Dissociation of alpha-macrofetoprotein and alpha-fetoprotein production during experimental injury.

The concentrations of two major fetal serum proteins of the rat, alpha-macrofetoprotein (AMF) and alpha-fetoprotein (AFP) are measured following administration of croton oil, carbon tetrachloride, galactosamine or ethionine, and after partial hepatectomy. Greatly elevated serum concentrations of AMF are found after croton oil injection, following oral administration of CCl4, and after partial hepatectomy, but not after ethionine or galactosamine. Elevations of AFP occur after administration of hepatotoxic agents during the stage of restitutive proliferation and after exposure to the hepatocarcinogen ethionine, but not after croton oil. Therefore, elevations of AMF and AFP are clearly dissociated under certain conditions. In situations such as partial hepatectomy, after which elevations in both AMF and AFP occur, AMF appears before AFP. Therefore, the production of AMF and AFP appear to be under completely independent regulation in the adult.     

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.     

The role of Kupffer cells in liver regeneration

The liver has a remarkable proliferative capacity after a partial hepatectomy. Previous studies have indicated that Kupffer cells have the potential to exert both stimulatory and inhibitory influences on hepatocyte proliferation. To elucidate the role of Kupffer cells in liver regeneration, mice were selectively depleted of Kupffer cells by injection of liposome-encapsulated dichloromethylene diphosphonate (lipo-MDP) at day 3 after a two-thirds hepatectomy. Results showed that liver regeneration was delayed after Kupffer cell-depletion. In control mice, hepatocyte growth factor (HGF) mRNA expressions were enhanced during liver regeneration and expressions of HGF were localized in fat-storing cells (Ito cells). In Kupffer cell-depleted mice, the number of HGF-expressing cells decreased in the regenerating liver, and expressions of HGF and its receptor (c-met) as well as other growth factors/cytokines were less prominent than in control mice. In contrast, expressions of TNF-alpha, another potent cytokine involved in liver regeneration, did not differ between Kupffer cell-depleted and control mice during the regeneration. Administration of TNF-alpha antibody did not reduce the expression of HGF or liver regeneration. These findings imply that Kupffer cells play a stimulatory role in liver regeneration by enhancing HGF expression via TNF-alpha-non-mediated mechanisms.     

Age-related variations in hepatic regeneration and erythropoietin production in the rat

Erythropoietin (Ep) is produced mainly by the liver and spleen during fetal and neonatal periods and by the kidney during adolescent and adult life. The liver is also an important extrarenal producer of Ep in the hypoxic, anephric adult animal. Subtotal hepatectomy results in a substantial elevation in serum Ep levels at 30-72 hours after hepatectomy in rats subsequently nephrectomized and rendered hypoxic. Ep production is related to the mass of regenerating liver with peak Ep production occurring during times of greatest tissue proliferation. Regenerative and erythropoietic responses to hepatectomy decline with advancing age. Rats undergoing repeated hepatectomies do not recover full liver mass but the initial rate of regeneration increases following each successive hepatectomy. Ep levels decline in anephric hypoxic rats undergoing multiple hepatectomies when compared to sham-operated controls.     

Alpha-fetoprotein: Cellular origin of a biological marker in rat liver under various experimental conditions

Summary Alpha₁-fetoprotein (AFP) was detected by serological, light and electron microscopic methods in various experimental models. These included (a) liver regeneration after partial hepatectomy or CCl₄ intoxication (mouse and rat); (b) liver intoxication by high doses of N-nitrosomorpholine (NNM) and chemical induction of hepatomas (rat). AFP levels varied greatly according to the animal species and strains used. Low and high AFP-producing species and strains were distinguished. In liver regeneration after hepatectomy or CCl₄ intoxication, cellular AFP was found in the cytoplasm of hepatocytes. In NNM-intoxicated livers, elevated AFP levels were associated with proliferation of canalicular epithelial cells in which AFP was localized. In early stages of hepatocarcinogenesis, significant AFP increase occurred after high-dose carcinogen feeding and AFP was also localized in proliferating canalicular epithelial cells. On low-dose NNM feeding, no cellular AFP was detected unless hepatomas had developed. At the stage of malignant conversion, distinct AFP staining and non-AFP staining hepatocellular carcinomas appeared in livers.     

Deficient liver regeneration after carbon tetrachloride injury in mice lacking type 1 but not type 2 tumor necrosis factor receptor.

Signaling by tumor necrosis factor type 1 receptor (TNFR-1) is required for the initiation of liver regeneration after partial hepatectomy. Using knockout mice that lack either TNFR-1 or TNFR-2, we determined whether signaling through TNF receptors is important for liver injury and hepatocyte proliferation induced by carbon tetrachloride (CCl4). Lack of TNFR-1 inhibited hepatocyte DNA synthesis after CCl4 injection. At 44 hours after the injection, replication of hepatocytes in TNFR-1 was 50% to 90% lower than in wild-type (WT) animals, depending on the dose injected. In WT animals, hepatocyte replication was essentially completed by 4 days after CCl4 injection, but replication at a low level persisted in TNFR-1 mice for at least 2 weeks. TNFR-1 knockout mice had little detectable NF-kappa B and STAT3 binding during the first 5 hours after CCl4, high plasma TNF, and reduced levels of plasma interleukin (IL)-6 and liver IL-6 mRNA. Injection of IL-6 30 minutes before CCl4 administration corrected the deficiency of hepatocyte replication at 44 hours and restored STAT3 binding to normal levels. In contrast, mice lacking TNFR-2 did not differ significantly from WT mice in NF-kappa B and STAT3 binding, IL-6 and TNF levels, or hepatocyte replication. Although AP-1 binding was induced in WT TNFR-1 and TNFR-2 knockout mice, binding in TNFR-2 knockouts was lower than in WT mice. C/EBP binding was much lower in TNFR-1 and TNFR-2 knockout mice than in WT mice. As assessed by morphological analysis and alanine aminotransferase levels, the acute injury caused by CCl4 appeared to be similar in the three groups of animals, but subsequent regeneration was impaired in mice lacking TNFR-1. We conclude that a TNFR-1 signaling pathway involving NF-kappa B, IL-6, and STAT3 is an important component of the hepatocyte mitogenic response induced by CCl4 injury in mouse liver.     

IL-12 induces specific cytotoxicity against regenerating hepatocytes in vivo.

Although impaired liver regeneration is thought to be a major cause of death in patients with fulminant hepatitis, the mechanisms are not well defined. Since IL-12 synthesis has been reported to be up-regulated in murine hepatitis virus infection, we studied the influence of continuous IL-12 stimulation on murine liver regeneration using flow cytometric and functional analyses. In non-hepatectomized mice, interestingly, the number of hepatic NK cells was significantly decreased on day 7, after six IL-12 injections, and day 14, after 13 IL-12 injections. The number of hepatic NKT cells was markedly increased on day 7 and day 14 of daily IL-12 treatment. The cytotoxic activity of hepatic lymphocytes against both YAC-1 and p815 cells was enhanced on day 2, after single IL-12 injection, and day 7, after six IL-12 injections. In contrast, hepatic lymphocytes isolated 24 h after partial hepatectomy with IL-12 pretreatment did not show any cytolytic activity against either YAC-1 cells or p815 cells. However, continuous IL-12 stimulation resulted in a significantly higher serum alanine aminotransferase (sALT) level 24 h after the partial hepatectomy as compared with sALT levels in mice subjected to either partial hepatectomy or IL-12 pretreatment alone. On the other hand, the expression of hepatic TNF-alpha mRNA was markedly enhanced by continuous IL-12 stimulation even 24 h after partial hepatectomy, as compared with that in non-treated mice and hepatectomy alone. Simultaneous administration of anti-tumor necrosis factor (TNF)-alpha mAb completely inhibited IL-12-induced in vivo enhancement of liver damage after partial hepatectomy. In conclusion, IL-12 induces the specific cytolytic activity against regenerating hepatocytes in vivo mainly through the enhancement of TNF-alpha synthesis.     

alpha-Fetoprotein (biology)]

The paper gives a historical overview of the origin and experimental analysis of alpha-fetoprotein (AFP) regulation and its reexpression in malignant tumors. It shows how a specific hepatoma-specific antigen that was later identified as the major protein component of embryonic serum was found and isolated during searches for murine hepatoma-specific antigens. Later on it was shown that AFP was synthesized by the yolk sac endoderm of the embryo and, later, by the fetal liver and its synthesis drastically diminishes in the adult animal liver. AFP synthesis restores in the moderately differentiated hepatomas and temporarily in the murine regenerating liver. The same regularity, but in other quantitative ratio, occurs in man. Analyzing the causes of AFP reexpression in the regenerating liver has indicated that suppression of AFP synthesis in the mature hepatocytes is reversible and controlled by the position of a hepatocyte in the liver plate: when the hepatocyte is included into the plate, it becomes fully differentiated with AFP suppression. Hepatocytic isolation from the plate leads to its "dedifferentiation" and AFP reexpression. AFP reexpression takes place in the perinecrotic cell layer in the liver poisoned by CCl4. Hepatocytic isolation from the liver and explantation into the cultured tissue results in strong reexpression of AFP while incorporation of the isolated hepatocytes in the three-dimensional extracellular matrix (ECM) leads to AFP suppression together with hepatocytic "maturation". Hepatocytic interaction with defective ECM does not suppress AFP synthesis. Our hypothesis suggests that disturbance of cell-ECM interactions during transformation and tumor progression is the main reason of AFT reappearance in liver tumors.     

Prolonged reduction of hepatocyte proliferative ability in rats after a single treatment with carbon tetrachloride.

Female Wistar rats were pretreated with I ml of carbon tetrachloride/kg of body weight or with olive oil. All the rats were given this dose of CCl4 20 or 40 days later. Liver regeneration as evaluated by 3H-thymidine incorporation into liver DNA and by the number of mitotic hepatocytes was markedly impaired in CCl4-pretreated rats when compared with olive oil-pretreated controls. DNA labelling reached only 83 and 59% and mitotic index 35 and 58% of control values, respectively, at 20-day and 40-day time intervals. The variables characteristic of liver damage did not parallel the changes in cell division. About 20% of hepatocytes were necrotic both in the CCl4-pretreated and in the control rats. The activity of serum alanine aminotransferase was higher in the CCl4-pretreated rats. Only serum aspartate aminotransferase activities were somewhat lower when compared to controls. Similarly, serum aminotransferases were much less affected by the pretreatment than the markers of regeneration when two low doses of CCl4 (0.125 ml/kg) were given to rats 20 days apart. The activities of microsomal enzymes aniline hydroxylase and pethidine demethylase were equal in control and in experimental rats 20 days after CCl4 pretreatment which indicated that the effects of CCl4 were not mediated by an overall decrease in cytochrome P-450 enzymes. In summary, a single pretreatment of rats with CCl4 induced changes in liver that lasted for 40 days and impaired liver regeneration when another dose of CCl4 was applied.     

肝再生中α2-巨球蛋白的表达动态分析

目的确定更多的与肝再生相关的基因.方法借助4～8正向抑制性消减文库(0-4-8-12短间隔连续部分肝切除)产生的α2-巨球蛋白基因片段,运用cDNA芯片分析了其mRNA在再生肝中的表达动态.结果肝再生中α2-巨球蛋白呈上调表达,而且变化幅度很大,至高点超过21倍.大部分肝切除中α2-巨球蛋白的表达高峰出现在8 h和16 h.短间隔连续部分肝切除中α2-巨球蛋白的表达呈现不同的变化趋势,4 h短间隔的连续肝切除诱导α2-巨球蛋白表达不断升高,而第二次的36 h短间隔的连续肝切除才能诱导α2-巨球蛋白表达显著升高.结论肝再生中α2-巨球蛋白可能在早期的正相急性反应中起重要作用,减少切除伤害带来的炎症,促进肝再生的顺利进行.     

Expression of NCAM in activated portal fibroblasts during regeneration of the rat liver after partial hepatectomy

In the portal tract of the regenerating liver after partial hepatectomy, vascular and bile ductular remodeling takes place in response to the portal hyperdynamic state and parenchymal hyperplasia. In order to reveal phenotypical changes in the portal fibroblasts, we immunohistochemically investigated neural cell adhesion molecules (NCAM) and alpha smooth muscle actin (alphaSMA) expression and the ultrastructural changes in them during liver regeneration. In the control rat liver, portal fibroblasts were negative for both NCAM and alphaSMA. They became positive for both markers two days after partial hepatectomy, increased in staining intensity, reached a maximum at three to four days, then decreased, being still clearly positive at 14 days. Under an electron microscope, portal fibroblasts from the regenerating liver had larger amounts of cytoplasm and rough endoplasmic reticulum than those from the control liver; thus they might be activated. Additionally, periportal hepatic stellate cells in the regenerating liver were activated with alphaSMA, but without NCAM. The present study has demonstrated that portal fibroblasts express NCAM and alphaSMA in the regenerating liver after partial hepatectomy via transformation into myofibroblasts following reconstruction of the portal tracts.     

Protection of hepatotoxic and lethal effects of CCl4 by partial hepatectomy

CCl4 is a hepatotoxic haloalkane, capable of producing hepatocellular fatty degeneration and centrilobular necrosis. Previous reports indicate induction of liver regeneration after 36-48 hr of CCl4 treatment, which is considered as a secondary effect. The present investigation was undertaken to evaluate the primary effects of CCl4 on hepatic DNA synthesis and to correlate liver regeneration with CCl4 toxicity. These studies were conducted in normal and actively regenerating livers using male Sprague-Dawley rats undergoing sham operation (SH), or partial (70%) hepatectomy (PH). Incorporation of 3H-thymidine (3H-T) in hepatocellular nuclear DNA and autoradiographic analyses of liver sections served as indices for hepatocellular regeneration. Initial experiments established that peak regeneration occurs at 2 days post-PH (PH2) and liver regeneration phases out by 7 days post-PH (PH7). SH and PH rats were challenged with a single ip dose of either corn oil vehicle or CCl4 at either 0.1 ml/kg (to represent subtoxic dose) or 2.5 ml/kg (to represent toxic dose). The low dose of CCl4 was not toxic and did not alter 3H-T incorporation and percentage labelled cells at 6 or 24 hours after administration to SH, PH2 or PH7 groups, indicating that there was no interference with PH-stimulated hepatocellular regeneration. The high dose of CCl4 was significantly hepatotoxic and lethal in SH rats, while in PH2 rats both hepatotoxic and lethal effects were significantly decreased. 3H-T incorporation as well as percentage labelled cells, highly stimulated by PH, were significantly decreased by high dose of CCl4. However, hepatocellular regeneration in PH2 rats treated with high dose of CCl4 was still significantly higher than SH or PH7 groups by virtue of the stronger stimulatory effect of PH. In PH7 rats, where hepatocellular regeneration had returned to the SH level, the hepatotoxic and lethal effects of the large dose of CCl4 were also restored. These findings show that the progressive phase of a single high dose of CCl4 injury which normally culminates in hepatotoxic and lethal effects is significantly mitigated by previously stimulated hepatocellular regeneration. High dose of CCl4 suppresses hepatocellular regeneration at early time points after administration in contrast to the smaller subtoxic dose of CCl4. By virtue of the much stronger stimulatory effect, PH results in the protection against the hepatotoxic and lethal effects of CCl4 despite the obtunding effects of the high dose on hepatocellular 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。     

Biosynthesis of ubiquinone-9 in the regenerating albino rat liver

The uptake of radioactivity from acetate-1-C¹⁴ into ubiquinone-9 in thin slices of regenerating albino rat liver was investigated on the 1st, 2nd, 3rd, 6th, 9th, 12th, and 15th day after partial hepatectomy. Marked acceleration of ubiquinone-9 biosynthesis was found during the first 3 days after removal of two-thirds of the liver. The maximal increase in biosynthesis (almost eightfold) was found 24 h after hepatectomy. This increase in the rate of ubiquinone-9 biosynthesis coincides with the time of increased mitotic activity and accelerated biogenesis of the mitochondria. A very small increase in the biosynthesis of ubiquinone-9 was found on the 12th and 15th days of regeneration.     

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.     

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.     

Regeneration and the immune system. II. Suppressor activities of lymphocytes activated in vivo by liver regeneration and their genetic control

The lymph node cells (LNC) activated in vivo by liver regeneration following partial hepatectomy of mice (primed lymph node cells) respond to regenerating liver cells in vitro with typical secondary immune response characteristics (Miyahara, S. et al., Eur. J. Immunol. 1983. 13: 878). These LNC activated in vivo suppress the proliferation of responder lymphocytes cultured with mitomycin C-treated regenerating syngeneic liver cells (sMLHLR). The suppressive activity was already present in LNC 4 days after partial hepatectomy and remained unchanged for at least 16 days. These primed LNC were effective not only on sMLHLR but also on syngeneic mixed lymphocyte culture (sMLR) and allogeneic mixed lymphocyte culture, of which responder cells share the I-A (I-B) subregions of the major histocompatibility complex (MHC) with primed LNC. At least one cell in the suppressor circuit is a T cell. The primed LNC restimulates in vitro with regenerating liver cells (in vitro reactivated primed LNC) suppressed the proliferation of syngeneic responder cells in sMLR, but not of cells from congeneic mice differing from the in vitro reactivated primed LNC at a cluster of genes linked to the Ig locus. Thus the suppressive activity of primed LNC is controlled by the I-A (I-B) subregions of the MHC and that of in vitro reactivated primed LNC by genes in the Ig region. The role of these suppressive cells in liver regeneration is discussed.     

外源精脒和精胺可诱导大鼠早期再生肝抗酶的表达

目的探讨精脒和精胺对大鼠早期再生肝抗酶(AZ)表达的影响,及在肝再生中的作用。方法外源多胺(溶于0.9%NaCl)皮下注射雄性SD大鼠(180～200g),进行部分肝切除(PH)诱导。采用RT-PCR和Western blotting方法进行PH后大鼠再生肝中AZ基因转录量和蛋白表达量的分析。结果对照组完整肝脏(PH后0h)中,AZ基因转录量、蛋白表达量较低,PH后均快速升高,3h达到峰值,5h出现明显下降,7h再次升高并达到峰值,之后缓慢下降。外源多胺处理后,两种剂量精脒(0.03mg/kg和0.15mg/kg)和精胺(0.06mg/kg和6mg/kg)处理组AZ mRNA及蛋白表达水平变化趋势与对照组相似,但低剂量处理组远远低于相应时间点高剂量处理组。精胺的作用效果更明显,作用时间更持久。结论外源多胺对大鼠早期再生肝AZ mRNA及蛋白表达具有剂量依赖性促进作用,而精胺的作用较强,精脒较弱。