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91.
Michael W. Fountain Robert Chiovetti Henri Kercret David O. Parrish Jere P. Segrest 《生物化学与生物物理学报:生物膜》1980,596(3):420-425
A method has been developed for the rapid separation of cells in suspension from non-cell associated lipid vesicles in various assays for vesicle-cell interaction. Separation is achieved on a discontinuous Ficoll-Paque gradient. Cells and free vesicles are totally separated, as evidenced by both radiolabelled vesicles, and vesicles containing the fluorescent dye 6-carboxyfluorescein. The main advantages of this method are the rapidity, efficacy, and gentleness of the separation. Viability of the cells remains consistently high (greater than 96%) throughout the separation. Since this method involves a one-step centrifugation, it precludes the necessity for repeated washings of cells which have been incubated with lipid vesicles. 相似文献
92.
John C. Angello Howard L. Hosick 《Biochemical and biophysical research communications》1982,107(3):1130-1137
Multicellular aggregates of tumorigenic mouse mammary epithelial cells contain a hyaluronate-rich matrix, both at the aggregate periphery and within the growing spheroid. It is proposed that the establishment of a hyaluronaterich matrix is essential to spheroid growth in vitro, and that the spheroid is a good model system for analysis of this aspect of early tumor development. 相似文献
93.
W.James Nelson Constantin E. Vorgias Peter Traub 《Biochemical and biophysical research communications》1982,106(4):1141-1147
A new method is described for the purification of the intermediate filament protein vimentin from Ehrlich ascites tumor cells using single-stranded DNA-cellulose affinity chromatography. The procedure is rapid and allows the large scale isolation of the protein. Partial characterization of vimentin shows that it has a molecular weight of 58000 and an apparent pI of 5.3. It can be degraded by the vimentin-specific, Ca2+-activated proteinase which results in the production of a characteristic set of degradation products. The vimentin also cross-reacts with the intermediate filament protein monoclonal antibody, α-IFA. 相似文献
94.
Thomas E. Gray David G. Thomassen Marc J. Mass J. Carl Barrett 《In vitro cellular & developmental biology. Plant》1983,19(7):559-570
Summary A cell culture system is described for the growth of rat tracheal epithelial (RTE) cells at clonal density. The system uses
normal, early passage RTE cells grown on feeder layers of lethally irradiated 3T3 cells. The RTE cells have a high colony
forming efficiency (5 to 10%) in culture, can be passaged up to 5 times, and are capable of more than 20 cumulative doublings
per colony forming cell. The epithelial nature of the cells was confirmed by cell and colony morphology, immunoperoxidase
staining of intracellular keratin, and cellular ultrastructural studies. The cytotoxic response of RTE cells to a variety
of carcinogens, including a direct acting chemical carcinogen, a physical carcinogen, and a series of polycyclic aromatic
hydrocarbons, was quantitated. A linear decrease in the logarithm of survival was observed with increasing doses ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), γ-irradiation, 7,12-dimethylbenz(a)anthracene, and a diol-epoxide of benzo(a)pyrene. No toxicity was observed after treatment with benzo(a)pyrene or 3-methylcholanthrene over the concentration range examined. In contrast, phorbol ester tumor promoters stimulated
cell growth markedly. Based on these and other studies, the RTE cell culture system represents a model system that will be
useful for quantitative studies of epithelial cell growth, differentiation, and carcinogenesis. 相似文献
95.
转移及非转移肿瘤移植后615小鼠血液流变学变化的研究 总被引:1,自引:0,他引:1
血道高转移瘤株FC、淋巴合并血道高转移瘤株U14、淋巴道高转移瘤株H22、非转移瘤株P615分别接种于336只纯系近交615小鼠.不同时间取血并处死动物,进行组织学及血液流变学检查.将转移瘤发展过程分为潜伏期、侵袭期、转移早、中、晚期,非转移瘤发展过程分为潜优期、增殖期、囊腔形成期及中心坏死期.本实验结果显示,不同转移能力及途径肿瘤发展的不同时期血液流变学变化规律不同,因而表明肿瘤侵袭、转移与血液流变学变化之间存在互为因果的紧密关系.其临床诊断及治疗意义被讨论. 相似文献
96.
D. Needham 《Cell biochemistry and biophysics》1991,18(2):99-121
Studies that examine the shear- and abrasion-sensitivity of proliferating cells are important in order to understand the behavior
of hybridoma cells in bioreactor culture and metastasizing cancer cells in the bloodstream. Little is known about the link
between morphology, structure, and mechanical properties of a given cell line, especially with respect to variations throughout
the cell cycle. In our experiments with GAP A3 hybridoma cells, distinct cell morphologies were identified and correlated
with phases of the cell cycle by video microscopic observation of synchronized cells, and of individual cells that were followed
throughout their cell cycle. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (apparent
cell viscosity) properties of single cells. As the cell cycle progressed at 37°C, an increase in cell volume from 1400 μm3 to 5700 μm3 was accompanied by an increase in apparent cell viscosity from 430 poise to 12,000 poise, consistent with an accumulation
of more cytoplasmic material in the “older” cells. Hybridomas are representative of the various leukemias derived from hemopoietic
cells, and even though as a whole, they appeared to be rather shear-insensitive, the wide range of property values demonstrates
that a given cell line cannot be characterized by a single value for any one property, and that properties must be related
to the cell cycle when considering proliferating cells. It is interesting to see if distinct stages in the metastatic sequence
of events might correlate with any of these physical features of the cell cycle, irrespective of cell type or cell line. For
example, the cytokinetic doublet could represent a fragile structure that may fail and produce cell death under fluid-shear
conditions that would not affect the cells at any other stage in the cell cycle. Identifying such cell cycle-dependent features
in metastasizing cancer cells could lead to a better understanding of the metastatic process and to possible clinical treatments
directed at making cells more shear- and abrasion-sensitive, and therefore, more likely to be killed by the natural hydrodynamic
forces of the circulatory system. 相似文献
97.
Summary Although an outwardly rectifying K+ conductance (I
K, A) is prominently expressed in human alveolar macrophages, the expression of this conductance in human monocyte-derived macrophages (HMDMs) is rare. We have analyzed the induction of the expression of I
K, A in voltage-clamped, in vitro differentiated HMDMs by a number of stimuli which produce either priming or activation of macrophages. Cultures were stimulated with lipopolysaccharide (LPS, 2 g/ml), interleukin 2 (IL-2, 100 U/ml), or combinations of LPS and either recombinant interferon-gamma (-IFN, 10 U/ml), phorbol myristate acetate (PMA, 0.01 or 1 g/ ml) and platelet activating factor (PAF, 20 ng/ml) for periods of up to 24 hr. Treatment of the cells with either LPS or IL-2 greatly enhanced the frequency of current expression. Treatment with either PMA or -IFN alone did not induce current expression; treatment of the cells with a combination of LPS and either PMA, -IFN, or PAF did not enhance current expression over that observed with LPS alone. The expression of the outwardly rectifying K+ current was observed in 36% (n=321) of the cells for cultures treated with LPS and 33% (n=55) of the cells for cultures treated with IL-2. The inactivating outward K+ current was absent in cells which were not treated with either LPS or IL-2. The kinetics of current activation and inactivation appeared identical to that previously described for the transient-inactivating outward current of the human alveolar macrophage. Cycloheximide (1 g/ml), an inhibitor of protein synthesis, completely suppressed LPS-induced current expression. No correlation was found between peak current amplitude and cell size in LPS-activated cells expressing the outwardly rectifying K+ current, indicating that current density was not held constant from cell to cell. The coupling of ion channel expression and secretion in individual HMDMs was studied using the reverse hemolytic plaque assay. Although an enhancement of K + current expression was observed following either LPS or IL-2 treatment, a quantitatively similar and uniform increase in the percentage of either IL-1 or lysozyme-secreting cells was not observed. The frequency of current expression in cells identified as secreting tumor necrosis factor- (TNF-), interleukin 1 (IL-1), or lysozyme was the same or decreased over that observed for nonsecreting cells. Thus, LPS treatment increases the number of K+ channels on HMDM membranes; however, K+ channel expression alone was not sufficient to give rise to enhanced secretion in LPS-activated macrophages. Enhanced K+ channel expression appears to be a part of the primary activation signal. K+-channel activation would hyperpolarize the membrane potential, potentially providing the driving force for calcium entry through voltage-independent pathways activated by the subsequent binding of soluble substances to membrane surface receptors, the secondary signal linked to secretion.This work was supported by NIH grant RO 1 GM36823. 相似文献
98.
Krishan K. Arora David M. Parry Peter L. Pedersen 《Journal of bioenergetics and biomembranes》1992,24(1):47-53
Hexokinase plays an important role in normal glucose-utilizing tissues like brain and kidney, and an even more important role in highly malignant cancer cells where it is markedly overexpressed. In both cell types, normal and transformed, a significant portion of the total hexokinase activity is bound to particulate material that sediments upon differential centrifugation with the crude mitochondrial fraction. In the case of brain, particulate binding may constitute most of the total hexokinase activity of the cell, and in highly malignant tumor cells as much as 80 percent of the total. When a variety of techniques are rigorously applied to better define the particulate location of hexokinase within the crude mitochondrial fraction, a striking difference is observed between the distribution of hexokinase in normal and transformed cells. Significantly, particulate hexokinase found in rat brain, kidney, or liver consistently distributes with nonmitochondrial membrane markers whereas the particulate hexokinase of highly glycolytic hepatoma cells distributes with outer mitochondrial membrane markers. These studies indicate that within normal tissues hexokinase binds preferentially to non-mitochondrial receptor sites but upon transformation of such cells to yield poorly differentiated, highly malignant tumors, the overexpressed enzyme binds preferentially to outer mitochondrial membrane receptors. These studies, taken together with the well-known observation that, once solubilized, the particulate hexokinase from a normal tissue can bind to isolated mitochondria, are consistent with the presence in normal tissues of at least two different types of particulate receptors for hexokinase with different subcellular locations. A model which explains this unique transformation-dependent shift in the intracellular location of hexokinase is proposed. 相似文献
99.
100.
Estrogen-stimulation of postconfluent cell accumulation and foci formation of human MCF-7 breast cancer cells 总被引:3,自引:0,他引:3
J F Gierthy D W Lincoln K E Roth S S Bowser J A Bennett L Bradley H W Dickerman 《Journal of cellular biochemistry》1991,45(2):177-187
Foci, nodules of cellular overgrowth, that appear after confluence are an in vitro characteristic of malignant transformation. A well-studied in vitro model of estrogen-dependent tumors is the MCF-7 cell line, derived from a pleural metastasis of a human breast adenocarcinoma. We report that cultivation of MCF-7 cells, using routine methods, results in extensive estrogen-stimulated postconfluent cell accumulation characterized by discrete three-dimensional arrays. Side view Nomarski optical sections revealed these to be principally multicellular foci with occasional domes and pseudoacinar vacuoles. This effect on MCF-7 cell growth occurs in media containing fetal bovine serum but not with calf serum or charcoal-dextran-treated fetal bovine serum unless supplemented with estrogens. Foci formation starts 5-6 days after confluence, and the number of foci generated is a function of the concentration of added estrogens. Foci formation is suppressed by the antiestrogens Tamoxifen and LY 156758. Addition of progesterone, testosterone, or dexamethasone had little or no effect, while various estrogens (ethinyl estradiol, diethylstilbestrol, and moxestrol) induced foci development. Clones derived from single cells of the initial MCF-7 population revealed a wide variance in estrogen-induced foci formation, demonstrating heterogeneity of this tumor cell line. The postconfluent cell growth of the estrogen receptor-deficient cell line, MDA-MB-231, contrasted with MCF-7 by developing an extensive multilayer morphology devoid of discrete structures. The tumorigenic potential of the MCF-7 cells used in our experiments was confirmed by their estrogen-dependent growth in immunosuppressed male BDF1 mice. These data suggest an estrogen receptor-based mechanism for the development of multicellular foci during postconfluent growth of MCF-7 cells. After confluence, foci, in contrast to the quiescent surrounding monolayer, retain proliferating cells. Focus formation, therefore, reflects the heterogeneous responsiveness of these cells to estrogens and should provide a model permitting in vitro comparisons between the progenitor cells of multicellular foci and the monolayer population. 相似文献