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1.
Altered growth kinetics precede calcium-induced differentiation in mouse epidermal cells 总被引:1,自引:0,他引:1
K. Elgjo H. Hennings O. P. F. Clausen 《In vitro cellular & developmental biology. Plant》1986,22(6):332-336
Summary Primary cultures of newborn mouse epidermal cells proliferate rapidly and with a high growth fraction for several months when
grown in medium with low calcium (0.02 to 0.1 mM). Addition of calcium to levels generally used in culture medium (1.2 mM) was followed by rapid changes in the pattern of proliferation. By using a combination of technics (a stathmokinetic method,
autoradiography, [3H]thymidine incorporation into DNA, DNA flow cytometry) it was found that cell flux was blocked for 5 to 6 h, followed by
a short rise in the mitotic rate at 10 h, and a gradual fall in all growth parameters until about 32 h after the calcium switch.
There was no accumulation of cells in any particular cell cycle phase. The results indicate that the calcium switch is followed
by a strong reduction in cell flux from G1 whereas the majority of the cells that had left G1 at the time of the switch completed one cell division before cessation of all proliferative activity. Both before and after
the switch the primary epidermal cultures consisted of one diploid and one tetraploid G1 DNA stemline that seemed to react in the same way to calcium.
This work reported in this paper was undertaken during the tenure of an American Cancer Society-Eleanor Roosevelt-International
Cancer Fellowship awarded by the International Union Against Cancer (K. E.). The project was supported by funds partly provided
by the International Cancer Research Data Bank Program of the National Cancer Institute, National Institutes of Health, Bethesda,
MD, under contract N01-C0-65341 (International Cancer Research Technology Transfer) and partly by the International Union
Against Cancer (O.P.F.C.). 相似文献
2.
Carol L. Williams Vanda A. Lennon Mark R. Pittelkow 《In vitro cellular & developmental biology. Plant》1989,25(5):397-401
Summary A time-dependent redistribution of microfilaments was observed in cultured human keratinocytes using a human monoclonal autoantibody
specific for myosin. Immunofluorescent staining revealed that 5 days after plating keratinocytes in either 0.1 mM or 2.0 mM
Ca++, myosin was distributed uniformly throughout the cytoplasm. At day 6, parallel arrays of myosin-containing microfilaments
were prominent in the cell peripheries. At day 7 the microfilaments formed circumferential rings. The distribution of the
microfilaments was disrupted by cytochalasin but not by colchicine, indicating that this novel distribution of myosin was
not dependent on colchicine-sensitive vimentin intermediate filaments. The time-dependent redistribution of myosin was not
influenced by cell population density, cell shape or cell cycle phase, except for mitotic cells in which myosin was distributed
diffusely through the cytoplasm. If, as suggested by Kolega (9), microfilaments align parallel to the direction of applied
tension, the redistribution of myosin-containing microfilaments in cultured keratinocytes may reflect the increased tension
between cells resulting from increasing strength of cell-cell junctions over time. In sectioned human skin, myosin was localized
in the peripheral cytoplasm of stratified epidermal cells. Tensions arising from the numerous desmosomal junctions between
cellsin vivo could account for this distribution of myosin.
Supported by grant NS-23537 (V. A. L.) from the National Institutes of Health, Bethesda, MD, and by the Mayo Foundation. C.
L. W. is recipient of the Kermit E. Osserman and Blanche McClure Fellowship, 1987, National Myasthenia Gravis Foundation. 相似文献
3.
Margareta Lirvall Pia Ljungqvist-Höddelius Åke Wasteson Karl-Eric Magnùsson 《Bioscience reports》1996,16(3):227-238
Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2±0.2 × 10–10 cm2/s, and 1.8±0.2 × 10–10 cm2/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3±0.3 × 10–10 cm2/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1±0.8 × 10–10 cm2/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.Abbreviations CAT
Catalase
- D
Lateral diffusion coefficient
- EDTA
Ethylenediaminetetraacetic acid
- EGF
Epidermal growth factor
- E-MEM
Eagle's minimum essential medium
- FCS
Fetal calf serum
- FRAP
Fluorescence recovery after photobleaching
- KRG
Krebs-Ringer phosphate buffer
- PBS
Phosphate-buffered saline
- R
Mobile fraction
- ROS
Reactive oxygen species
- SEM
Standard error of the mean
- SOD
Superoxide dismutase
- UVA
Ultraviolet light-A (315-400 nm)
- UVB
Ultraviolet light-B (280-315 nm) 相似文献
4.
This study was implemented to test the Episkin model of reconstructed epidermis in the evaluation of the efficacy of cosmetic or dermopharmaceutical products on cutaneous energy metabolism. The energy metabolism is evaluated by measuring the concentration of intracellular ATP by a method using an ultrasensitive bioluminescent reaction. The work presented compares results obtained in reconstructed epithelium and monolayer primary cultures of human keratinocytes.After application of a hydrosoluble product, the increase in intracellular ATP is identical in a monolayer culture of keratinocytes (+239±18% versus control) and in Episkin (+248±21% versus control). An emulsion was also tested on the two models. It is only possible to test the emulsion at a dilution of under 0.05% on a keratinocyte culture, and this means that the real efficacy of the product is underestimated (+145±18% versus control). The three-dimensional model enables the application of the undiluted emulsion, and the results show an increase in intracellular ATP of +420±80% versus control: products in final formulation can be tested in normal conditions of use.Abbreviations BPE
bovine pituitary extract
- DMEM
Dulbecco's modified Eagle's medium
- EDTA
ethylene diamine tetraacetic acid
- EGF
epidermal growth factor
- K-SFM
keratinocytes serum free medium
- MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
- O/W
oil in water
- PBS
phosphate-buffered saline 相似文献
5.
Jeffery R. Cook Robert G. van Buskirk 《In vitro cellular & developmental biology. Animal》1996,32(5):300-306
Summary Laminin synthesis and deposition are concomitant with the development of a basal lamina between the human epidermis and the
underlying dermis. One of the challenges in tissue engineering of human epidermal models is to develop substrates and conditions
that encourage the development of a basement membrane. The purpose of this study was to determine if actin filaments and/or
microtubules are involved in the synthesis/secretion of laminin by normal human epidermal keratinocytes (NHEK)in vitro. NHEK synthesize and secrete laminin subunits B1, B2, and M but little, if any, of laminin subunit A. Data indicate that
disruption of microfilaments by the destabilizing agent, cytochalasin D, had no apparent effect on the relative synthesis
rates of most cytosolic proteins as, revealed by one-dimensional sodium dodecyl sulfate (SDS) gel electrophoresis. This drug,
however, increased laminin B2 synthesis several fold over untreated controls. This enhanced synthetic rate was independent
of the type of collagen, matrix on which the NHEK were grown. Similar increases in synthesis of the M and B1 laminin chains
were not observed. To determine if this increase in synthesis lead to increases in laminin B2 secretion, laminin B2 was immunoprecipitated
from both the apical and basal domains of NHEK cells grown on microporous membranes. While more laminin B1, B2, and M were
secreted basally than apically, an observation consistent with laminin’s role in basal lamina formation, cytochalasin D had
no apparent effect on either basal or apical laminin B2 secretion. Experiments with the microtubule destabilizer, nocodazole,
showed no similar effects on laminin synthesis and/or secretion. We conclude that (a) disruption of the actin network in NHEK
selectively increases the synthesis of laminin B2, (b) the secretion of laminin B2 from NHEK cells is not governed by either
the microfilamentous cytoskeleton or the amount of laminin synthesized by NHEK, and (c) disruption of the microtubular network
does not alter laminin synthesis or secretion. 相似文献
6.
In the present investigation we studied the metabolism of 1α,25-dihydroxy-[1β-3H] vitamin D3 (3H-1,25(OH)2D3) in culture-grown human keratinocytes (CHK). Our results showed that the cellular uptake of 3H-1,25(OH)2D3, upon incubation with CHK, occurred very rapidly; and it paralleled a decrease in the concentration of 3H-1,25(OH)2D3 in the medium. The amount of 3H-calcitroic acid, on the other hand, increased slowly in the medium, while the concentration of 3H-calcitroic acid in the cell remained undetectable during the whole period of incubation. When the cells were preincubated with 1,25(OH)2D3 (10?8M), conversion of 3H-1,25(OH)2D3 to 3H-calcitroic acid increased almost twofold, indicating that 1,25(OH)2D3 catalyzed its own catabolism. © 1995 Wiley-Liss, Inc. 相似文献
7.
J. Donald Weaver G. Stetten J. W. Littlefield 《In vitro cellular & developmental biology. Animal》1991,27(8):670-675
Summary During experiments concerning the introduction of oncogenes into normal human keratinocytes, we observed long-lived colonies
arising spontaneously at the same low frequency in control cultures as in those transfected with Ha-rasEJ or activated c-myc
or both. Two of these were karyotyped early in their life span and showed additional chromosomal material on the short arm
of chromosome 9 in one case and of chromosome 18 in the other, whereas the parental cells had a normal karyotype. This indicates
the presence of a partial trisomy in each line, although the origin of the extra chromosomal material is not known. A similarly
long-lived human keratinocyte line containing an isochromosome of the long arm of chromosome 8 has been described elsewhere.
Together these results suggest that the spontaneous occurrence of long-lived lines is more common in human keratinocytes than
in fibroblasts and that a triple dose of one or more genes may be the initial event in this process.
This work was supported by grant CA16754 from the National Cancer Institute, Bethesda, MD. 相似文献
8.
Niels C. Krejci Lynne Smith Rebecca Rudd Robert Langdon Joseph McGuire 《In vitro cellular & developmental biology. Animal》1991,27(12):933-938
Summary To investigate the regulation of epithelial differentiation, normal human epidermal keratinocytes were cultured floating on
the surface of culture medium without attachment to a solid substrate. Keratinocytes spread out on the surface of the medium,
proliferated and differentiated either into several flat lacy sheets 1 to 3 cells thick (on medium containing 0.15 mM calcium) or formed one single aggregate of cells from 5 to 15 cells in thickness on medium containing 1.15 mM calcium. The cell aggregates demonstrated a pattern of ordered epithelial differentiation. Levels of progressive differentiation
resembling the structure of normal human epidermis were identified by light microscopy, immunohistochemistry, and electron
microscopy. Differentiation proceeded from cells at the air side toward cells at the medium side with basal appearing cells
on the air side and keratinocytes expressing filaggrin and involucrin on the side toward the medium. These results demonstrate
that organized epithelial differentiation can occur in the absence of extracellular matrix. In contrast with other air-liquid
interface cultures, epithelial differentiation in the absence of extracellular matrix progresses from air towards medium. 相似文献
9.
Su-Chin C. Liu Mary J. Eaton Marvin A. Karasek 《In vitro cellular & developmental biology. Plant》1979,15(10):813-822
Summary Using gels of acid-soluble, collagen as a culture surface, trypsin-released keartinocytes from 0.1-mm, split-thickness sections
of newborn foreskin may be plated with high efficiency and subcultured at a 1∶5 split a 2- to 3-week intervals for three subpassages.
When plated at a density of 3.2×104 cells per cm2, keratinocytes attach to the gel with an efficiency of over 70%; after a lag phase of 3 days, the cells multiply exponentially
with a doubling time of 60 hr. Cultures reach a growth-plateau phase at a density of 47.7×104 cells per cm2. Both hydrocortisone and epidermal growth factor (EGF) stimulate slightly the growth of primary cultures; both factors are
required for proliferation of the 2nd and further passage of keratinocytes. As the cultures reach, confluence multilayers,
of stratified cells are formed and cells of squamous morphology are spontaneously released from the surface. When the released
cells and the attached cells are pulsed with [3H]-histidine and [14C]-leucine, a higher ratio of histidine to leucine is observed in the released cells indicating the biochemical onset of maturation.
Orange G-Aniline Blue staining of the released cells show some of the cells to be completely keratinized. Fibrous proteins
extracted from the cultured cells and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis display the characteristic
stratum corneum proteins of 60,000 and 66,000 daltons.
Supported in part by Grants AM 14121 and AM 19595 of the U.S. Public Health Service. 相似文献