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71.
72.
Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: Modulation by culture conditions 总被引:5,自引:0,他引:5
Clark T. Bishop Zermeena Mirza James D. Crapo Bruce A. Freeman 《In vitro cellular & developmental biology. Plant》1985,21(4):229-236
Summary Culture conditions modulating cell damage from xanthine plus xanthine oxidase-derived partially reduced oxygen species were
studied. Porcine thoracic aorta endothelial cells and porcine lung fibroblasts were maintained in monolayer culture. Cells
were prelabeled with51Cr before xanthine plus xanthine oxidase exposure. Endothelial cells showed 30 to 100% more lysis than fibroblasts and thus
seemed more sensitive to this oxidant stress. The effect of cell culture age, as indicated by population doubling level (PDL),
was examined. Response of low PDL endothelial cells and fibroblasts subjected to oxidant stress was compared with the response
of PDL 15 cells. Both low PDL endothelial cells and fibroblasts responded differently to the lytic effect of xanthine oxidase-derived
free radicals than did higher PDL cells. Specific activities of the antioxidant enzymes catalase, managanese superoxide dismutase,
copper-zinc superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured in both low
and high PDL fibroblasts and endothelial cells. Antioxidant enzyme specific activities could only partially explain the differences
in response to oxidant stress between fibroblasts and endothelial cells and between low and high PDL cells. Cell culture medium
composition modulated the rate of production, and relative proportions of xanthine plus xanthine oxidase-derived partially
reduced species of oxygen, i.e. superoxide, hydrogen peroxide, and hydroxyl radical. Serum content of medium was important
in modulating free radical generation; superoxide production rates decreased 32%, H2O2 became undetectable, and hydroxyl radical generation decreased 54% in the presence of 10% serum. The medium protein and iron
content also modulated free radical generation. The data suggest that cell culture media constituents, cell type, and cell
culture age greatly affect in vitro response of cells subjected to oxidant stress.
Research supported by American Lung Association Fellowship Training Grant and Research Training Grant, the R. J. Reynolds
Corporation, and National Institutes of Health Grants HL29784 and 1 HL 23805. 相似文献
73.
Receptor-mediated regulation of calcium mobilization and cyclic GMP synthesis in neuroblastoma cells 总被引:3,自引:0,他引:3
In neuroblastoma N1E 115 cells, carbachol, histamine and PGE1 elevated cyclic GMP content and, induced the efflux of preloaded 45Ca2+, the release of membrane-bound Ca2+ measured by fluorescent CTC, and the increase in [Ca2+]i as measured by Quin 2 fluorescence. The time course of the responses, the absolute requirement of extracellular Ca2+, the inhibition by receptor blockers, and the concentration dependency on histamine were all similar between these responses. The observation indicates that the mobilization of Ca2+, especially the increase of [Ca2+]i, may be intimately linked to the synthesis of cyclic GMP in the cells. 相似文献
74.
Interference by DDT and cyclodiene types of insecticides with chloroplast-associated reactions 总被引:1,自引:0,他引:1
The effects of DDT, some of its analogs, and selected cyclodiene insecticides on isolated spinach (Spinacea oleracea L.) thylakoids were identified, characterized, and compared to responses induced by selected herbicides. Except for endrin, the insecticides inhibited light-induced electron transport, altered chlorophyll fluorescence transients, and competitively displaced [14C]atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine], a known photosystem II inhibitor, from the membranes. The insecticides appeared to act at, or near B, the secondary electron acceptor of photo-system II. Binding of DDT and dieldrin was estimated at 900 and 2200 molecules, respectively, per photosynthetic unit (490 chlorophyll molecules). The insecticides also inhibited valinomycin-induced swelling of the thylakoid membrane. Whereas inhibition of electron transport can be attributed to interaction by the insecticides with a proteinaceous component of the thylakoid membrane, interference with the action of valinomycin may involve interaction with lipoidal constituents of the membrane. 相似文献
75.
A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 106 cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 105 cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 106 cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture. 相似文献
76.
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
77.
H. Christopher Wilson Nadine C. Milos 《In vitro cellular & developmental biology. Plant》1987,23(5):323-331
Summary This study investigates the nutritional requirements ofXenopus laevis neural crest cells and melanophores developing in vitro. A comparison is made between the growth and differentiation of cells
in serum-containing medium and a chemically defined, serum-free medium that we have designed. Our chemically defined medium
is more efficient than serum-supplemented medium in promoting proliferation of these cells. Several supplements are required
to enhance culture development. These include insulin, α-melanocyte stimulating hormone, somatotropin, luteotrophic hormone,
linoleic acid, uridine, and putrescine. In addition, collagen and fibronectin provide the most conductive environment tested
for cell migration and adhesion.
This work was supported by establishment and major equipment grants from the Alberta Heritage Foundation for Medical Research
to N. C. M. Nadine C. Milos is a Heritage Medical Research Scholar of the Alberta Heritage Foundation for Medical Research. 相似文献
78.
Summary New cell lines, designated as ML-DmDl≈10, were established from dissociated imaginal discs ofDrosophila melanogaster. The culture medium was prepared by mixing in a 1:1 ratio Cross and Sang’s M3(BF) medium, supplemented with 10% heat inactivated
fetal bovine serum (FBS), with the supernatant of a primary embryonic cell culture made in the M3(BF) medium and supplementing
this mixture with insulin. One cell line was established in the medium containing larval hemolymph instead of the primary
culture supernatan, and another was established in fresh M3(BF) medium supplemented with insulin and FBS. In these mediums,
imaginal disc cells first formed aggregates and cellular vesicles within a few weeks followed by the proliferation of thin-layered
cells around them after about 1 mo. Ten cell lines have so far been established from two kinds of imaginal discs and disc
mixtures. The ploidy of these cell lines was predominantly diploid. Population doubling time was about 50 to 70 h at 3 to
10 mo. after initiation of the culture. When the cell aggregates formed in vitro were implanted in metamorphosing larvae,
they differentiated at high frequency into adult cuticular strutures in the early phase of the primary culture. This differentiation
of aggregates was also observed, though at low frequency, in a culture maintained by dilution-transfer for 6 to 15 mo. in
vitro. 相似文献
79.
碱性蛋白酶生产菌——地衣芽孢杆菌经丝裂霉素C或紫外线处理,均可诱导释放噬菌体,电镜观察表明噬菌体头部外廓呈六边形,有不收缩的尾部(头部40nm×40nm,尾部107×5,7nm),噬菌体BLL1 DNA对限制酶Eco R Ⅰ,HindⅢ和Bam H Ⅰ敏感,分别切割成8,15,和9个片段,经电泳法测定。噬菌体DNA分子量约相当于23.4kb,根据解链温度计算出噬菌体的G+C含量约为31.2摩尔%,噬菌体提纯的外壳蛋白经SDS-聚丙烯酰胺凝胶电泳呈现5条主带,其分子量分别约为78000,72000,55000,39000,35000。 相似文献
80.
Summary Newborn rat adipocyte precursors, isolated from inguinal fat pads of 2 day-old NBR rats proliferate and undergo adipose differentiation
in defined medium in the absence of serum when cultivated on polylysine coated dishes in DME-F12 medium supplemented with
fibronectin, insulin, transferrin and FGF. After 7 days in culture in these conditions, 90% of the cells have undergone differentiation
as measured by the increase of G3PDH specific activity and by the accumulation of triglycerides in their cytoplasm. In contrast,
the cells cultivated in the presence of 10% fetal bovine serum, have a limited ability to differentiate. These results indicate
that newborn rat adipocyte precursors from inguinal fat pads do not require the presence of an undefined adipogenic factor
in order to differentiate in culture. In contrast, proliferation and differentiation are dependent on the presence of insulin
in the culture medium. Moreover, the data presented in this paper show that the rat adipocyte precursor culture represents
a rapid and reproducible system for investigating the processes of adipose tissue development and for studying the negative
and positive regulators of the adipose differentiation in a controlled environment.
This work was supported by grants from the Juvenile Diabetes Foundation, File #185221 and from the National Institutes of
Health 1 PO1 CA37589.
Editor’s Statement This paper extends to primary cultures the serum-free methods previously applied to studies of adipocyte
differentiation in established lines. The observation that serum can block differentiation in this system suggests the existence
of previously unrecognized circulating plasma or platelet factors affecting adipocyte differentiation, and the model developed
provides an assay for the identification of these factors. 相似文献