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1.
Hiroyoshi Hoshi Mikio Kan Wallace L. McKeehan 《In vitro cellular & developmental biology. Plant》1987,23(10):723-732
Summary Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on
human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase
mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented
medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth
factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed
that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported
attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined
as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically
stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte
number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could
be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned
medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein
(1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The
results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification
and characterization of additional normal hepatocyte growth factors.
This work was supported by NIH grant DK35310.
Editor’s statement Many investigators have struggled with the special problems associated with culture of differentiated hepatocytes.
In this paper attention is given to the specific growth factor requirements for fetal human hepatocytes. The observation that
factors from hepatoma conditioned medium or neural extracts enhanced the growth of the cells may indicate that additional
growth factors are to be identified that are important in the survival and proliferation of hepatocytes, and may also indicate
that the malignant transformation of these cells may involve the production of autocrine growth stimulators. 相似文献
2.
The content of reduced glutathione and of glutathione disulfide as well as the activities of glutathione reductase, glutathione peroxidase, glutathione S-transferases, catalase and superoxide dismutases were determined in human hepatoma Hep 3B cells in relation to free-radical toxicity in order to appreciate the defense capacities of these cells compared to data on normal hepatocytes. When Hep 3B cells were exposed to lindane, a known inducer of free-radical production, superoxide dismutase activity appeared as the best-adapted cellular parameter for early detection of the resulting free-radical toxicity.Abbreviations AAS
atomic absorption spectrometry
- CDNB
1-chloro-2,4-dinitrobenzene
- DMEM
Dulbecco's modified Eagle medium
- GPx
glutathione peroxidase
- G.Red
glutathione reductase
- GSH
reduced glutathione
- GSSG
glutathione disulfide
- GST
glutathione S-transferases
- Prot
proteins
- SOD
superoxide dismutase 相似文献
3.
Anne L. Holleran Donald A. Briscoe Gary Fiskum Joanne K. Kelleher 《Molecular and cellular biochemistry》1995,152(2):95-101
A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of14C-glutamine to14CO2 and of14C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle -ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label -ketoglutarate carbon 5, [2-14C]glucose, [1-14C]acetate and [5-14C]glutamine. The analysis indicated that the probability of flux of TCA cycle -ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of -ketoglutarate through -ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [14C]glutamine to14CO2, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through -ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of -ketoglutarate was demonstrated by measuring incorporation of [5-14C]glutamine into14C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells. 相似文献
4.
Vincent S. Wu Craig V. Byus 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1984,804(1):89-99
We investigated the ability of intracellular ornithine to alter both the biosynthesis of putrescine and the activity of ornithine decarboxylase in Reuber H35 hepatoma cells in culture incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA). In confluent cultures of H35 cells, the addition of TPA (1.6 μM) caused the activity of ornithine decarboxylase to increase by more than 100-fold within 4 h. When exogenous ornithine (0.1–1.0 mM) was added to the culture medium with TPA, a marked dose-dependent increase in the production of putrescine was observed. The activity of ornithine decarboxylase in the same cultures incubated with ornithine decreased in a similar dose-dependent manner. The addition of arginine (0.1–1.0 mM) (but not lysine or histidine) to the H35 cells in culture concomitant with TPA also led to a relative increase in putrescine biosynthesis and a decrease in ornithine decarboxylase activity compared to cultures not receiving the amino acids. A similar response to exogenous ornithine and TPA was observed in a series of less confluent rapidly growing cultures which were in culture for a shorter period of time. The confluent cultures possessed a basal level of arginase (55 units/mg protein) which increased approx. 2-fold upon treatment with TPA. The intracellular concentration of ornithine in the unstimulated cells was in the order of 0.02–0.03 mM. Upon incubation of the cells with exogenous ornithine or arginine, the intracellular pools of these amino acids increased 4- to 8-fold. 相似文献
5.
We have previously identified an Msp I site at the 5′ end of the rat albumin gene whose undermethylation is necessary but not sufficient for stable albumin expression in rat hepatoma cells [1]. We have also shown that the extinction of albumin expression in somatic hybrids is not the result of methylation at this site, since for two different crosses, rapid extinction was found to occur in the absence of any de novo methylation of the previously active gene[2]. In the present study, we examine albumin expression and albumin gene methylation for independent hybrid clones isolated from crosses between albumin expressing rat hepatoma cells and cells of two different non-expressing lines. The cells from hybrid clones of both crosses are characterized by stable extinction of albumin expression. Moreover, we find that de novo methylation of the “extinguished” albumin gene can occur in somatic hybrids, but only some weeks after the gene has ceased to be expressed. 相似文献
6.
Roeland van Wijk Lydie Tichonicky Jaques Kruh 《In vitro cellular & developmental biology. Plant》1981,17(10):859-862
Summary Exposure of HTC cells to sodium butyrate caused inhibition of growth. The site of growth inhibition was studied by time-lapse
cinematography and [3H]thymidine incorporation studies. Evidence is presented that sodiunm butyrate affected the cell cycle at a specific point
immediately after mitosis. Inasmuch as it does not modify the interphase duration after its removal, butyrate may be used
for HTC synchronization.
This work was supported by l'Institut Nationale de la Santé et de la Recherche Médicale and la Centre Nationale de la Recherche
Scientifique (L. T. and J. K.). 相似文献
7.
8.
Akira Niwa Katsuhiko Yamamoto Kenji Sorimachi Yosihiro Yasumura 《In vitro cellular & developmental biology. Plant》1980,16(11):987-993
Summary The rat hepatoma cell line, H4-II-E, was grown serially over a I-year period and about 30 passages in arginine-, glutamine-,
and tyrosine-deprived and ornithine-supplemented Eagle's mininum essential medium with no supplements other than biotin. The
adapted cel line, R-Y121B, proliferates in the above mentioned medium with a doubling time of about 4 days and maintains hepatic
“marker” enzymes such as tyrosine aminotransferase, phenylalanine hydroxylase, and all the enzymes of the urea cycle.
This work was supported in part by Grant-in-Aid for Cancer Research 301050 and Science Research Grant 337013 from the Ministry
of Education, Science and Culture, Japan. 相似文献
9.
Rat hepatoma cells accumulate considerably less 2-aminoisobutyrate after cultivating in the absence of serum the change in rate of aminoisobutyrate uptake takes place within 1 h of serum starvation. Starvation of amino acids by contrast raises aminoisobutyrate uptake in the presence or absence of serum, but the cells are much less responsive to amino acid supply than to availability of serum. Phosphate (10 mM) reduced aminoisobutyrate uptake by cells grown in serum to that exhibited by serum-starved cells. Aminoisobutyrate uptake by cells grown in serum was reduced by glycine, proline, alanine, serine, glutamine, methylaminoisobutyrate and 2-aminonorbornane-2-carboxylate, the effects of methylaminoisobutyrate and 2-aminonorbornane-2-carboxylate being additive. However, similar inhibition phenomena were not seen for cells deprived of serum where aminoisobutyrate uptake tended to a relatively constant level insensitive to inhibitory influences, yet substantially greater than that arising by simple diffusion. The comparative insensitivity of our hepatoma line when starved of serum to competition and repression phenomena is in contrast to findings of others. Our results also suggest a lack of clear delineation of specificities for the A and L transport systems as usually defined. 相似文献
10.
Peter G.W. Plagemann Richard Marz Robert M. Wohlhueter Jon C. Graff John M. Zylka 《生物化学与生物物理学报:生物膜》1981,647(1):49-62
6-Mercaptopurine and 6-thioguanine strongly inhibited the zero-trans entry of hypoxanthine into Novikoff rat hepatoma cells which lacked hypoxanthine/guanine phosphoribosyltransferase, whereas 8-azaguanine had no significant effect. 6-Mercaptopurine was transported by the hypoxanthine carrier with about the same efficiency as its natural substrates (Michaelis-Menten constant = 372 ± 23 μM; maximum velocity = 30 ± 0.7 pmol/μl cell H2O per s). 8-Azaguanine entry into the cells, on the other hand, showed no sign of saturability and was not significantly affected by substrates of the hypoxanthine/guanine carrier. The rate of entry of 8-azaguanine at 10–100 μM amounted to only about 5% of that of hypoxanthine transport and was related to its lipid solubility in the same manner as observed for various substances whose permeation through the plasma membrane is believed to be non-mediated. Only the non-ionized form of 8-azaguanine (pKa = 6.6) permeated the cell membrane.Studies with wild type Novikoff cells showed that permeation into the cell was the main rate-determining step in the conversion of extracellular 8-azaguanine to intracellular aza-GTP and its incorporation into nucleic acids. In contrast, 6-mercaptopurine was rapidly transported into cells and phosphoribosylated; the main rate-determining step in its incorporation into nucleic acids was the further conversion of 6-mercaptopurine riboside 5'-monophosphate. 相似文献