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91.
Roberts ML Wells DJ Graham IR Fabb SA Hill VJ Duisit G Yuasa K Takeda S Cosset FL Dickson G 《Human molecular genetics》2002,11(15):1719-1730
The ability to transfer the dystrophin gene stably to the skeletal muscle of DMD patients is a major confounding issue in establishing an effective gene therapy for this disease. To overcome this problem, we have examined the ability of muscle fibres from mdx mice to act as in situ factories of retroviral vector production. Tibialis anterior (TA) muscles from 4-week-old mdx mice were injected with an adenoviral vector expressing LacZ within a retroviral expression cassette (AdLZIN). Retroviral vector production was induced by the inclusion of two additional adenoviral vectors expressing retroviral gag-pol (AdGagPol) and 10A1 env genes (Ad10A1). Upon introduction of infected muscles into cell culture, colonies of beta-galactosidase-expressing myotubes formed only in cultures where the muscle was injected with AdLZIN, AdGagPol and Ad10A1, but not from muscle injected with AdLZIN only. Muscles from mdx/nude mice producing retroviral vector displayed a 4.6-fold increase in beta-galactosidase-positive myofibres after 1 month, compared with contralateral muscle in the same animal injected with AdLZIN and AdGagPol only. By constructing a hybrid adeno-retroviral vector expressing a truncated micro-dystrophin construct (AdmicroDyIN), we were able to partially correct the mdx dystrophic phenotype. AdmicroDyIN-mediated expression of micro-dystrophin in mdx TA muscle restored the formation of the dystrophin-associated glycoprotein complex and significantly reduced the level of muscle degeneration over uninjected controls. By stimulating in situ production of retroviral vector expressing micro-dystrophin, we achieved 92%+/-6% transduction of myofibres in the TA muscle by 4 weeks. Strikingly, by 3 months post injection, micro-dystrophin was still expressed to high levels in nearly all the myofibres of the TA muscle. By comparison, there was a pronounced drop in the levels of micro-dystrophin expressed by muscles injected with AdmicroDyIN only. Finally, using a novel PCR approach, we detected reverse-transcribed, integrated proviral sequences in TA muscle genomic DNA by 4 weeks post injection, the levels of which were found to increase after 3 months. 相似文献
92.
Special report. Steriod receptors in breast cancer. 总被引:5,自引:0,他引:5
E R DeSombre P P Carbone E V Jensen W L McGuire S A Wells J L Wittliff M B Lipsett 《The New England journal of medicine》1979,301(18):1011-1012
93.
Systemic distribution of woodchuck hepatitis virus in the tissues of experimentally infected woodchucks 总被引:6,自引:0,他引:6
To better assess the extent of the tissue tropism of mammalian hepadnaviruses, 10 tissues from each of six woodchucks were examined for the presence and state of woodchuck hepatitis virus (WHV) nucleic acids 15 months after experimental WHV infection. The tissues examined were peripheral blood lymphocytes, lymph node, spleen, bone marrow, thymus, pancreas, kidney, ovary, testis, and liver. Tissue samples from three chronically infected animals and three animals with serologic patterns of recovery (serum: WHsAg-, anti-WHs+, anti-WHs+, WHV DNA-) from acute WHV infection were analyzed in parallel by in situ hybridization and Southern and Northern blot techniques. WHV nucleic acids were detected in several individual tissues from each animal examined, although not all tissues in every animal contained WHV. Substantial differences were observed among the various tissues and animals with respect to the frequency, level, and intratissue distribution of WHV nucleic acids, as well as the presence of different viral genomic forms. Active WHV DNA replication was present only in the liver and spleen of the chronically infected animals. No evidence of ongoing WHV DNA replication was found in any of the tissues from the recovered animals. WHV DNA was homogeneously distributed among all hepatocytes in the livers of the chronic carriers. By contrast, WHV DNA in all the extrahepatic tissues, and in the livers of the recovered animals, was detected only in scattered foci of cells. 相似文献
94.
Detailed investigation of factors influencing amplification efficiency and allele drop-out in single cell PCR: implications for preimplantation genetic diagnosis 总被引:16,自引:0,他引:16
Preimplantation genetic diagnosis (PGD) of single gene disorders relies on PCR-based tests performed on single cells (polar bodies or blastomeres). Despite the use of increasingly robust protocols, allele drop-out (ADO; the failure to amplify one of the two alleles in a heterozygous cell) remains a significant problem for diagnosis using single cell PCR. In extreme cases ADO can affect >40% of amplifications and has already caused several PGD misdiagnoses. We suggest that an improved understanding of the origins of ADO will allow development of more reliable PCR assays. In this study we carefully varied reaction conditions in >3000 single cell amplifications, allowing factors influencing ADO rates to be identified. ADO was found to be affected by amplicon size, amount of DNA degradation, freezing and thawing, the PCR programme, and the number of cells simultaneously amplified. Factors found to have little or no affect on ADO were local DNA sequence, denaturing temperature (94 or 96 degrees C) and cell type. Consideration of the causal factors identified during this study should permit the design of PGD protocols that experience little ADO, thus improving the accuracy of PGD for single gene disorders. 相似文献
95.
The primary interest of our laboratory is understanding how the signals that a T cell receives influence its behavior during
an immune response. Recently, we have focused our attention on the examination of T cell responses at an individual cell level.
This article outlines our approach, presents our initial findings, and discusses the implications of these findings relative
to our current models of T cell activation. 相似文献
96.
Pence BW Gaynes BN Whetten K Eron JJ Ryder RW Miller WC 《Journal of acquired immune deficiency syndromes (1999)》2005,40(4):434-444
BACKGROUND: Substance abuse (SA) and mental illness (MI) commonly co-occur with HIV infection in the United States and have important implications for clinical management of HIV/AIDS. Yet SA/MI often go untreated due in part to a lack of practical, validated screening tools. SETTING: HIV clinic in academic medical center. METHODS: The 16-item SA/MI Symptoms Screener (SAMISS) targets SA/MI in HIV-positive patients. Consecutive consenting HIV-positive patients completed the SAMISS and then a reference standard diagnostic tool, SCID, the Structured Clinical Interview for DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition). RESULTS: Twenty percent of participants (29/148) had an SA diagnosis and 41% (59/143) had an MI diagnosis in the past year on the SCID; 48% (68/143) had 1 or both. Thirty-seven percent (55/148) screened positive for SA and 69% (99/143) screened positive for MI on the SAMISS. The SAMISS had 86% (95% CI: 68%-96%) sensitivity and 75% (66%-82%) specificity for SA and 95% (86%-99%) sensitivity and 49% (38%-60%) specificity for MI. Patients with SA were likely to show up as false positives for MI and vice versa. CONCLUSION: The SAMISS functioned well as a first-line screening tool for SA/MI in this HIV clinic population. It missed few cases and was easily incorporated into a busy clinical setting. Persons screening positive require a more rigorous confirmatory psychiatric evaluation. 相似文献
97.
F. R. Wells 《International journal of experimental pathology》1972,53(4):445-456
The vascular response to the α-toxin of Clostridium perfringens type A, was observed topographically in the cremaster muscle of the rat, in terms of exudation, labelling of damaged vessels by circulating carbon, and in addition histologically for patency of the vascular plexus.It was confirmed that the permeability response is biphasic. The short-lived immediate phase corresponds to that of venular labelling, and the delayed phase reaches its peak rather later than the corresponding phase of capillary labelling. The intensity and extent of these responses are determined by the degree of injury, but their shape and timing, especially in the immediate phase, vary almost as consistently with the duration of exposure to circulating dye or carbon.After the standard dose of toxin, vascular patency is largely unaffected until 24 hours. Apparently irreversible vascular occlusion occurs rather earlier with larger doses. A three-fold reduction of the standard dose proportionately reduces both exudation and capillary labelling but leaves immediate venular labelling unaffected, suggesting that the latter is not dose dependent and therefore non-specific. Prolongation of moderate venular labelling into the middle of the delayed phase may occur at this dosage. Its absence after the standard dose suggests that delayed inhibition of venular reactivity may be occurring.Irregular labelling of venules and small veins persists throughout the delayed phase with doses 2·4 or more times the standard dose. A brief ultramicroscopic survey revealed appearances in both venules and capillaries at 1-2 hours after injury closely comparable to those which have been described for Cl. oedematiens toxin at 6-24 hours.In rats given carbon during the delayed phase, the effective vascular patency 10 minutes later includes half of the labelled capillaries up to 10 μm in diameter. This proportion is little affected by toxin dose, but intensely so when the carbon clearance time is increased, suggesting that such injured microvessels may be a major source of plasma protein exudation. 相似文献
98.
99.