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1.
Normand Marceau Micheline Noël Julien Deschênes 《In vitro cellular & developmental biology. Plant》1982,18(1):1-11
Summary Hepatocytes isolated from neonatal (NN) and adult (AD) rats were seeded on fibronectin coated substratum and cultured in arginine-free
medium supplemented with various combinations of insulin, dexamethasone, triiodothyronine (T3), albumin, and transferrin, in presence or absence of fibronectin depleted serum (FDS). The main finding is that in response
to certain hormone mixtures, both NN and AD hepatocytes can be stimulated to proliferate, as revealed by an increase in cell
number, a [3H]thymidine incorporation into nuclei, and extractable DNA as well as the appearance of mitotic figures. Moreover, this proliferative
activity is associated with changes in hepatocyte ploidy. However, the proliferative response of NN hepatocytes to hormone
action is much different from that of AD hepatocytes, and the addition of FDS amplifies this activity in NN but inhibits it
in AD hepatocyte cultures. Measurements of tyrosine aminotransferase and lactate dehydrogenase activities indicate a good
preservation of NN and AD hepatocyte functional integrity under certain culture conditions. A good maintenance of albumin
production in NN and AD hepatocyte cultures requires the presence of dexamethasone, whereas theα-fetoprotein production in NN hepatocyte cultures is reduced quite rapidly under most conditions. Noα-fetoprotein is detectable in AD hepatocyte cultures.
Part of this work was presented at the 31st Annual Meeting of the Tissue Culture Association, St. Louis, MO, June 1980. 相似文献
2.
G Lescoat N Pasdeloup B Kneip C Guguen-Guillouzo 《European journal of cell biology》1987,44(1):128-134
Fetal hepatocytes were cultured alone or in association with primitive biliary cells (RLEC) in the presence or absence of dexamethasone. Cell-cell contacts were established 3 h or five days after hepatocyte seeding and their effects on hepatocyte growth and functional activities were evaluated in the presence or absence of dexamethasone. Establishment of cellular interactions with RLEC in coculture decreased hepatocyte growth, while it stimulated production of alpha-fetoprotein, albumin and transferrin. Addition of dexamethasone to coculture inhibited alpha-fetoprotein secretion and maintained the synthesis rate of albumin and transferrin together with an additional inhibition of DNA synthesis. The levels of mRNAs corresponding to the three proteins were also measured. We observed that the levels of alpha-fetoprotein, albumin and transferrin secretion in cocultures maintained in the presence or absence of dexamethasone were well correlated with the relative amounts of their corresponding mRNAs. Consequently, it may be assumed that the primitive mechanism involved in the increased functional activity of fetal hepatocytes in coculture is of pretranslational origin. Furthermore, the present data provide evidence that heterotypic interactions and dexamethasone act as distinct modulators of growth and maturation of fetal rat hepatocytes. 相似文献
3.
D Magielska-Zero A Guzdek J Bereta A Kurdowska K Cieszka A Koj 《Acta biochimica Polonica》1988,35(4):287-295
Dexamethasone and insulin stimulate production of several plasma proteins in primary cultures of adult rat hepatocytes but inhibit their production in primary cultures of Morris hepatoma cell line 7777W. The acute phase response elicited in cultured cells by crude cytokines from activated rat peritoneal macrophages is considerably higher in hepatocytes in the presence of hormones, and especially of dexamethasone. In hepatoma cells the hormones enhance the cytokine-induced formation of fibrinogen and cysteine proteinase inhibitor but are without significant effect on suppression of albumin and alpha-fetoprotein synthesis by macrophage supernatants. 相似文献
4.
Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes 总被引:4,自引:0,他引:4
Perumana R. Sudhakaran Stamatis C. Stamatoglou R. Colin Hughes 《Experimental cell research》1986,167(2):505-516
Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and secretion of alpha-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata, and its synthesis by hepatocytes seeded onto a mixed substratum of laminin and fibronectin is down-regulated by fibronectin in a dose-related manner. Similarly, type IV collagen synthesis is less when the cells are seeded on the homologous matrix protein substratum than on heterologous substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as alpha-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured. 相似文献
5.
Monolayer cultures of newborn rat hepatocytes were initiated in the presence or absence of dexamethasone, and fibronectin was analysed by indirect immunofluorescence microscopy using a specific antiserum raised in rabbit. Dexamethasone-treated hepatocytes produce a well defined extracellular matrix of fibronectin that begins to form as early as 24 h after treatment. Non-treated hepatocytes exhibit very little immunofluorescence staining. Moreover, examinations by phase contrast microscopy reveal a very good preservation of the hepatocyte typical epithelial morphology and a drastic inhibition of fibroblast growth in the treated cultures. 相似文献
6.
Modulation of fetal and neonatal rat hepatocyte functional activity by glucocorticoids in co-culture 总被引:2,自引:0,他引:2
Fetal and neonatal rat hepatocytes were cultured alone or in association with another liver epithelial cell type, in a medium with or without hydrocortisone. Secretion of albumin and alpha-fetoprotein decreased in pure hepatocyte culture, whereas in co-culture it remained stable for several days. Furthermore, addition of hydrocortisone to the co-culture medium induced a rapid increase in albumin production which was maintained at a high level. In contrast, alpha-fetoprotein production was inhibited. At the same time, an abundant extracellular material was secreted between and around hepatocyte colonies. The results demonstrate that the reciprocal relation between albumin and alpha-fetoprotein production which occurs during in vivo perinatal hepatocyte maturation is also observed in vitro. Both cell-cell contacts and glucocorticoids play a key role in this process. It appears that fetal and neonatal hepatocytes can maturate when maintained in a co-culture system. 相似文献
7.
8.
Dexamethasone and dimethylsulfoxide as distinct regulators of growth and differentiation of cultured suckling rat hepatocytes 总被引:6,自引:0,他引:6
Dexamethasone can promote the differentiation of different tissues in vivo while dimethylsulfoxide is a commonly used inducer of differentiation in various tumor cell types in culture. In the present study, the effects of dexamethasone and dimethylsulfoxide on growth and functional activities of cultured differentiating suckling rat hepatocytes stimulated with various combinations of EGF, insulin, and glucagon were evaluated. Hepatocytes stimulated with EGF and either insulin or glucagon entered S phase and mitosis after a lag period of 24 h. These hormonal factors thus provide simple combinations of hepatocyte-growth regulators. Dexamethasone in the presence of EGF and glucagon inhibited the initiation of DNA synthesis and mitosis, but it had no effect on EGF-insulin stimulated cultures. Such a differential effect of dexamethasone was observed at concentrations ranging from 4 nM to 200 microM. alpha-Fetoprotein, albumin, and tyrosine aminotransferase were used as typical markers of hepatocyte differentiation status. Irrespective of the combinations of growth-promoting factors used, dexamethasone inhibited alpha 1-fetoprotein production and maintained albumin production and tyrosine aminotransferase inducibility. In contrast, dimethylsulfoxide at 2% inhibited hepatocyte growth and supported the maintenance of the production of both alpha 1-fetoprotein and albumin, independent of the hormonal growth regulators used. On this basis, dexamethasone and dimethylsulfoxide act as distinct modulators of growth and maturation of cultured differentiating suckling rat hepatocytes. 相似文献
9.
Regulation of plasma fibronectin biosynthesis by glucocorticoids in chick hepatocyte cultures 总被引:2,自引:0,他引:2
D Nimmer G Bergtrom H Hirano D L Amrani 《The Journal of biological chemistry》1987,262(21):10369-10375
10.
Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones 总被引:5,自引:0,他引:5
Carmen de Juan Manuel Benito Alberto Alvarez Isabel Fabregat 《Experimental cell research》1992,202(2):495-500
Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones. 相似文献
11.
Sustained production of plasma proteins, notably albumin, is a reliable indicator of the differentiated state of hepatocytes. In this work, we have developed a fetal hepatocyte culture system where studying the regulation of albumin expression in proliferating liver cells. Our results show that under proliferative conditions (i.e., in the presence of EGF) fetal hepatocytes maintain albumin production above control quiescent non-treated cells. Glucagon and noradrenaline have no effect on the proliferation induced by EGF in cultured fetal hepatocytes; however, they act synergistically with the growth factor, increasing intracellular albumin levels. The maximum response is obtained by treatment of cells with EGF and noradrenaline. The stimulatory noradrenergic effect is mimicked by agents that increase cyclic AMP levels (forskolin plus IBMX). However, vasopressin or phorbol esters have no effect on albumin production, neither alone nor in combination with EGF. Dexamethasone, which does not alter the proliferative induction of EGF, increases albumin content. This effect is independent of the proliferative status of the cells and is not enhanced by glucagon, noradrenaline, or cyclic AMP increasing agents. The hormonal changes observed in albumin production partially correlate with changes in mRNA levels. This is the first time that cyclic AMP increasing agents are shown to act synergistically with EGF, increasing the expression of this liver specific gene. 相似文献
12.
Neil C. Talbot Vernon G. Pursel Caird E. Rexroad Jr. Thomas J. Caperna Anne M. Powell Roger T. Stone 《In vitro cellular & developmental biology. Animal》1994,30(12):851-858
Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary
cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver
cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small
biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing
gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and
β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder
cell co-culture may be useful for the sustainable culture of hepatocytes from other species. 相似文献
13.
Regulation of albumin gene expression in a series of rat hepatocyte cell lines immortalized by simian virus 40 and maintained in chemically defined medium. 总被引:18,自引:5,他引:13
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A series of simian virus 40 (SV40)-immortalized hepatocyte cell lines were characterized for albumin production, the regulation of albumin production, and the expression of other liver-specific genes. This series of cell lines is particularly useful for studying the regulation of hepatocyte gene expression because the cell lines express liverlike levels of a number of liver-specific functions and do so while growing in a chemically defined medium. SV40-immortalized hepatocyte cell lines were derived from colonies of albumin-producing epithelial cells that arose after primary hepatocytes maintained in chemically defined medium were transfected with SV40 DNA. Some cell lines secreted albumin at levels equal to or greater than those secreted by freshly plated primary hepatocytes, and all but one line continued to produce albumin for more than 20 passages. The variation in albumin secretion among cell lines reflected differences in the amount of albumin produced per cell and not in the percentage of albumin-producing cells in each line. The characterization of selected cell lines showed that albumin production was regulated by cell density during the growth cycle. Albumin production in most cell lines was also regulated by dexamethasone; however, one cell line continued to produce high levels of albumin when the cells were grown in medium lacking dexamethasone, demonstrating that although glucocorticoid can induce albumin production in some cell lines, it is not required for high levels of albumin production by all cells in culture. Regulation of albumin production measured at the level of protein secretion was paralleled by changes in steady-state levels of a 2.3-kilobase albumin RNA. Albumin-producing SV40-immortalized hepatocytes secreted a variety of other plasma proteins, including transferrin, hemopexin, and the third component of complement. These cells also expressed tyrosine aminotransferase activity that was inducible by dexamethasone. Alpha-fetoprotein production was not detected in any of the cell lines examined. 相似文献
14.
The effect of dexamethasone on albumin production by fetal rat hepatocytes in culture 总被引:2,自引:0,他引:2
G C Yeoh V J Brighton D A Angus M Kraemer J Vassy M T Chalemeau 《European journal of cell biology》1985,38(1):157-164
Hepatocytes derived from 15 and 19-day gestation rats synthesize and secrete albumin during culture. Albumin secretion is maintained when the culture medium is supplemented with dexamethasone but declines in its absence. The fall in secretion rate correlates with the level of albumin messenger RNA in the respective cultures. Even when dexamethasone is present, the level of albumin production in 19-day gestation hepatocytes is 6 to 7 times greater than that observed in hepatocytes derived from 15-day gestation rats. Immunocytochemical studies were undertaken to establish whether the difference in secretion rate was due to a difference in the amount of albumin produced by all the hepatocytes of the respective cultures or whether there were fewer hepatocytes which were capable of synthesizing albumin in the less mature liver. The results indicate that albumin production is reduced in all hepatocytes when cultured in the absence of dexamethasone. 相似文献
15.
Effect of hepatocyte-stimulating factor and glucocorticoids on plasma fibronectin levels. 总被引:6,自引:1,他引:5
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We evaluated the effects of hepatocyte-stimulating factor (HSF) and a glucocorticoid (dexamethasone) on changes in the levels, in vivo and in vitro, of plasma fibronectin (Fn), a glycoprotein that is synthesized and secreted by hepatocytes. In turpentine-treated chickens, plasma levels of Fn, which peaked at 48 h (whereas fibrinogen levels were maximum at 72 h) rose 200-250% over basal levels, whereas albumin levels decreased by 20-40%. Corticosterone levels in serum samples taken between 5 and 48 h after injection revealed a 124% increase in hormone levels at 24 h in turpentine-treated chickens. We also showed that circulating HSF levels were maximal 8 to 12 h after injection and that HSF activity, as assessed by molecular-exclusion chromatography, was eluted in the 30-45 kDa range. Addition of either serum-derived HSF or dexamethasone (2 nM) to chick hepatocyte cultures resulted in a 130-150% increase in secreted Fn as well as in fibrinogen. When HSF and dexamethasone were added together, a 360-489% increase in the secreted levels of both proteins was found. Chicken mononuclear phagocytic cells treated with lipopolysaccharide secreted an HSF activity that was eluted in two peaks, a minor peak at approximately 70 kDa and a major peak in the 25-40 kDa range. Addition of mononuclear-cell-derived HSF resulted in a greater increase in Fn levels than did the addition of serum HSF. These findings indicate that Fn, like fibrinogen, is an acute-phase protein, the production of which, at least in chickens, is stimulated by HSF and glucocorticoids in an additive manner. 相似文献
16.
Studies of fibronectin synthesized by cultured chick hepatocytes 总被引:3,自引:0,他引:3
We have adapted a chick embryo liver cell system for studying the synthesis of proteins secreted by hepatocytes. In primary liver cell cultures maintained for several days in arginine-deficient medium containing ornithine (0.7 mM) and carbamyl phosphate (1 mM), only hepatocytes demonstrated normal morphological and biosynthetic characteristics, indicating that they possessed a functional ornithine cycle as a source of arginine production. Non-parenchymal liver cells, such as fibroblasts, which lack the ornithine cycle were excluded. Hepatocytes in arginine-deficient or arginine-containing medium synthesized fibronectin (Fn) over several days at a constant rate of 3 micrograms +/- 1 microgram/mg cell protein per day, with fibronectin representing approximately 3% of the total secreted hepatocyte proteins during any culture period after the first 24 h. Pulse-chase experiments indicated that Fn synthesis and secretion was relatively rapid (t1/2 = 45 min) and represented approximately 95% of the intracellularly labelled Fn. This Fn is secreted predominantly as a 450 kD dimer with a subunit size that is indistinguishable from the plasma form as assessed by one-dimensional electrophoretic analysis. Continuous exposure of hepatocytes to insulin caused a moderate decrease (26%) in Fn synthesis, whereas there was no effect of short-term exposure. In contrast, dexamethasone stimulated Fn production 2-3-fold, consistent with its known ability to stimulate hepatocyte production of acute phase proteins. Under these conditions, electrophoretic analyses showed that an increased quantity of intact hepatocyte Fn was produced having the same molecular size of plasma Fn. 相似文献
17.
18.
Han J. Moshage Hans J.W. de Haard Hans M.G. Princen Sing Hiem Yap 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1985,824(1):27-33
Corticosteroids are known to stimulate the synthesis of a number of liver-specific proteins. The reports regarding the effect of glucocorticoid on albumin synthesis in vivo and in vitro are controversial. In an attempt to determine the mechanism by which glucocorticoid exerts its influence on hepatic albumin synthesis and to find an explanation for the conflicting data, we have studied the effect of dexamethasone disodium phosphate on albumin synthesis and albumin messenger RNA as determined by the molecular hybridization technique in hepatocytes in rat in vivo and in suspension culture. In hepatocyte suspension culture, addition of 0.48 μM dexamethasone in medium at zero time led to a significant increase (20%) in incorporation of labeled precursor into albumin as compared to control experiments; this was accompanied by a maintainance of the initial level of full-length albumin mRNA for a 9 h period. In hepatocytes cultured without dexamethasone in the medium there was a progressive loss of albumin mRNA content. Despite this finding, dexamethasone was not able to increase the albumin mRNA content in hepatocyte to a level higher than the initial value. Moreover, administration of this hormone either intraperitoneally or intravenously into rats did not lead to enhanced cell-free albumin synthesis or to an increased level of albumin mRNA. These findings suggest that glucocorticoid does not play an essential role in the regulation of albumin synthesis in vivo. In vitro, however, glucocorticoid leads to a preservation of the initial level of albumin mRNA and thus plays a role in the control of spontaneous dedifferentiation of liver cells in culture. 相似文献
19.
Glucocorticosteroids and cyclic AMP induce carbamoylphosphate synthetase (ammonia) (CPS) in rat hepatocytes. Using an enzyme immunoassay applied to hepatocyte cultures fixed in situ, it has been demonstrated that the capacity of hepatocytes to synthesize CPS in the presence of both hormones is present as soon as the cells become recognizable as hepatocytes. Immunochemical staining of the cultures shows that hepatocytes do not acquire or express the capacity to accumulate CPS at high rates synchronously. The average levels of CPS per hepatocyte that are observed upon hormone treatment are approx 50-fold lower in embryonic than in adult hepatocytes, corresponding with an approx 10-fold lower synthetic capacity (per gram hepatocytes) and an approx 5-fold smaller size of embryonic compared to adult hepatocytes. Carbamoylphosphate synthetase levels are therefore a good parameter in studies that aim to establish the mechanisms that underly the ontogenesis of the hepatic phenotype. 相似文献
20.
Proliferation of fetal rat hepatocytes in response to growth factors and hormones in primary culture 总被引:4,自引:0,他引:4
Fetal rat hepatocytes (day 19 of gestation) multiply in primary culture in arginine-free, hydrocortisone-containing chemically defined medium MX-82 supplemented either with epidermal growth factor (EGF) or insulin or both. In contrast, hepatocytes did not multiply under similar culture conditions using Dulbecco's minimum essential medium (DMEM). Cells underwent two divisions within 10 days in cultures maintained in MX-82 medium without a medium change, and cells grew to increased final cell densities when the medium was renewed every third day. When the medium MX-82 was enriched by the addition of lipids, intermediary metabolites, and trace metals (medium MX-83), cells grew to higher densities. In the absence of the growth factors, cells became quiescent and subsequently could be induced to synthesize DNA in response to EGF. With the increasing numbers of cells per dish, the growth response of the hepatocytes diminished. Levels of hepatocyte-specific albumin and alpha-fetoprotein mRNAs at day 0 were similar to those observed at day 10 in primary fetal rat hepatocyte cultures and were maintained at higher levels in medium MX-83 than in medium MX-82. 相似文献