共查询到20条相似文献,搜索用时 15 毫秒
1.
Characteristics of taurine transport in rat hepatocytes maintained in primary culture for 24 h (cultured hepatocytes) have been investigated. The uptake of [3H] taurine by cultured hepatocytes at 2 degrees C was unsaturable, whereas that at 37 degrees C consisted of unsaturable and saturable processes. The saturable transport system was sodium-dependent and consisted of two processes with low and with high affinities. The latter process (Km, 76.9 microM; Vmax, 0.256 nmole/mg protein/min; activation energy (EA), 37.8 kcal mol-1) was competitively inhibited by 2,4-dinitrophenol and ouabain, as well as by taurine analogues such as hypotaurine and guanidinoethyl sulphonate. The Vmax and EA values found in cultured hepatocytes at 37 degrees C were 6.0 and 6.8 times higher than those found in freshly isolated hepatocytes. These results indicate that taurine transport in hepatocytes in primary culture consisted of unsaturable, and saturable, sodium and energy-dependent carrier-mediated transport processes, respectively. The facilitation of the latter transport system by primary culture of hepatocytes is also suggested. 相似文献
2.
James E. Klaunig Randall J. Ruch Peter J. Goldblatt 《In vitro cellular & developmental biology. Plant》1985,21(4):221-228
Summary Rainbow trout (Salmo gairdneri) hepatocytes were isolated using a two-step perfusion through the portal vein. A typical perfusion yielded 2.92×106 liver cells with a mean viability of 96.3%. Hepatocytes comprised 93.4% of the total cell isolate. Survival of hepatocytes
in suspension culture was dependent on fetal bovine serum concentration and temperature of incubation. Serum concentrations
of 5, 10, and 20% produced the highest survival during primary culture. Hepatocyte survival was in inverse proportion to the
incubation temperature. Trout hepatocyte DNA synthesis and mitosis decreased during the culture period. Cytochromep
450 activity decreased rapidly during the first 2 d of culture and then remained low but measurable during the remaining 8 d
of culture. Culture temperature also influenced thep
450 activity with lower temperatures producing greater activity. Morphologic changes occurred in the cells during culture. Isolated
hepatocytes self-aggregated, forming strands and clumps that increased in size with time in culture. Junctional complexes
between cells were evident within the aggregates. Nuclear atypia, increases in size and number of autophagic vacuoles, and
the appearance of bundles of intermediate filaments also were observed with increased time in culture.
This work was supported in part by an American Cancer Society Grant (Ohio Division, Inc.) and an NIH Biomedical Research Support
Grant 5507RR05700010. 相似文献
3.
Terusama Hatahara Jerome M. Seyer 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,716(3):431-438
Hepatocytes were obtained from rat liver and maintained in primary culture for periods up to 14 days. Collagen synthesis was maximal after 3–5 days and declined thereafter. The rate of collagen production was appox. one-tenth that observed by the rat skin fibroblasts of the same animals after 3–5 passages. Type I procollagen, the major macromolecular collagenous species, was identified as a 450 000 dalton molecule which was converted to 120 000 dalton, denatured, reduced procollagen chains. Prior pepsin digestion of the native procollagen released 95 000 dalton collagen chains identified as α1(I) and α2(I) by co-migration with carrier rat skin type I collagen chains. The production of type III procollagen was also tentatively identified by DEAE-cellulose chromatography. This material was isolated and identified with type-specific antibodies developed against the amino-terminal extension peptide of bovine skin type III procollagen. The relative distribution of type I:type III procollagen was estimated at 7:3 similar to the ratio previously found in whole rat liver. No evidence of type IV or type V procollagen biosynthesis was observed. These results suggest that rat hepatocytes in primary culture are capable of interstitial type I and type III collagen biosynthesis in a ratio similar to that found in their parent hepatic tissue in situ. They also suggest that the less abundant type IV (basement membrane-associated) or type V are nor major collagenous products of these cells. 相似文献
4.
Isolation and culture of adult hepatocytes from liver biopsies 总被引:4,自引:0,他引:4
Summary Hepatocytes were isolated from liver biopsies of rats, guinea pigs, rabbits, dogs, and humans. The procedure is based on cannulation
of large veins in the cut face of the biopsy, followed by collagenase perfusion. Yields averaged 19×106 viable hepatocytes/g liver. Viability averaged 84%, as determined by trypan blue dye exclusion. Cultures were prepared from
the isolated hepatocytes and were found to be comparable in morphology andn-demethylase activity to hepatocyte cultures prepared by the in situ perfusion of the liver. The development of this method
should facilitate comparative studies of the cytotoxicity, genotoxicity, and metabolism of foreign chemicals in primary hepatocyte
cultures.
These studies were supported by Grant 5-ROI-ES01597-02 from the National Institute of Environmental Health Sciences and Regional
Research Project CA-D*-ETX-3634-RR(NE115). Dog liver biopsies were provided by Dr. W. Spangler at the Laboratory for Energy-Related Health Research.
Unused parts of human liver biopsies were provided by Drs. N. Pimstone and B. Ruebner at the Sacramento Medical Center. 相似文献
5.
Kohji Miyazaki M.D. Ryosaburo Takaki Fumio Nakayama Shoichiro Yamauchi Akitoshi Koga Satoru Todo 《Cell and tissue research》1981,218(1):13-21
Summary Biopsy tissue of adult human liver was gently dissociated with collagenase followed by Dispase. By repeated low g centrifugation, a large number of almost pure, viable hepatocytes was obtained. This is the first report of a successful procedure for obtaining adult human hepatocytes for study in tissue culture. The isolated cells have the typical morphology of liver parenchyma, and these characteristics persist throughout the period of culturing. Evidence of their function is indicated by albumin synthesis. This procedure is now being used to study human hepatocyte functions in vitro and the effects of a variety of agents including carcinogens and viruses. 相似文献
6.
Cell density dependent morphological changes in adult rat hepatocytes during primary culture 总被引:2,自引:0,他引:2
T Koji P K Nakane M Murakoshi K Watanabe H Terayama 《Cell biochemistry and function》1988,6(4):237-243
In order to gain morphological insights about the cell density dependency, hepatocytes cultured at a low cell density (less than about 0.1 X 10(5) nuclei (cm2)-1) and at a high cell density (greater than about 1 X 10(5) nuclei (cm2)-1) were examined ultrastructurally 24 h after plating (just prior to the beginning of DNA synthesis). The results were as follows: (i) glycogen rosettes disappeared completely in low density culture as compared with sections from an intact liver. In contrast, glycogen rosettes were still present in high density culture. (ii) Polysomes seemed increased in low density culture in comparison with those seen in sections from an intact liver and from the high density culture. (iii) In low density culture, the shape of mitochondria deviated from that of hepatocytes in an intact liver and the mitochondria often lost a characteristic close contact with rough endoplasmic reticulum (rough ER). (iv) In low density culture, bundles of filamentous structure were detected, which were not found in an intact liver or high density culture. The following features were found only in high density culture; (v) numerous villous cytoplasmic protrusions developed along the area facing adjacent cells, and seemed to intertwine with each other, and (vi) between the hepatocytes, only abortive junctions were found. These results indicate that the hepatocytes cultured at a low density express most of the characteristics of the hepatocytes in a regenerating liver and the features of the cells cultured at a high density are very similar to those of the hepatocytes in sections from an intact liver. 相似文献
7.
Changes in the expression of cell adhesion molecule and albumin genes were investigated in primary cultures of rat hepatocytes with and without poly- N-p -vinylbenzyl-D-lactonamide (PVLA) coating of the dishes. In PVLA-coated cultures, hepatocytes aggregated into spheroids and expressed liver cadherin and albumin mRNAs at higher levels. In uncoated cultures, hepatocytes revealed low levels of cadherin and albumin mRNAs, but higher levels of integrin alpha-1 mRNA. The changes in mRNA levels of liver cadherin and integrin alpha-1 coordinated well with those in spheroid and monolayer formation of hepatocytes, respectively. These results suggest that, in the PVLA-coated culture, hepatocytes expressed cadherin at higher levels to promote cell-cell adhesion and further maintain the differentiated function, such as albumin secretion, for prolonged times. 相似文献
8.
Hans Hirsiger Urs Giger Urs A. Meyer 《In vitro cellular & developmental biology. Plant》1984,20(3):172-182
Summary Monolayer cultures were prepared from hepatocytes of 15 d chick embryos and maintained at high cell density in a chemically
defined medium. In the absence of growth stimulatory conditions DNA synthesis was observed only during the first 10 to 16
h of culture. Thus, after a 12 h exposure to [3H]thymidine ([3H]dThd, 4 to 16 h) 9.1±1% (
,n=4) of the hepatocyte nuclei were labeled. Labeled mitotic nuclei, up to late telophase, were regularly observed in these
cultures. Beyond 16 h less than 2% labeled nuclei were found (12 h of [3H]dThd), which indicates that the hepatocytes entered proliferative quiescence. DNA synthesis of “resting” hepatocytes was
stimulated by insulin and, only slightly, by hydrocortisone, glucagon, or fetal bovine serum. Triiodothyronine (T3), or the nucleoside inosine (i) did not stimulate. Combination of insulin (I) with hydrocortisone (H), T3 (T), or glucagon (G) resulted in a more than additive effect. Nearly maximal stimulation occurred with the combinations IHT
and ITG. Labeling increased at 10 ng/ml of each component and was maximal at 1 to 10 μg/ml. A lag period of 8 to 10 h after
hormone administration (IHiTG, 10 μg/ml) was observed before nuclear labeling increased. Within the subsequent 10 h a considerable
proportion of the hepatocytes (up to 30% or more) entered DNA synthesis. Mitotic activity (with nuclei in prophase up to late
telophase) also was stimulated. An increase of both total DNA and protein content was measured in several experiments. Hormonal
stimulation of hepatocyte DNA synthesis and mitotic activity was associated with decreased β-naphthoflavone-mediated induction
of cytochrome P450. A causal relationship between these two phenomena remains to be established. It is suggested that chick
embryo hepatocyte cultures are a useful tool for studies on hepatocyte proliferation and differentiation.
The present study is based on original observations by Dr. F. R. Althaus (presently at the Institute of Pharmacology and Biochemistry,
University of Zürich, Switzerland). This contribution of his and his incisive criticism are acknowledged.
The study was supported by Grant 3.893.81 from the Swiss National Research Foundation. 相似文献
9.
草鱼肝细胞的分离与原代培养 总被引:1,自引:0,他引:1
目的以草鱼(Ctenopharyngodon idellus)肝细胞为实验对象,在不同条件下进行原代培养,以探讨适合草鱼肝细胞生长的最佳条件及培养方法,用于饲料营养与非营养物质对草鱼肝细胞代谢、损伤作用机制的研究。方法采用温胰蛋白酶消化法和红细胞裂解液分离、纯化肝细胞,MTT法测定细胞增殖率,并测定不同时期培养液上清液中LDH、Alb和BUN的含量,分析肝细胞生长状况。结果采用0.25%浓度的温胰蛋白酶消化法,消化20min,分步收集肝细胞,经台盼蓝染色检测和血球计数板计数,活细胞数≥99%。结论在含10%胎牛血清、10μg/mL胰岛素的M199培养基中,以接种浓度1.7×106cell/mL左右为宜,置于27℃、4.5%CO2浓度的恒温培养箱中,可成功培养草鱼原代肝细胞。 相似文献
10.
Roger G. Ulrich Danielle G. Aspar Clay T. Cramer Rolf F. Kletzien Leonard C. Ginsberg 《In vitro cellular & developmental biology. Plant》1990,26(8):815-823
Summary Isolation and culture techniques for hepatocytes from whole livers of the cynomolgus monkey,Macaca fascicularis, are described. Hepatocytes were isolated by two-step perfusion of livers, using collagenase with hyaluronidase; fructose
and trypsin inhibitor were included to reduce cell loss. Yields from a single liver average 4×109 cells with viabilities of 90.8±5.7%. Cells, plated on collagen substrates, were assessed for changes in morphology and various
marker enzyme activities over a period of 7 d in culture. Cells exhibited a morphology similar to that observed for this species
in vivo; little change in attached and spread cells was observed over the length of time monitored. Enzyme activities for
catalase, succinate dehydrogenase, and tyrosine aminotransferase were observed to decrease significantly (though considerable
activity remained), whereas acid phosphatase and 5′-nucleotide phosphodiesterase remained unchange. Activity of cytochrome
P-450 reductase was observed to increase slightly for the first 2 d, then decrease to about 60% of initial levels. Activity
of α-mannosidase was stable for 4 d but was observed to be increased at Day 7. Cells were observed to retain metabolic responsiveness
demonstrated by glucose production by both gluconeogenesis and glycogenolysis in response to glucagon stimulation. The monkey
hepatocytes obtained by methods described here thus retain hepatocellular morphology and activity through at least 1 wk in
culture without medium or culture modification. 相似文献
11.
Sung Mun Yang Doo Hoon Lee Jung Keug Park 《Biotechnology and Bioprocess Engineering》2000,5(2):99-105
Cell-cell interaction and the extracellular matrix (ECM) are believed to play essential roles duringin vitro culturing of primary hepatocytes in the control of differentiation and in the maintenance of tissue specific functions. The
objective of this study was to examine the effects of degree of cell-cell contact (DCC) on liver specific function of rat
primary hepatocytes. Hepatocyte aggregates with various degrees of cell-cell contact,i.e., dispersed cells, longish aggregate, rugged aggregate, and smooth spheroid were obtained at 1, 5–6, 15–20, and 36–48 hrs,
respectively in suspension cultures grown in spinner flasks embedded in Caalginate bead and collagen gel in order. The smooth
spheroids displayed a decrease in viability and functional activities. This may result from mass transfer limitation and shear
damage caused by agitation during aggregation. The rugged aggregate showed a higher viability and albumin secretion rate than
the dispersed cells or the other aggregates. This result indicates the possible enhancement of a bioartificial liver's (BAL)
performance using primary hepatocytes and the reduction in time to prepare a BAL through optimization of the immobilization
time. 相似文献
12.
Jian Zhong Tong Sophie Sarrazin Doris Cassio Frdric Gauthier Fernando Alvarez 《Biology of the cell / under the auspices of the European Cell Biology Organization》1994,81(1):77-81
Summary— Human hepatocytes cultured with a hormonally defined medium on non-adherent poly-(2-hydroxyethyl methacrylate) coated surface were able to form spheroids. The maintenance of liver-specific functions was assessed by following secretion of albumin, transferrin and α-antitrypsin that were still detectable after 4 months of spheroidal culture. Moreover, cytochrome P-450 IA was induced by methylcholanthrene for up to 2 weeks. This cell system is very promising for long-term in vitro studies of human hepatocyte functions. 相似文献
13.
Brian A. Laishes Gary M. Williams 《In vitro cellular & developmental biology. Plant》1976,12(7):521-532
Summary The conditions for obtaining representative, primary adult rat hepatocyte cultures were explored. The methods applied included
enzymatic liver perfusion which was nondestructive to hepatocytes, the prevention of aggregation of dissociated cells and
the selective attachment of viable cells. These procedures yielded a recovery of 50% of the liver cells which gave rise to
cultures representing 14% of the total liver cells. The cultures were composed of homogeneous epithelial-like cells cytologically
similar to hepatocytes and possessed a number of liver-specific enzymes. There was virtually no cell division initially and
most cells died between 24 and 48 hr. Insulin enhanced the attachment of the liver cells, altered their morphology, but did
not prolong cell survival.
This study was supported by grant no. BC 133 from the American Cancer Society. 相似文献
14.
We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine. 相似文献
15.
Adult rat hepatocytes were cultured for 15 days on type I collagen-coated permeable membranes in a hormonally defined Waxman's modified medium supplemented with very low concentrations of insulin, glucagon and dexamethasone. Phase contrast examination showed that 15-day-old cultures still formed a regular monolayer of polygonal cells. In similarly aged cultures, intracellular glycogen was abundant and evenly distributed, while steatosis remained very limited. Scanning and transmission electron microscopy showed that well developed bile canaliculi could be observed on the lateral side of the hepatocyte membrane after 4 days of incubation and persisted for 2 weeks. These canalicular structures probably originated from coalescence of membrane invaginations observed in 1-day-old cultures. Transmission electron microscopy showed that the ultrastructure of the cells was very close to that of normal rat hepatocytes in the intact liver. These results suggest that rat hepatocytes cultured under these experimental conditions are able to develop and maintain tissue-specific cytochemical and morphological properties for at least 15 days. 相似文献
16.
Influence of medium composition and culture conditions on glutathione S-transferase activity in adult rat hepatocytes during culture 总被引:2,自引:0,他引:2
Y. Vandenberghe D. Ratanasavanh D. Glaise A. Guillouzo 《In vitro cellular & developmental biology. Plant》1988,24(4):281-288
Summary Glutathione S-transferase (GST) activity was measured in adult rat hepatocytes during either pure culture or coculture with
another rat liver cell type in various media. Addition of nicotinamide, selenium, or dimethylsulfoxide, deprivation of cyst(e)ie
and the use of two complex media were tested. Whatever the conditions used, after a constant decrease during the first 24
h, GST remained active over the whole culture period (1–2 wk). However, various patterns were observed: GST activity either
remained relatively stable to approximately 50% of the initial value or showed a moderate or strong increase. The highest
values were found in pure hepatocyte cultures maintained in the presence of nicotinamide or dimethylsulfoxide. Similar changes
were observed using 1-chloro-2,4-dinitrobenzene or 1,2-dichloro-4-nitrobenzene as substrates for GST. Addition of 10−4
M indomethacin resulted in 37 to 60% inhibition of enzyme activity. Thus, these results demonstrate that GST remained expressed
during culture but its levels markedly varied depending on the medium composition and type and age of culture.
Y. V. was supported by Instituut voor Wetenschappel?k Onderzoek in Landbouw en Nijverheid. This work was supported by INSERM. 相似文献
17.
Formation of cylindrical multicellular aggregate (cylindroid) and expression of liver specific functions of primary rat hepatocytes 总被引:2,自引:0,他引:2
Hiroshi Mizumoto Masashi Hayakami Kohji Nakazawa Hiroyuki Ijima Kazumori Funatsu 《Cytotechnology》1999,31(1-2):69-75
In our studies of the development of a hybrid artificial liver, we investigated the formation of cylindrical multicellular
aggregate (cylindroid) of primary rat hepatocytes on a pressed sheet of polyurethane foam (pressed–PUF) as a culture substratum.
Hepatocytes formed cylindroids by attaching to a pressed–PUF surface, peeling off from the surface and aggregating. The diameter
and length of most cylindroids were approximately 200–500 μm and 500 μm–2 mm, respectively. The activities of liver specific
functions (albumin secretion and ammonia metabolism) of hepatocyte cylindroids were equivalent to or higher than those of
hepatocyte spheroids. These results suggest that hepatocyte cylindroids can maintain highly differentiated functions longer
than hepatocyte spheroids, and that a PUF/cylindroid culture may be effective to develop of a hybrid artificial liver.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
18.
Brian A. Laishes Gary M. Williams 《In vitro cellular & developmental biology. Plant》1976,12(12):821-832
Summary Primary monolayer cell cultures of adult rat hepatocytes underwent change in morphology and substantial cell loss between
1 and 3 days postinoculation. Dexamethasone-supplementation (1μM) of the culture medium maintained the polygonal epithelial morphology of the hepatocytes and increased longevity such that
over 80% of the cells survived for 3 days and at least 30% for 8 or 9 days. This enhancement of survival was obtained up to
48 hr postinoculation, but the earlier the time of dexamethasone supplementation the greater the effect. Removal of dexamethasone
resulted in a decrease in longevity. The positive effect of dexamethasone on longevity was observed following dexamethasone
replacement of insulin in supplemented cultures, but the combination of insulin and dexamethasone resulted in poorer survival
than with dexamethasone alone. The results are interpreted to indicate that dexamethasone provided a requirement of the in
vitro environment for survival and suggest that elaboration of a complex medium is required to maintain hepatocytes in culture.
This study was supported by an Alexander Ralston Peacock Memorial Grant for Cancer Research (No. BC-133A) from the American
Cancer Society. 相似文献
19.
Vernon E. Steele Julia T. Arnold 《In vitro cellular & developmental biology. Plant》1985,21(12):681-687
Summary Nasal turbinate epithelial cells were isolated from rats, rabbits, and humans using either a surgical or an in situ enzyme
incubation technique. The culture conditions that permit optimal cell attachment and selective growth of the nasal epithelial
cells were determined. These conditions will permit the long-term culture of these cells where typically 20 to 30 population
doublings were observed. Differences between rat and human nasal epithelial cells were seen in substrate requirements, colony-forming
efficiency, and response to fetal bovine serum and bovine serum albumin. These methodology and results will permit mechanistic
studies of normal and abnormal cellular function and comparative response studies between nasal epithelial cells from rats
and humans.
This work was supported under U.S. Environmental Protection Agency contract 68-02-4032. 相似文献
20.
Yumi Kono Suyun Yang Eve A. Roberts 《In vitro cellular & developmental biology. Animal》1997,33(6):467-472
Summary To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of
collagen gel, called a “collagen gel sandwich.” Maintenance of hepatocellular functions in this system was compared with that
of identical hepatocyte preparations cultured on dry-collagen coated dishes or co-cultured with rat liver epithelial cells.
Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period
of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin
secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion
could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer
of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture.
These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes
in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves
differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system
is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro. 相似文献