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31.
Novel proteins with binding specificity for DNA CTG repeats and RNA CUG repeats: implications for myotonic dystrophy 总被引:7,自引:6,他引:7
While an unstable CTG triplet repeat expansion is responsible for myotonic
dystrophy, the mechanism whereby this genetic defect induces the disease
remains unknown. To detect proteins binding to CTG triplet repeats, we
performed bandshift analysis using as probes double- stranded DNA fragments
having CTG repeats [ds(CTG)6-10] and single- stranded oligonucleotides
having CTG repeats ss(CTG)8 or RNA CUG triplet repeats (CUG)8. The source
of protein was nuclear and cytoplasmic extracts of HeLa cells, fibroblasts
and myotubes. Proteins binding to the double-stranded DNA repeat
[ds(CTG)6-10], were inhibited by nonlabeled ds(CTG)6-10, but not by a
non-specific DNA fragment (USF/AD-ML). Another protein binding to ssCTG
probe and RNA CUG probe was inhibited by nonlabeled (CTG)8 and (CUG)8.
Nonlabeled oligos with different triplet repeat sequences, ss(CAG)8 or
ss(CGG)8, did not inhibit binding to the ss(CTG)8 probe. However, when
labeled as probes, the (CAG)8 and (CGG)8 bound to proteins distinct from
the CTG proteins and binding was inhibited by nonlabeled (CAG)8 or (CGG)8
respectively. The protein binding only to the RNA repeat (CUG)8 was
inhibited by nonlabeled (CUG)8 but not by nonlabeled single- or
double-stranded CTG repeats. Furthermore, the CUG-BP exhibited no binding
to an RNA oligonucleotide of triplet repeats of the same length but having
a different sequence, CGG. The CUG binding protein was localized to the
cytoplasm, whereas dsDNA binding proteins were localized to the nuclear
extract. Thus, several trinucleotide binding proteins exist and their
specificity is determined by the triplet sequence. The novel protein,
CUG-BP, is particularly interesting since it binds to triplet repeats known
to be present in myotonin protein kinase mRNA which is responsible for
myotonic dystrophy.
相似文献
32.
During attempts to generate Sendai virus-specific T-cell hybridomas, a number of autoreactive T-cell hybridomas were produced. These hybrids secrete IL-2 in response to class II-positive syngeneic cells in the absence of added Sendai virus. Class II molecules on the stimulator cells are involved since monoclonal anti-class II antibodies block the reaction, and mapping studies using recombinant mouse strains show the response is class II restricted. Hybridomas adapted to grow in serum-free medium do not respond to syngeneic cells in the absence of serum; however, addition of fetal calf serum (FCS) restores the response. The component of FCS responsible for the stimulation of the hybridomas has been partially purified and characterized, and its mode of action investigated. These findings suggest that the putative 'autoreactive' T-cell hybridomas reported by others may be specific for a component of the culture medium rather than being truly self-reactive. 相似文献
33.
Cariogenicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by allelic exchange. 总被引:7,自引:10,他引:7 下载免费PDF全文
Streptococcus mutans produces several enzymes which metabolize sucrose. Three glucosyltransferase genes (gtfB, gtfC, and gtfD) and a single fructosyltransferase gene (ftf) encode enzymes which are important in formation of exopolysaccharides. Mutants of S. mutans V403 carrying single and multiple mutations of the gtfB, gtfC, gtfD, and ftf genes recently have been constructed by allelic exchange in our laboratory. Using selected strains from this panel of mutants, we examined the importance of water-insoluble glucan, water-soluble glucan, and fructan production in cariogenicity while controlling for the effects of strain and species variability. Genetic and biochemical characterization of mutants and assays of glucosyltransferase and fructosyltransferase activities were performed to ensure that the phenotypes of strains coincided with deficiencies predicted by genotype. The young gnotobiotic rat model of cariogenicity was used to assess virulence of the wild-type strain and isogenic mutants. Mutant strains were less virulent than the wild type in almost every location examined for caries on tooth surfaces and level of involvement of lesions (depth and severity). Inactivation of either gtfB and gtfC or ftf dramatically reduced virulence; the subsequent inactivation of gtfD did not enhance the effect of reduced virulence. 相似文献
34.
Pneumolysin induces the salient histologic features of pneumococcal infection in the rat lung in vivo. 总被引:11,自引:0,他引:11
C Feldman N C Munro P K Jeffery T J Mitchell P W Andrew G J Boulnois D Guerreiro J A Rohde H C Todd P J Cole 《American journal of respiratory cell and molecular biology》1991,5(5):416-423
Streptococcus pneumoniae infections are common, but how they cause host tissue injury and death is incompletely understood. Immunization with pneumolysin, a thiol-activated toxin produced by the pneumococcus, partially protects animals during subsequent infection. The mechanism by which pneumolysin contributes to disease is not known. The aim of the present investigation was to determine the histologic changes induced by recombinant pneumolysin in the rat lung and to compare them with the changes induced by live organisms. Injection of either toxin (200 or 800 ng) or bacteria into the apical lobe bronchus was associated with the development of a severe lobar pneumonia restricted to the apical lobe. The changes induced by the toxin were greater at the higher concentration, and changes were most severe in those animals in which there was partial ligation of the apical lobe bronchus. The pneumonitis was less severe following injection of a modified toxin with decreased hemolytic activity, generated by site-directed mutagenesis of the cloned pneumolysin gene, indicating that this property of the toxin was important in generating pulmonary inflammation. There was still considerable pneumonitis after injection of a modified toxin with decreased capacity to activate complement. 相似文献
35.
Bersinger NA; Brandenberger A; Berger E; Baumann CK; Birkhauser MH 《Human reproduction (Oxford, England)》1998,13(7):1962-1967
We have previously observed the repeated presence of low but detectable
amounts of the trophoblast marker pregnancy-specific beta1-glycoprotein
(SP1) in the serum of some women undergoing in-vitro fertilization (IVF)
treatment around the time of oocyte retrieval. The occurrence of these
signals seemed to be restricted to a defined group of patients which also
showed a lower pregnancy success rate in a preliminary study. To test our
hypothesis we have analysed 173 consecutive cycles leading to an embryo
transfer. Fifty-four cycles (31%) had a serum SP1 level of at least 0.1
ng/ml between days embryo transfer -5 and embryo transfer (group A). Five
pregnancies were obtained in this group (pregnancy rate = 9.3%), while in
group B, defined by the absence of detectable SP1 before embryo transfer
(119 cycles), 36 ongoing pregnancies were achieved (30.3%). Ten of the 41
pregnancies were achieved in 33 first-time non-pregnant patients undergoing
further attempts during the study period. Again the pregnancy rate was
higher in the first-time group B women (9/23 versus 1/10 for group A).
Patients tended to remain in their groups A or B, the latter being
associated with a better immediate as well as subsequent chance for
pregnancy. Group A cycles had a significantly lower endometrial thickness
two days before oocyte retrieval than group B (P = 0.0011). We postulate
that the presence of an unknown, maternal and progesterone- or follicle
stimulating hormone-independent factor in some patients could stimulate
tonic ectopic SP1 synthesis and at the same time negatively influence
endometrial development.
相似文献
36.
Tissue sections and cultured lymphocytes from rabbits clinically affected following experimental infection with Alcelaphine herpesvirus-1 (AHV-1) were assessed for the presence of viral DNA by in situ hybridization with the cloned major HindII repeat sequence of this virus. Small numbers of virus-infected cells were consistently detected only in submandibular lymph nodes, while other tissues showed no evidence of viral DNA. Virus titration in culture suggested that there were higher titres of virus in the lymph nodes, spleen and lung of infected animals than in the kidney or peripheral blood lymphocytes and confirmed the low level of virus in these animals. Substantially more viral DNA was detected by in situ hybridization in lymphocytes following at least 24 h of culture, suggesting that viral replication is normally repressed by the host. 相似文献
37.
Differential regulation of leucocyte L-selectin (CD62L) expression in normal lymphoid and inflamed extralymphoid tissues. 总被引:2,自引:0,他引:2 下载免费PDF全文
AIMS: To study tissue expression of L-selectin, a leucocyte cell surface molecule that is considered to be involved in adhesion to certain endothelia, particularly in peripheral lymph nodes and during inflammation, and is shed upon leucocyte activation. METHODS: Leucocytes were examined by immunohistochemistry and double immunofluorescence staining in various lymphoid sites and normal and inflamed extralymphoid tissues. RESULTS: L-selectin was present on mantle zone B lymphocytes in different lymphoid sites, including in intestinal lymphoid tissue, but was absent on germinal centre B cells. Splenic white pulp B cells also expressed L-selectin. The proportion of T lymphocytes expressing L-selectin depended on the site under study, being greatest in peripheral lymph nodes (mean 48% of T cells positive), and lower in mucosal lymphoid sites and spleen (9 and 11% positive, respectively). Non-lymphocytic L-selectin staining was observed on follicular dendritic cells in tonsils and on macrophages in thymus. L-selectin positive leucocytes were rare in normal extralymphoid tissues, and relatively few were seen in most inflammatory settings. However, in rejecting renal transplants, a higher proportion (30%) of leucocytes expressed L-selectin. CONCLUSIONS: Overall, the results indicate how the degree of L-selectin expression by leucocytes in particular tissues may reflect a requirement for L-selectin expression for entry into those tissues and the activation state of leucocytes once localised there. 相似文献
38.
Early cellular responses to intradermal injection of Kveim suspension in normal subjects and those with sarcoidosis. 总被引:2,自引:0,他引:2 下载免费PDF全文
In a detailed controlled study of the cellular response to Kveim suspension in vivo we used immunohistological and histochemical methods to examine cryostat sections of immature Kveim biopsy specimens in subjects with sarcoidosis and normal controls. Changes seen at 48 hours, at which time papular reactions have sometimes been reported, are described. Eight cases of sarcoidosis previously confirmed by a positive Kveim test were studied, in five of whom the test remained positive; plus two subjects with sarcoidosis studied prospectively; and four healthy controls. There were two main features of the 48 hour response: collagen disruption with associated histiocytes, which showed increased acid phosphatase activity; and perivascular infiltrates of lymphocytes and small groups of dendritic cells. The T4:T8 ratios in the infiltrates were similar to those found in the peripheral blood of the subjects, and few lymphocytes showed evidence of activation. T lymphocytes were also seen free in the dermis and migrating to the epidermis. Small juxtacapillary clumps of dendritic cells, identified by NA1/34 (= OKT6; Langerhans' cells) and RFD1 (interdigitating cell) monoclonal antibodies, were found. The Langerhans' cells in the epidermis were, however, normal in number and distribution. These features, which were found in all groups, are not consistent with pre-existing hypersensitivity to Kveim suspension in sarcoidosis. Subsequent differences between sarcoid and normal subjects in the development of granulomas in the Kveim response may therefore relate to the different handling of the foreign material by the cells affected, rather than to differences in the early non-specific recruitment of the cells to the test site. 相似文献
39.
Seven murine lymphomas and three plasmacytomas were examined for the distribution of the theta antigen, immunoglobulin determinants, the receptor for Fc portion of antigen—antibody complexes and the receptor for the third component of complement. The theta-bearing tumours lacked C3 and Fc receptors and easily detectable surface immunoglobulin. The theta-negative lymphomas, while being morphologically lymphocytic, lacked all but the Fc receptors. One of the plasmacytomas possessed clearly detectable surface immunoglobulin. All three lacked the receptors for Fc and C3. 相似文献
40.