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101.
Lindenbaum M Perkins E Csonka E Fleming E Garcia L Greene A Gung L Hadlaczky G Lee E Leung J MacDonald N Maxwell A Mills K Monteith D Perez CF Shellard J Stewart S Stodola T Vandenborre D Vanderbyl S Ledebur HC 《Nucleic acids research》2004,32(21):e172
Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK− and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed. 相似文献
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Isolation of nuclei from label-retaining cells and measurement of their turnover rates in rat colon 总被引:3,自引:0,他引:3
Kim SJ Cheung S Hellerstein MK 《American journal of physiology. Cell physiology》2004,286(6):C1464-C1473
We describe here a new technique for isolating nuclei from long-term label-retaining cells (LRCs), a subpopulation enriched with stem cells from colon, and for measuring their proliferation rates in vivo. A double-label approach was developed, combining the use of bromodeoxyuridine (BrdU) and 2H2O. Male Fisher 344 rats were administered BrdU in drinking water continuously for 28 wk. BrdU was then discontinued (BrdU washout), and animals (n = 33) were switched to 2H2O in drinking water and killed after 2, 4, and 8 wk. Nuclei from BrdU-positive cells (LRCs) were collected by flow cytometry. The percentages of LRCs were 7 and 3.8% after 4 and 8 wk of BrdU washout, respectively. Turnover rates of LRCs were measured on the basis of deuterium incorporation from 2H2O into DNA of LRC nuclei, as determined by mass spectrometry. The proliferation rate of the LRCs collected was 0.330.90% per day (half-life of 77210 days). Significant contamination from other potentially long-lived colon cells was excluded. In conclusion, this double-labeling method allows both physical isolation of nuclei from colon epithelial LRCs and measurement of their in vivo proliferation rates. Use of this approach may allow better understanding of mechanisms by which agents induce or protect against colon carcinogenesis. carcinogenesis; deuterated water; long-term label-retaining cells; stable isotopes 相似文献
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Sibanda W Pillay V Danckwerts MP Viljoen AM van Vuuren S Khan RA 《AAPS PharmSciTech》2004,5(1):128-141
A Plackett-Burman design was employed to develop and optimize a novel crosslinked calcium-aluminum-alginatepectinate oilisphere
complex as a potential system for the in vitro site-specific release ofMentha piperita, an essential oil used for the treatment of irritable bowel syndrome. The physicochemical and textural properties (dependent
variables) of this complex were found to be highly sensitive to changes in the concentration of the polymers (0%–1.5% wt/vol),
crosslinkers (0%–4% wt/vol) and crosslinking reaction times (0.5–6 hours) (independent variables). Particle size analysis
indicated both unimodal and bimodal populations with the highest frequency of 2 mm oilispheres. Oil encapsulation ranged from
6 to 35 mg/100 mg oilispheres. Gravimetric changes of the crosslinked matrix indicated significant ion sequestration and loss
in an exponential manner, while matrix erosion followed Higuchi's cube root law. Among the various measured responses, the
total fracture energy was the most suitable optimization objective (R
2 =0.88, Durbin-Watson Index=1.21%, Coefficient of Variation (CV)=33.21%). The Lagrangian technique produced no significant
differences (P>.05) between the experimental and predicted total fracture energy values (0.0150 vs 0.0107 J). Artificial Neural Networks,
as an alternative predictive tool of the total fracture energy, was highly accurate (final mean square error of optimal network
epoch≈0.02). Fused-coated optimized oilispheres produced a 4-hour lag phase followed by zero-order kinetics (n>0.99), whereby
analysis of release data indicated that diffusion (Fickian constantk
1=0.74 vs relaxation constantk
2=0.02) was the predominant release mechanism. 相似文献
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Carvajal-Carmona LG Ophoff R Service S Hartiala J Molina J Leon P Ospina J Bedoya G Freimer N Ruiz-Linares A 《Human genetics》2003,112(5-6):534-541
We report a comparative genetic characterization of two population isolates with parallel demographic histories: the Central Valley of Costa Rica (CVCR) and Antioquia (in northwest Colombia). The analysis of mtDNA, Y-chromosome and autosomal polymorphisms shows that Antioquia and the CVCR are genetically very similar, indicating that closely related parental populations founded these two isolates. In both populations, the male ancestry is predominantly European, whereas the female ancestry is mostly Amerind. In agreement with their isolation, the Amerindian mtDNA diversity of Antioquia and the CVCR is typical of ethnically-defined native populations and is markedly lower than in other Latin American populations. A comparison of linkage disequilibrium (LD) at 18 marker pairs in Antioquia and the CVCR shows that markers in LD in both populations are located at short genetic distances (相似文献