The calcium-dependence of the stimulation of neonatal rat hepatocyte DNA synthesis and division by epidermal growth factor, glucagon and insulin

A low concentration (10(-11) mol/l) of epidermal growth factor (EGF) and/or an equimolar (10(-14) mol/l) mixture of glucagon and insulin stimulated DNA synthesis in hepatocytes in 4-day-old primary cultures of neonatal rat liver. EGF seems to have acted by inducing quiescent hepatocytes to begin cycling, while the glucagon-insulin combination seems to have acted mainly by shortening the cell cycle time. Incubation in low calcium medium blocked untreated hepatocytes in the G1 phase of their cycle and prevented EGF and the glucagon-insulin mixture from stimulating DNA synthesis. Nevertheless, hepatocytes in calcium-deficient medium did respond to these agents, as they reached a late stage of prereplicative development before being blocked: in fact, they initiated DNA synthesis soon after the addition of calcium. EGF, but not the glucagon-insulin combination, also enabled the already cycling hepatocytes (but not the newly activated ones) to overcome the block imposed by the extracellular calcium deficiency after a delay of several hours.     

肝细胞生长因子基因修饰的间充质干细胞治疗肝损伤的研究进展

减轻肝脏损伤、促进肝脏修复和再生始终是肝脏疾病研究中的重点。间充质干细胞(MSCs)是众多具有组织修复和再生能力细胞中的明星细胞,合成的多种细胞因子经旁分泌途径发挥调控细胞生存,调节炎症反应,促进血管再生和减轻纤维化等多种生物学效应,肝细胞生长因子(HGF)便是重点细胞因子之一。基于HGF的信号调控作用,再结合MSCs的干细胞优势,HGF基因修饰间充质干细胞(HGF-MSCs)作为一种干细胞治疗新策略能够发挥“1+1>2”的效果。本文就HGF-MSCs在减轻和修复肝损伤中的研究进展作综述。     

Characterization of a rat liver epithelial cell line to detect inhibitors of metabolic cooperation

Summary A normal rat liver epithelial cell line, with phenotype characteristics of “oval” cells (WB-F344), was examined for its ability to perform gap-junctional intercellular communication as measured by metabolic cooperation. To test for gap-junctional intercellular communication, 6-thioguanine-sensitive cells were cocultivated with 6-thioguanine-resistant cells. It was found that the recovery of 6-thioguanine-resistant cells depended on the densities of the 6-thioguanine-sensitive cells. Higher densities of 6-thioguanine-sensitive cells reduced the recovery of 6-thioguanine-resistant cells. These observations demonstrate that rat liver epithelial cells could metabolically cooperate, implying they could perform gap-junctional intercellular communication. Two tumor-promoting organochlorine pesticides, aldrin and dieldrin, were potent inhibitors of metabolic cooperation for these cells, but 12-0-tetradecanoyl-phorbol-13-acetate and teleocidin, known mouse skin tumor promoters, were not significantly effective in inhibiting metabolic cooperation. The results suggest that these cells might provide the basis for an in vitro assay specifically to study liver tumor promoters. Research was sponsored by a grant from the Air Force Office of Scientific Research, Air Force Systems Command, USAF, under grant AFOSR-86-0084. The U. S. Government is authorized to reproduce and distribute reprints for government purposes notwithstanding any copyright notation thereon.     

Characterization of cytokeratin 19-positive hepatocyte foci in the regenerating rat liver after 2-AAF/CCl<Subscript>4</Subscript> injury

Partial hepatectomy or carbon tetrachloride (CCl₄) injury, following treatment of rats with 2-acetylaminofluorene (2-AAF) to inhibit proliferation of hepatocytes, induces proliferation of oval cells and possibly their differentiation into nodular foci of hepatocytes when higher doses of 2-AAF are used. Unfortunately, immunohistochemistry in previous studies failed to show oval cell markers in these foci, and thereby to demonstrate the precursor–product relationship between oval cells and hepatocytes. Immunohistochemistry on livers of rats treated with high dose 2-AAF/CCl₄ was used. We found 7.6% of the hepatocyte foci were positive for an oval cell marker cytokeratin 19 (CK-19). These foci were positive for alpha-fetoprotein, less positive for carbamoylphosphate synthetase 1, and more positive for laminin in the basement membrane lining. Rarely present transitional foci had weaker expression of CK-19 and discontinuous laminin. Focal hepatocyte differentiation of oval cells was characterized by cell hypertrophy, membranous CK-19, and positive hepatocyte nuclear factor 4 (HNF-4). HNF-4⁺ small oval cells surrounding CK-19⁺ foci were frequently seen, suggesting that a paracrine mechanism(s) may be responsible for the enlargement of CK-19⁺ foci. In conclusions, oval cells appear to differentiate to CK-19⁺ foci and then to CK-19⁻ foci in the high dose 2-AAF/CCl₄ model. This study was supported by grants from National Science Council (94-2314-B-002-256 and 95-2314-B-002-099), Executive Yuan, Taiwan.     

Treatment with leucine stimulates the production of hepatocyte growth factor in vivo

Hepatocyte growth factor (HGF) has pleiotropic effects. Up-regulation of HGF activity in vivo may be beneficial. Branched-chain amino acids (BCAAs) are known to modulate various cellular functions. When starved rats received intraperitoneal injections of valine, leucine or isoleucine, only leucine treatment increased both hepatic and circulating levels of HGF in a dose-dependent manner, up to 1.5 and 2.3 times higher, respectively, than in controls. When young growing rats with free access to food were injected with leucine once a day for a week, HGF levels and liver weights were significantly higher than those of control rats. Furthermore, 1 week of leucine treatment of adult rats resulted in elevated serum albumin levels with an increase in HGF levels. Taken together with our previous report showing that leucine stimulates HGF production by hepatic stellate cells in culture, leucine, among BCAAs, may induce an increase in HGF production by the liver in vivo.     

Characteristics of hepatocyte proliferation in the regenerating rat liver with hydroxyurea synchronization of the proliferative processes

5-week old rats underwent a partial hepatectomy (2/3 of the liver was removed). The operated rats were repeatedly injected hydroxyurea, within 9-24 or 12-24 hours following operation. Proliferation of hepatocytes and non-parenchymal cells in the regenerating liver was studied. Five or six injections of hydroxyurea increased the labelling index of hepatocytes up to 62.5% and induced proliferation of bile duct cells significantly.     

Axotomy-dependent urokinase induction in the rat facial nucleus: possible stimulation of microglia by neurons

The phenomenon in which urokinase-type plasminogen activator (uPA) is induced in the axotomized facial nucleus suggests an interaction between injured motoneurons and microglia. We examined the relation of neurons and microglia to the induction of uPA in vitro. The amount of uPA released from a co-culture of neurons and microglia was much greater than the addition of that from each alone, suggesting the occurrence of an interaction between the two. The analysis of conditioned neuronal medium (CNM)-effects on microglia and conditioned microglial medium (CMM)-effects on neurons revealed that microglia enhance uPA release in response to CNM, rather than vice versa. Characterization of the CNM-effect on microglia demonstrated that CNM enhances not only uPA release but also the specific activity of acid phosphatase and 5'-nucleotidase in microglia. The profile of microglial activation caused by CNM was quite different from that caused by lipopolysaccharide (LPS)-activation. These results suggest that a specific soluble constituent(s) derived from neurons activates microglia by a mechanism different from LPS. As a candidate molecule for the microglial activation, brain-derived neurotrophic factor was detected in the CNM. Thus, uPA induction in the axotomized facial nucleus may be explained by a neuronal stimulus leading to uPA induction in microglia.     

Synergistic induction of hepatocyte growth factor in human skin fibroblasts by the inflammatory cytokines interleukin-1 and interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for wound healing. HGF expression markedly increases in wounded skin and is mainly localized in dermal fibroblasts. HGF expression level in human dermal fibroblasts in vitro, however, is low and thus may be stimulated by some factors in the process of wound healing. Candidates of the factors are inflammatory cytokines released by polymorphonuclear and mononuclear cells infiltrating the wounded area, but HGF production in human dermal fibroblasts is only slightly induced by interleukin (IL)-1, tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. We here report that a combination of IL-1beta and IFN-gamma or a combination of TNF-alpha and IFN-gamma very markedly induced HGF production. The synergistic effect of the former was more marked than that of the latter. Synergistic effects of IL-1beta and IFN-gamma were observed at more than 10 pg/ml and 10 IU/ml, respectively, and were detectable as early as 12 h after addition. Neither IFN-alpha nor IFN-beta was able to replace IFN-gamma. HGF mRNA expression was also synergistically upregulated by IL-1beta and IFN-gamma. IL-1beta plus IFN-gamma-induced synergistic production of HGF was potently inhibited by treatment of cells with the extracellular signal-regulated kinase (ERK) kinase inhibitor PD98059 and the p38 inhibitor SB203580 but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Taken together, our results indicate that a combination of IL-1beta and IFN-gamma synergistically induced HGF production in human dermal fibroblasts and suggest that activation of ERK and p38 but not of JNK is involved in the synergistic effect.     

Effect of bromodeoxyuridine on the proliferation and growth of ethyl methanesulfonate-exposed P3 cells: Relationship to the induction of sister-chromatid exchanges

Although sister-chromatid exchange (SCE) analysis is recognized as an indicator of exposure to DNA-damaging agents, the results of these analyses have been confounded by the use of bromodeoxyuridine (BrdUrd) to differentially label the sister chromatids. Not only does BrdUrd itself induce SCE, it also modulates the frequency of SCE induced by certain DNA-damaging agents. In order to examine this effect of BrdUrd on SCE frequency, an indirect method which lends itself to measurements both with and without BrdUrd was employed. Human teratocarcinoma-derived (P3) cells were exposed to ethyl methanesulfonate (EMS) and cultured with increasing concentrations of BrdUrd for lengths of time corresponding to one, two, and three generations of cell growth. At each time point, the distribution of nuclei among the phases of the cell-cycle and cell growth were evaluated for each concentration and chemical. A statistical model was employed which tested both for the main effects of chemicals and culture times and for interactions between these factors. Both EMS and BrdUrd significantly affected the percentages of nuclei within the cell-cycle. Exposure to EMS resulted in decreases in the percentages of nuclei in G0 + G1 and increases in the G2 + M compartment. Exposure to BrdUrd affected the size of the G0 + G1 compartment as well as the percentage of S-phase nuclei. Cell growth was reduced as a consequence of increasing EMS concentration and as a function of BrdUrd concentration; the effects of these chemicals were more readily apparent at the later time points. Most importantly, for both the cell-cycle kinetics data and the cell growth data, no evidence of an interaction between the effects of EMS and the effects of BrdUrd was detected statistically. These results may be interpreted to mean that while both EMS and BrdUrd affect the induction of SCE, under the conditions of this experiment, the effects are additive rather than interactive.Abbreviations: EMS, ethyl methanesulfonate - BrdUrd, bromodeoxyuridine - BrdUTP, bromodeoxyuridine triphosphate - dCTP, deoxycytidine triphosphate - SCE, sister-chromatid exchange - P3, human teratocarcinoma derived - HBSS, Hank's Balanced Salt Solution - HOUR, culture time - REP, replicate     

In vivo transfer of hepatocyte growth factor gene accelerates proliferation of hepatic oval cells in a 2-acetylaminofluorene/partial hepatectomy model in rats

To clarify the effect of hepatocyte growth factor (HGF) on proliferation of hepatic oval cells, we transferred HGF gene into liver of the Solt-Farber rat model. Male Fisher 344 rats were infected with a recombinant adenovirus carrying the cDNA for HGF (pAxCAHGF) from tail vein. HGF mRNA showed its peak at 4 days, and diminished thereafter. The total and proliferating cell nuclear antigen-positive hepatic oval cells were significantly elevated in HGF-transferred rats, in which stem cell factor and c-kit mRNA increased at each time point. Our results suggest that in vivo transfer of the HGF gene into liver accelerates proliferation of hepatic oval cells in the Solt-Farber model in rats.     

The effects of aging on phosphofructokinase induction during lymphocyte mitogenesis in relation to DNA and protein synthesis

The incorporation of radiolabeled leucine into phytohemagglutinin-stimulated human lymphocytes increases by 9 hours after mitogen addition in the young whereas this process is delayed by two-fold in the aged (18 hours). Once induced, the leucine incorporation is about 56% less in the aged as compared to the young. The induction of phosphofructokinase (PFK) catalytic activity mimics the induction of protein synthesis in both young (9 hours) and aged (18 hours) subjects also taking twice as long to induce in the aged and attaining much lower levels of induction with increasing subject age. The increase of thymidine incorporation in mitogen-stimulated cells does not occur until 12 hours after the increase in leucine incorporation in both the young (21 hours) and aged (30 hours) which also represents a 9 hour age-related delay in induction. The marked increase in protein synthesis rate occurs in a concerted manner with the induction of glycolysis and the delay and impairment in protein biosynthesis in the aged appears to relate to the similar age-related findings for glycolytic enzyme induction. The mitogen-induced increase in DNA synthesis is a later event and the age-related delay in DNA synthesis induction may be secondary to the delay in the induction of protein synthesis. Other enzyme-dependent processes besides DNA synthesis and glycolysis may also be secondary to a primary slowing of protein synthesis in the aged and related to the delayed cell cycle time frequently observed in aged subjects.     

P13K—Akt—mTORC1信号途径在细胞生长增殖中的调控作用

P13K-AKT—mTORCl信号途径在细胞生长增殖中起重要调控作用,P13K-Akt—mTORl信号途径能够调节细胞周期相关蛋白基因的表达来调控细胞的增殖;同时,P13K—Akt-mTORl信号途径也能够调控细胞的生长和大小;P13K-Akt-mTORCl信号途径的异常活化与肿瘤发生紧密相关。就P13K—AKT-mTORCl信号途径在细胞生长增殖中的作用作一综述。     

The relation between inhibition of glycolysis and stimulation of oxygen uptake due to glucagon in livers from rats in different metabolic conditions

The relation between the effects of glucagon on oxygen consumption and glycolysis in livers from rats under different metabolic conditions was examined. Respiration of substrate-free perfused livers with different glycolytic fluxes, induced by changes in the pattern of food intake, responds differently to the infusion of 1 nM glucagon. The increases in oxygen uptake caused by 1 nM glucagon correlate reasonably well with the absolute decreases in glycolysis. The degree of inhibition of glycolysis is approximately constant (58 per cent) for all metabolic conditions. When no recovery of glycolysis occurs upon cessation of glucagon infusion, the same happens with oxygen consumption, which remains stimulated. It is concluded that in livers with no appreciable biosynthetic activities, the action of glucagon on respiration and glycolysis may be interpreted in terms of an interaction of interpreted in terms of an interaction of cytosolic and mitochondrial ATP generating processes.     

缝隙连接在同型半胱氨酸介导的自发性高血压大鼠血管平滑肌细胞增殖中的作用

目的:探讨缝隙连接(GJ)是否参与同型半胱氨酸(Hcy)介导的自发性高血压(SHR)大鼠血管平滑肌细胞(VSMCs)增殖及可能的分子机制。方法:原代培养SHR VSMCs,细胞分四组:①对照组,②Hcy组,③缝隙连接阻断剂(18α-GA)组和④Hcy+18α-GA组。MTT法及流式细胞仪检测细胞的增殖活性,免疫荧光技术观察细胞中Cx43、Cx40蛋白表达及定位,Western blot法检测细胞中Cx43、Cx40蛋白表达,染料示踪分子传递法(划痕标记染料传输法)检测细胞的缝隙连接功能。结果:①与对照组相比,Hcy组MTT法测得A值及细胞周期测得S值增高(P0.05),18α-GA组降低(P0.05);与Hcy组比,TGFβ-1+18α-GA组A值及S值均降低(P0.05)。②免疫荧光技术检测细胞中Cx43、Cx40蛋白表达呈阳性,两者共定位于胞浆。③与对照组相比,Hcy组Cx43、Cx40蛋白表达增强(P0.05),18α-GA组Cx43、Cx40表达均减弱(P0.05);与Hcy组比,Hcy+18α-GA组Cx43、Cx40表达均减弱(P0.05)。④与对照组相比,Hcy组缝隙连接功能明显增强(P0.05),18α-GA组缝隙连接功能明显减弱(P0.05),与Hcy组比,Hcy+18α-GA组缝隙连接功能显著降低(P0.05)。结论:Hcy通过上调SHR VSMCs Cx43和Cx40的蛋白表达,引起缝隙连接通讯功能的增强,促进了SHR VSMCs增殖。     

Stimulation of rat hepatocyte proliferation in vitro and in vivo by factors derived from the bovine small intestinal mucosa

Summary A factor with a molecular weight of less than 1 kDa in the mucosa of the bovine small intestine (low molecular weight factor or LMW factor) stimulated DNA synthesis in rat hepatocytes in primary culture. This factor only showed its activity when it was added with a larger factor with a molecular weight of 30 kDa that was also found in the same tissue (high molecular weight factor or HMW factor). The LMW factor probably acts to enhance the action of a hepatotrophic growth factor, since EGF and HGF can substitute for the HMW factor. The action of the LMW factor was not due to the actions of low molecular weight substances such as norepinephrine, estradiol, triiodothyronine, and putrescine, which enhance the action of EGF or HGF, since substantial amounts of these substances were not found in the extract. When intraperitoneally administered into rats, after two-thirds hepatectomy, the LMW factor enhanced hepatocyte proliferation without the administration of the HMW factor. In the regenerating liver, a hepatotrophic growth factor(s), which acts synergistically with the LMW factor, might be properly provided, but the supply of the LMW factor might be below the level that maximally stimulates hepatocyte proliferation.