals is different from adults.     

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.