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961.
Eun Bae Kim Jee Soo Son Qian Kun Zhang Nam Kyung Lee Sung Hee Kim Jin Huk Choi Sang Kee Kang Yun Jaie Choi 《Current microbiology》2010,61(1):29-36
Genetic engineering of Lactococcus lactis to produce a heterologous protein may cause potential risks to the environment despite the industrial usefulness of engineered
strains. To reduce the risks, we generated three auxotrophic recombinant L. lactis subsp. lactis IL1403 strains expressing a heterologous protein, BmpB, using thyA- and alr-targeting integration vectors: ITD (thyA
−
alr
+
bmpB
+), IAD (thyA
+
alr
−
bmpB
+), and ITDAD (thyA
−
alr
−
bmpB
+). After construction of integration vectors, each vector was introduced into IL1403 genome. Integration of BmpB expression
cassette, deletion of thyA, and inactivation of alr were verified by using PCR reaction. All heterologous DNA fragments except bmpB were eliminated from those recombinants during double crossover events. By using five selective agar plates, we also showed
thymidine auxotrophy of ITD and ITDAD and d-alanine auxotrophy of IAD and ITDAD. In M17G and skim milk (SYG) media, the growth of the three recombinants was limited.
In MRS media, the growth of IAD and ITDAD was limited, but ITD showed a normal growth pattern as compared with the wild-type
strain (WT). All the recombinants showed maximal BmpB expression at an early stationary phase when they were cultivated in
M17G supplemented with thymidine and d-alanine. These results suggest that auxotrophic recombinant L. lactis expressing a heterologous protein could be generated to reduce the ecological risks of a recombinant L. lactis. 相似文献
962.
Marina Vacotto Melina Rapacioli Vladimir Flores Sara Fiszer de Plazas 《Neurochemical research》2010,35(10):1609-1619
It is known that the NMDA-R NR1 subunit is needed for the receptor activity and that under hypoxia the evolution toward apoptosis
or neuronal survival depends on the balance NR2A/NR2B subunits. This paper analyzes the effect of acute hypoxia on the above
mentioned subunits mRNAs during development. The mean percentage of NR1+ neurons displayed the higher plasticity during development
while the NR2A+ neurons the higher stability. Acute hypoxia increased the mean percentage of NR1+ and NR2B+ neurons at ED12
but only that of NR1+ neurons at ED18. Acute hypoxia increased the levels of expression of NR1 and NR2B mRNAs at ED12 without
changes in the NR2A mRNA. During early stages there is a higher sensitivity to change the subunits mRNA levels under a hypoxic
treatment. At ED12 acute hypoxia increased the probability of co-expression of the NR1–NR2A and NR1–NR2B subunits combinations,
the level of NR1 and NR2B and the ratio NR2B/NR2A. These conditions facilitate the evolution towards apoptosis. 相似文献
963.
Casoni GL Ulivi P Mercatali L Chilosi M Tomassetti S Romagnoli M Ravaglia C Gurioli C Gurioli C Zoli W Silvestrini R Poletti V 《The International journal of biological markers》2010,25(4):229-235
Idiopathic pulmonary fibrosis (IPF) is difficult to diagnose because of numerous interstitial lung diseases with similar symptoms. As serum DNA has proven useful for early lung cancer detection, we aimed to define the relevance of this marker in discriminating IPF from other fibrotic and nonfibrotic/nonmalignant lung diseases. DNA was quantified in 191 subjects: 64 healthy individuals, 58 patients with IPF, 17 patients with nonspecific pulmonary fibrosis (13 idiopathic nonspecific interstitial pneumonia, 4 chronic hypersensitivity pneumonitis), and 52 patients with other diffuse/nonmalignant lung diseases. The median value of free DNA in IPF patients was 61.1 ng/mL (range 7.1-405), which was significantly higher than that of healthy donors (median 6.8, range 2.2-184) (p<0.001) and that of patients with other diffuse/nonmalignant lung diseases (median 28.0, range 4.2-281) (p=0.004). The area under the ROC curve was 0.926 (95% CI 0.879-0.973) when IPF patients were compared with healthy donors, and 0.702 (95% CI 0.609-0.796) when a comparison was made with non-IPF pulmonary diseases. In conclusion, we observed significantly higher levels of free circulating DNA in patients with IPF than in those with other fibrotic or diffuse/nonmalignant lung diseases. 相似文献
964.
Parthasarathy Chandrakesan Ishfaq Ahmed Tariq Anwar Yu Wang Shubhashish Sarkar Pomila Singh Sara Peleg Shahid Umar 《The Journal of biological chemistry》2010,285(43):33485-33498
Utilizing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we measured hyperplasia and NF-κB activation during progression (days 6 and 12 post-infection) and regression (days 20–34 post-infection) phases of TMCH. NF-κB activity increased at progression in conjunction with bacterial attachment and translocation to the colonic crypts and decreased 40% by day 20. NF-κB activity at days 27 and 34, however, remained 2–3-fold higher than uninfected control. Expression of the downstream target gene CXCL-1/KC in the crypts correlated with NF-κB activation kinetics. Phosphorylation of cellular IκBα kinase (IKK)α/β (Ser176/180) was elevated during progression and regression of TMCH. Phosphorylation (Ser32/36) and degradation of IκBα, however, contributed to NF-κB activation only from days 6 to 20 but not at later time points. Phosphorylation of MEK1/2 (Ser217/221), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr182) paralleled IKKα/β kinetics at days 6 and 12 without declining with regressing hyperplasia. siRNAs to MEK, ERK, and p38 significantly blocked NF-κB activity in vitro, whereas MEK1/2-inhibitor (PD98059) also blocked increases in MEK1/2, ERK1/2, and IKKα/β thereby inhibiting NF-κB activity in vivo. Cellular and nuclear levels of Ser536-phosphorylated (p65536) and Lys310-acetylated p65 subunit accompanied functional NF-κB activation during TMCH. RSK-1 phosphorylation at Thr359/Ser363 in cellular/nuclear extracts and co-immunoprecipitation with cellular p65-NF-κB overlapped with p65536 kinetics. Dietary pectin (6%) blocked NF-κB activity by blocking increases in p65 abundance and nuclear translocation thereby down-regulating CXCL-1/KC expression in the crypts. Thus, NF-κB activation persisted despite the lack of bacterial attachment to colonic mucosa beyond peak hyperplasia. The MEK/ERK/p38 pathway therefore seems to modulate sustained activation of NF-κB in colonic crypts in response to C. rodentium infection. 相似文献
965.
Achieving successful vascularization remains one of the main problems in bone tissue engineering. After scaffold implantation, the growth of capillaries into the porous construct may be too slow to provide adequate nutrients to the cells in the scaffold interior and this inhibits tissue formation in the scaffold core. Often, prior to implantation, a controlled cell culture environment is used to stimulate cell proliferation and, once in place, the mechanical environment acting on the tissue construct is determined by the loading conditions at the implantation site. To what extent do cell seeding conditions and the construct loading environment have an effect on scaffold vascularization and tissue growth? In this study, a mechano-biological model for tissue differentiation and blood vessel growth was used to determine the influence of cell seeding on vascular network development and tissue growth inside a regular-structured bone scaffold under different loading conditions. It is predicted that increasing the number of cells seeded homogeneously reduces the rate of vascularization and the maximum penetration of the vascular network, which in turn reduces bone tissue formation. The seeding of cells in the periphery of the scaffold was predicted to be beneficial for vascularization and therefore for bone growth; however, tissue formation occurred more slowly during the first weeks after implantation compared to homogeneous seeding. Low levels of mechanical loading stimulated bone formation while high levels of loading inhibited bone formation and capillary growth. This study demonstrates the feasibility of computational design approaches for bone tissue engineering. 相似文献
966.
967.
968.
969.
Pasi Tavi Topi Korhonen Sandra L. Hänninen Joseph D. Bruton Sara Lööf Andras Simon Håkan Westerblad 《Journal of cellular physiology》2010,223(2):376-383
Quiescent satellite cells sit on the surface of the muscle fibres under the basal lamina and are activated by a variety of stimuli to disengage, divide and differentiate into myoblasts that can regenerate or repair muscle fibres. Satellite cells adopt their parent's fibre type and must have some means of communication with the parent fibre. The mechanisms behind this communication are not known. We show here that satellite cells form dynamic connections with muscle fibres and other satellite cells by F‐actin based tunnelling nanotubes (TNTs). Our results show that TNTs readily develop between satellite cells and muscle fibres. Once developed, TNTs permit transport of intracellular material, and even cellular organelles such as mitochondria between the muscle fibre and satellite cells. The onset of satellite cell differentiation markers Pax‐7 and MyoD expression was slower in satellite cells cultured in the absence than in the presence of muscle cells. Furthermore physical contact between myofibre and satellite cell progeny is required to maintain subtype identity. Our data establish that TNTs constitute an integral part of myogenic cell communication and that physical cellular interaction control myogenic cell fate determination. J. Cell. Physiol. 223: 376–383, 2010. © 2010 Wiley‐Liss, Inc. 相似文献
970.
Gianluca Storci Pasquale Sansone Sara Mari Gabriele D'Uva Simona Tavolari Tiziana Guarnieri Mario Taffurelli Claudio Ceccarelli Donatella Santini Pasquale Chieco Kenneth B. Marcu Massimiliano Bonafè 《Journal of cellular physiology》2010,225(3):682-691
Extracellular and intracellular mediators of inflammation, such as tumor necrosis factor alpha (TNFα) and NF‐kappaB (NF‐κB), play major roles in breast cancer pathogenesis, progression and relapse. SLUG, a mediator of the epithelial–mesenchymal transition process, is over‐expressed in CD44+/CD24? tumor initiating breast cancer cells and in basal‐like carcinoma, a subtype of aggressive breast cancer endowed with a stem cell‐like gene expression profile. Cancer stem cells also over‐express members of the pro‐inflammatory NF‐κB network, but their functional relationship with SLUG expression in breast cancer cells remains unclear. Here, we show that TNFα treatment of human breast cancer cells up‐regulates SLUG with a dependency on canonical NF‐κB/HIF1α signaling, which is strongly enhanced by p53 inactivation. Moreover, SLUG up‐regulation engenders breast cancer cells with stem cell‐like properties including enhanced expression of CD44 and Jagged‐1 in conjunction with estrogen receptor alpha down‐regulation, growth as mammospheres, and extracellular matrix invasiveness. Our results reveal a molecular mechanism whereby TNFα, a major pro‐inflammatory cytokine, imparts breast cancer cells with stem cell‐like features, which are connected to increased tumor aggressiveness. J. Cell. Physiol. 225: 682–691, 2010. © 2010 Wiley‐Liss, Inc. 相似文献