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91.
CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays 总被引:10,自引:0,他引:10
Kreher CR Dittrich MT Guerkov R Boehm BO Tary-Lehmann M 《Journal of immunological methods》2003,278(1-2):79-93
The frequency and the cytokine signature of antigen-specific T cells in the blood reflect the magnitude and the quality of T cell immunity in vivo. Recently, cytokine enzyme-linked immunospot (ELISPOT) assays performed on freshly isolated peripheral blood mononuclear cells (PBMC) emerged as a promising tool for monitoring these key parameters, providing direct feedback information on the efficacy of vaccinations and immune therapies. However, performing ELISPOT assays with freshly isolated cells is not readily feasible in the context of clinical trials. The ability to obtain valid ELISPOT data on cryopreserved samples would greatly enhance ex vivo immune monitoring capabilities. We have therefore systematically studied antigen-specific T cell responses in freshly isolated PBMC and after cryopreservation. Four healthy donors were selected that displayed T cell responses to six recall antigens. The antigen reactive T cells were defined as CD4 or CD8 cells, and their cytokine effector class was established measuring interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-5. The donors were bled at three different time points, and their PBMC were tested fresh and after freeze-thawing. The results showed that the frequencies and type 1/type 2 cytokine signatures of recall antigen-specific CD4 and CD8 cells are unaffected after cryopreservation. In contrast to these data obtained on human PBMC, cryopreservation of murine spleen cells causes a decrease in cytokine secretion. 相似文献
92.
In Hypocrea jecorina (anamorph: Trichoderma reesei) multiple gene deletions are limited by the number of readily available selection markers. We have therefore constructed
a blaster cassette which enables successive gene knock-outs in H. jecorina. This 3.5 kb pyr4 blaster cassette contains the H. jecorina pyr4 marker gene encoding orotidine-5′-monophosphate (OMP) decarboxylase flanked by two direct repeats of the Streptoalloteichus hindustanus bleomycin gene (Sh ble), which facilitate the excision of the blaster cassette by homologous recombination after each round of deletion. Functionality
of this pyr4 blaster cassette was demonstrated by deletion of the glk1 encoding glucokinase and hxk1 encoding hexokinase. 1.4–1.8 kb of the non-coding flanking regions of both target genes were cloned into the respective blaster
cassettes and transformation of a pyr4 negative H. jecorina strain with the two cassettes resulted in 10–13% of the transformants in the deletion of one of the two kinase genes. For
excision of the pyr4 blaster cassettes, Δglk1 strains were selected for growth in the presence of 5-fluoroorotic acid. Recombination between the two Sh ble elements resulted in uridine auxotrophic strains which retained their respective glucokinase negative phenotype. Subsequent
transformation of one of these auxotrophic Δglk1 strains with the hexokinase blaster cassette resulted in pyr4 prototrophic strains deleted in both glk1 and hxk1. Δglk1 strains showed reduced growth on d-glucose and d-fructose whereas Δhxkl strains showed reduced compact growth on d-glucose but were unable to grow on d-fructose as carbon source. The double Δglk1Δhxk1 deletion strain was completely unable to grow on either d-glucose or d-fructose.
Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers DQ068384 (H. jecorina glk1) and DQ068385 (H. jecorina hxk1). 相似文献
93.
94.
Stöhr H Marquardt A Nanda I Schmid M Weber BH 《European journal of human genetics : EJHG》2002,10(4):281-284
The RFP-TM protein family was first described in Caenorhabditis elegans as hypothetical transmembrane proteins containing a conserved 350-400 amino acid domain including the invariant peptide motif RFP. The VMD2 gene underlying Best disease was shown to represent the first human member of the RFP-TM protein family. More than 97% of the disease-causing mutations are located in the N-terminal RFP-TM domain implying important functional properties. Here, we have identified three novel VMD2-related human genes (VMD2L1, VMD2L2 and VMD2L3) demonstrating a high degree of conservation in their respective RFP-TM domains. Each of the VMD2-like proteins has a unique C-terminus that lack similarity to other proteins or motifs. By FISH analysis, VMD2L1 was localised to chromosome 19p13.2-p13.12, VMD2L2 to 1p32.3-p33 and VMD2L3 to 12q14.2-q15. RT-PCR analyses revealed tissue-restricted expression of the three genes with both VMD2L1 and VMD2L2 abundantly transcribed in colon. VMD2L1 is present in the retinal pigment epithelium while VMD2L3 shows predominant expression in skeletal muscle. 相似文献
95.
Röcken C Radun D Glasbrenner B Malfertheiner P Roessner A 《Virchows Archiv : an international journal of pathology》1999,434(1):95-100
We report on a 58-year-old Caucasian woman who went to a general practitioner about recurrent abdominal pain, night sweats
and weight loss of a few weeks’ duration. Once gynaecological disease had been ruled out, the patient was admitted to hospital
with severe abdominal pain and intestinal obstruction and a right-sided hemicolectomy was performed. Following the investigation
of osteolytic lumbar vertebrae, 18 months after visiting the general practitioner the patient was finally found to be suffering
from generalized AA-amyloidosis secondary to gastrointestinal tuberculosis. This had been misinterpreted as Crohn’s disease.
Re-examination of the specimens from the right-sided hemicolectomy demonstrated that scanty deposits of AA-amyloid were present
9 months after the first presentation. AA-amyloid can thus be present in serious inflammatory disease even during the first
9 months after the initial clinical presentation.
Received: 23 June 1998 / Accepted: 19 August 1998 相似文献
96.
Bernhard F. X. Reber Benno Schindelholz 《Pflügers Archiv : European journal of physiology》1996,432(5):893-903
Bradykinin and caffeine were used as two different agonists to study inositol 1,4,5-trisphosphate (IP3)-sensitive and caffeine/ryanodine-sensitive intracellular Ca2+ release in the outgrowing neurites of nerve-growth-factor (NGF)-treated rat phaeochromocytoma cells (PC12). Changes in neuritic
intracellular free Ca2+ ([Ca2+]i) in single cells were measured after loading with a 1:1 mixture of the acetoxymethyl (AM) ester of the Ca2+-sensitive dyes Fura-red and Fluo-3, in combination with confocal microscopy. Bradykinin-induced Ca2+ release was blocked by U73211, a specific phospholipase C inhibitor. Caffeine-induced Ca2+ release was very low in neurites at rest. It increased after the cells were preloaded with Ca2+. The Ca2+ signal evoked at high concentrations of bradykinin (>500 nM) arose from a trigger zone in the proximal part of the neurite,
as a bi-directional wave towards the growth cone and cell body. The speed of neuritic Ca2+ waves was reduced in cells loaded with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-tetraacetic acid/AM. Preloading of Ca2+ stores led to increased bradykinin-induced Ca2+ release, as seen for caffeine, and faster Ca2+ wave speeds. Caffeine evoked a simultaneous [Ca2+]i rise along the neurites of Ca2+ preloaded cells. Higher Ca2+ signal amplitudes and faster Ca2+ wave speeds, but no longer-lasting IP3-induced [Ca2+]i signals, correlated with increased caffeine-induced Ca2+ release in the neurites. At low concentrations of bradykinin (<1.0 nM), the Ca2+ signals ceased to propagate as complete Ca2+ waves. Instead, repetitive stochastic Ca2+ release events (neuritic Ca2+ puffs) were observed. Neuritic Ca2+ puffs spread across only a few microns, at a slower speed than neuritic Ca2+ waves. These Ca2+ puffs represent elementary Ca2+ release units, whereby the released Ca2+ ions form these elementary events into the shape of a Ca2+ wave.
Received: 16 April 1996 / Received after revision and accepted: 13 May 1996 相似文献
97.
Peter Sandner Bernhard Gess Konrad Wolf Armin Kurtz 《Pflügers Archiv : European journal of physiology》1996,431(6):905-912
There is accumulating evidence from in vitro experiments that the gene expression of the vascular endothelial growth factor
(VEGF) is, like that of the erythropoietin (EPO) gene, regulated by the oxygen tension and by divalent cations such as cobalt.
Since the information about the regulation of VEGF gene expression in vivo is rather scarce, this study aimed to examine the
influence of hypoxia and of cobalt on VEGF gene expression in different rat organs and to compare it with that on EPO gene
expression. To this end male Sprague-Dawley rats were exposed to carbon monoxide (0.1% CO), hypoxia (8% O2 ) or to cobalt chloride (12 and 60 mg/kg s.c.) for 6 h. mRNA levels for VEGF- 188, -164, and -120 amino acid isoforms in
lungs, hearts, kidneys and livers were semiquantitated by RNase protection. For these organs we found a rank order of VEGF
mRNA abundance of lung >> heart > kidney = liver. EPO mRNA levels were semiquantitated in kidneys and livers. Hypoxia, CO
and cobalt increased EPO mRNA levels 60-fold, 140-fold and 5-fold, respectively, in the kidneys, and 11-fold, 11-fold and
3-fold, respectively, in the livers. None of these manoeuvres caused significant changes of VEGF mRNA in lung, heart or kidneys.
Only in the livers did hypoxia lead to a significant (50%) increase of VEGF mRNA. These findings suggest that, in contrast
to the in vitro situation, the expression of the VEGF gene in normal rat tissues is rather insensitive to hypoxia. In consequence,
the in vivo regulation of the VEGF and the EPO genes appear to differ substantially, suggesting that the regulation of the
VEGF and EPO genes may not follow the same essential mechanisms in vivo.
Received: 31 July 1995/Received after revision: 20 November 1995/Accepted: 27 November 1995 相似文献
98.
Carsten Fülber Dan E. Demco Ofer Weintraub Bernhard Blümich 《Macromolecular chemistry and physics.》1996,197(2):581-593
The transverse nuclear 1H magnetization decay in poly(styrene-co-butadiene) (SBR) is investigated by editing 13C NMR spectra. This technique allows for the assignment of localized 1H dynamical information by discriminating the chemical sites based on their chemical shift in the 13C dimension. Here, the homo- and heteronuclear dipolar couplings contribute to the 1H NMR relaxation giving additional information to a homonuclear experiment. In this heteronuclear 2D experiment two prominent peaks are observed in the 13C dimension, which correspond to CH and CH2 groups, respectively. The decay rate in the 1H dimension is found for both groups to scale with the crosslink density. An additional ultra-fast magnetization decay is reported. The effect of the carbon black filler is investigated for this component. The analysis of the 13C NMR edited transverse 1H magnetization relaxation is a useful tool in combining high resolution NMR spectra with information on molecular dynamics, providing insight into crosslink density and filler effects. 相似文献
99.
Zankl A Neumann L Ignatius J Nikkels P Schrander-Stumpel C Mortier G Omran H Wright M Hilbert K Bonafé L Spranger J Zabel B Superti-Furga A 《American journal of medical genetics. Part A》2005,(1):61-67
Platyspondylic lethal skeletal dysplasia (PLSD) Torrance type (PLSD-T) is a rare skeletal dysplasia characterized by platyspondyly, brachydactyly, and metaphyseal changes. Generally a perinatally lethal disease, a few long-term survivors have been reported. Recently, mutations in the carboxy-propeptide of type II collagen have been identified in two patients with PLSD-T, indicating that PLSD-T is a type 2 collagen-associated disorder. We studied eight additional cases of PLSD-T and found that all had mutations in the C-propeptide domain of COL2A1. The mutational spectrum includes missense, stop codon and frameshift mutations. All non-sense mutations were located in the last exon, where they would escape non-sense-mediated RNA-decay. We conclude that PLSD-T is caused by mutations in the C-propeptide domain of COL2A1, which lead to biosynthesis of an altered collagen chain (as opposed to a null allele). Similar mutations have recently been found to be the cause of spondyloperipheral dysplasia, a non-lethal dominant disorder whose clinical and radiographical features overlap those of the rare long-term survivors with PLSD-T. Thus, spondyloperipheral dysplasia and PLSD-T constitute a novel subfamily within the type II collagenopathies, associated with specific mutations in the C-propeptide domain and characterized by distinctive radiological features including metaphyseal changes and brachydactyly that set them apart from other type 2 collagenopathies associated with mutations in the triple-helical domain of COL2A1. The specific phenotype of C-propeptide mutations could result from a combination of diminished collagen fibril formation, toxic effects through the accumulation of unfolded collagen chains inside the chondrocytes, and alteration of a putative signaling function of the carboxy-propeptide of type 2 collagen. 相似文献
100.