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41.
Discriminatory power and reproducibility of novel DNA typing methods for Mycobacterium tuberculosis complex strains 下载免费PDF全文
Kremer K Arnold C Cataldi A Gutiérrez MC Haas WH Panaiotov S Skuce RA Supply P van der Zanden AG van Soolingen D 《Journal of clinical microbiology》2005,43(11):5628-5638
In recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110 restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods for Mycobacterium tuberculosis were investigated using the strain collection of the interlaboratory study of Kremer et al. This strain collection contains 90 M. tuberculosis complex and 10 non-M. tuberculosis complex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods for M. tuberculosis and that VNTR typing is the epidemiological typing method of choice for the near future. 相似文献
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de la Rosa G Longo N Rodríguez-Fernández JL Puig-Kroger A Pineda A Corbí AL Sánchez-Mateos P 《Journal of leukocyte biology》2003,73(5):639-649
Distinct subsets of dendritic cells (DCs) are present in blood, probably "en route" to different tissues. We have investigated the chemokines and adhesion molecules involved in the migration of myeloid (CD11c(+)) and plasmacytoid (CD123(+)) human peripheral blood DCs across vascular endothelium. Among blood DCs, the CD11c(+) subset vigorously migrated across endothelium in the absence of any chemotactic stimuli, whereas spontaneous migration of CD123(+) DCs was limited. In bare cell migration assays, myeloid DCs responded with great potency to several inflammatory and homeostatic chemokines, whereas plasmacytoid DCs responded poorly to all chemokines tested. In contrast, the presence of endothelium greatly favored transmigration of plasmacytoid DCs in response to CXCL12 (stromal cell-derived factor-1) and CCL5 (regulated on activation, normal T expressed and secreted). Myeloid DCs exhibited a very potent transendothelial migration in response to CXCL12, CCL5, and CCL2 (monocyte chemoattractant protein-1). Furthermore, we explored whether blood DCs acutely switch their pattern of migration to the lymph node-derived chemokine CCL21 (secondary lymphoid-tissue chemokine) in response to microbial stimuli [viral double-stranded (ds)RNA or bacterial CpG-DNA]. A synthetic dsRNA rapidly enhanced the response of CD11c(+) DCs to CCL21, whereas a longer stimulation with CpG-DNA was needed to trigger CD123(+) DCs responsive to CCL21. Use of blocking monoclonal antibodies to adhesion molecules revealed that both DC subsets used platelet endothelial cell adhesion molecule-1 to move across activated endothelium. CD123(+) DCs required beta(2) and beta(1) integrins to transmigrate, whereas CD11c(+) DCs may use integrin-independent mechanisms to migrate across activated endothelium. 相似文献
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Summary We have cloned the Cephalosporium acremonium pyr4 gene by cross-hybridization with the equivalent gene from Neurospora crassa, the closest relative from which this gene is available. The C. acremonium pyr4 gene complements an E. coli pyrF mutant lacking orotidine-5-phosphate decarboxylase (OMPdecase), and most probably does not contain introns. Maxicell analysis in E. coli shows that it encodes a 46 kDa polypeptide. The C. acremonium OMPdecase contains a highly conserved pentadecapeptide characteristic for this category of enzyme. Extensive sequence comparison suggests an important role of this region in enzymatic activity. 相似文献
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General morphology and capsid fine structure of African swine fever virus particles 总被引:11,自引:0,他引:11
JoséL. Carrascosa JoséM. Carazo Angel L. Carrascosa Narciso García Antonio Santisteban Eladio Viñuela 《Virology》1984,132(1):160-172
The structure of African swine fever virus particles has been examined by electron microscopy. The analysis of virions prepared by negative staining, thin sectioning, and freeze-drying and shadowing showed that the virus particle was composed of several concentric structures with an overall icosahedral shape. The inner region of the virus particles was a nucleoid that was surrounded by a membrane covered by the capsid. The capsid had side-to-side dimensions of 172 to 191 nm and was built up by capsomers arranged in an hexagonal lattice. Computer-filtered electron micrographs of either negatively stained or freeze-dried and shadowed capsids revealed capsomers with a hexagonal outline and a hole in the center. The intercapsomer distance ranged from 7.4 to 8.1 nm. The triangulation number of the capsid was estimated to be 189 to 217, indicative of 1892 to 2172 capsomers. Extracellular African swine fever virus particles had an external membrane that resembled the cytoplasmic unit membrane. 相似文献
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Luis C. Antn Sol Ruiz Elena Barrio Guillermo Marqus Angel Snchez Fernando Vivanco 《European journal of immunology》1994,24(3):599-604
The covalent binding reaction of the third component of complement (C3) with rabbit IgG immune aggregates has been studied by enzymic digestion of C3b-IgG adducts. In these adducts C3b was radioactively labeled in the free thiol group generated during activation of the internal thioester of C3. Trypsin digestion of 14C-labeled C3b-IgG adducts degrades C3b to a small antibody-bound 14C-labeled C3 fragment (14C-C3frg), whereas the antibody remains unaltered. Papain digestion of trypsin-treated 14C-C3frg-IgG complexes generated Fc and Fab fragments bearing equivalent amounts of covalently bound 14C-C3frg (43% and 40%, of the total C3 present in the aggregates, respectively). Hydroxylamine treatment of the 14C-C3frg-Fab and 14C-C3frg-Fc complexes released a 14C-C3frg of similar size (about 3–4 kDa) in which the N-terminal residue was the radiolabeled Cys1010. A fragment with the same radioactive N terminus and characteristics was obtained by sequential trypsin and papain digestion of purified C3 labeled with iodo–[14C] acetamide. Affinity-purified 14C-C3frg-Fc complexes digested with pepsin generated a mixture of radioactive peptides, most probably complexes formed by 14C-C3frg and Cγ2 or the hinge digestion products, and 14C-C3frg-pFc' complexes. The latter was also immunoprecipitated with anti-Fc-Sepharose from the pepsin digestion supernatants of 14C-labeled-C3b-IgG complexes. Taken together these data indicate that, during complement activation through the alternative pathway by IgG immune aggregates, C3 is not bound to a single site on the antibody molecule. Both Fab and Fc regions of IgG are equally efficient targets for C3 anchorage. In addition, the data confirm the pFc' as a region of C3 attachment within the Fc portion, and strongly suggest that C3b is bound either to the Cγ2 domain or the hinge or both. 相似文献
50.
Paredes AM Ferreira D Horton M Saad A Tsuruta H Johnston R Klimstra W Ryman K Hernandez R Chiu W Brown DT 《Virology》2004,324(2):373-386
Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a "pore"-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell. 相似文献