全文获取类型
收费全文 | 57648篇 |
免费 | 3819篇 |
国内免费 | 23篇 |
出版年
2023年 | 298篇 |
2022年 | 665篇 |
2021年 | 1435篇 |
2020年 | 835篇 |
2019年 | 1094篇 |
2018年 | 1470篇 |
2017年 | 1221篇 |
2016年 | 2020篇 |
2015年 | 3065篇 |
2014年 | 3360篇 |
2013年 | 4388篇 |
2012年 | 5066篇 |
2011年 | 4880篇 |
2010年 | 3000篇 |
2009年 | 2573篇 |
2008年 | 3603篇 |
2007年 | 3445篇 |
2006年 | 3094篇 |
2005年 | 2829篇 |
2004年 | 2620篇 |
2003年 | 2506篇 |
2002年 | 2165篇 |
2001年 | 407篇 |
2000年 | 305篇 |
1999年 | 413篇 |
1998年 | 487篇 |
1997年 | 351篇 |
1996年 | 328篇 |
1995年 | 305篇 |
1994年 | 299篇 |
1993年 | 297篇 |
1992年 | 179篇 |
1991年 | 190篇 |
1990年 | 195篇 |
1989年 | 148篇 |
1988年 | 144篇 |
1987年 | 117篇 |
1986年 | 103篇 |
1985年 | 139篇 |
1984年 | 126篇 |
1983年 | 110篇 |
1982年 | 109篇 |
1981年 | 112篇 |
1980年 | 104篇 |
1979年 | 99篇 |
1978年 | 70篇 |
1977年 | 77篇 |
1976年 | 60篇 |
1975年 | 59篇 |
1973年 | 56篇 |
排序方式: 共有10000条查询结果,搜索用时 328 毫秒
991.
Emanuela Anna Roselli Silvia Lazzati Federico Iseppon Massimiliano Manganini Livia Marcato Marzia Bruna Gariboldi Federico Maggi Francesca Romana Grati Giuseppe Simoni 《Cytotherapy》2013,15(11):1340-1351
Background aimsFirst-trimester chorionic villi (CV) are an attractive source of human mesenchymal stromal cells (hMSC) for possible applications in cellular therapy and regenerative medicine. Human MSC from CV were monitored for genetic stability in long-term cultures.MethodsWe set up a good manufacturing practice cryopreservation procedure for small amounts of native CV samples. After isolation, hMSC were in vitro cultured and analyzed for biological end points. Genome stability at different passages of expansion was explored by karyotype, genome-wide array-comparative genomic hybridization and microsatellite genotyping.ResultsGrowth curve analysis revealed a high proliferative potential of CV-derived cells. Immunophenotyping showed expression of typical MSC markers and absence of hematopoietic markers. Analysis of multilineage potential demonstrated efficient differentiation into adipocytes, osteocytes, chondrocytes and induction of neuro-glial commitment. In angiogenic experiments, differentiation in endothelial cells was detected by in vitro Matrigel assay after vascular endothelial growth factor stimulation. Data obtained from karyotyping, array-comparative genomic hybridization and microsatellite genotyping comparing early with late DNA passages did not show any genomic variation at least up to passage 10. Aneuploid clones appeared in four of 14 cases at latest passages, immediately before culture growth arrest.ConclusionsOur findings indicate that hCV-MSC are genetically stable in long-term cultures at least up to passage 10 and that it is possible to achieve clinically relevant amounts of hCV-MSC even after few stages of expansion. Genome abnormalities at higher passages can occasionally occur and are always associated with spontaneous growth arrest. Under these circumstances, hCV-MSC could be suitable for therapeutic purposes. 相似文献
992.
Maria C Romay Mark J Millard Jeffrey C Glaubitz Jason A Peiffer Kelly L Swarts Terry M Casstevens Robert J Elshire Charlotte B Acharya Sharon E Mitchell Sherry A Flint-Garcia Michael D McMullen James B Holland Edward S Buckler Candice A Gardner 《Genome biology》2013,14(6):R55
Background
Genotyping by sequencing, a new low-cost, high-throughput sequencing technology was used to genotype 2,815 maize inbred accessions, preserved mostly at the National Plant Germplasm System in the USA. The collection includes inbred lines from breeding programs all over the world.Results
The method produced 681,257 single-nucleotide polymorphism (SNP) markers distributed across the entire genome, with the ability to detect rare alleles at high confidence levels. More than half of the SNPs in the collection are rare. Although most rare alleles have been incorporated into public temperate breeding programs, only a modest amount of the available diversity is present in the commercial germplasm. Analysis of genetic distances shows population stratification, including a small number of large clusters centered on key lines. Nevertheless, an average fixation index of 0.06 indicates moderate differentiation between the three major maize subpopulations. Linkage disequilibrium (LD) decays very rapidly, but the extent of LD is highly dependent on the particular group of germplasm and region of the genome. The utility of these data for performing genome-wide association studies was tested with two simply inherited traits and one complex trait. We identified trait associations at SNPs very close to known candidate genes for kernel color, sweet corn, and flowering time; however, results suggest that more SNPs are needed to better explore the genetic architecture of complex traits.Conclusions
The genotypic information described here allows this publicly available panel to be exploited by researchers facing the challenges of sustainable agriculture through better knowledge of the nature of genetic diversity. 相似文献993.
994.
Paola Locatelli Fernanda Daniela Olea Anna Hnatiuk Diana Sepúlveda Juan Manuel Pérez Sáez Rafael Argüello Alberto Crottogini 《Cytotherapy》2013,15(2):163-170
Background aimsGiven the close similarity between ovine and human cardiomyocytes, sheep models of myocardial infarction and heart failure are increasingly used in studies of stem cell-mediated heart regeneration. In these studies, mesenchymal stromal cells (MSCs) are frequently employed. To enhance the paracrine effects of these MSCs, ex vivo transfection with genes encoding growth factors has been proposed. Although viral vectors exhibit higher transfection efficiency than plasmids, they entail the risks of uncontrolled transgene expression and immune reactions that preclude repeated administration. Our aim was to optimize the efficiency of plasmid-mediated transfection of ovine MSCs, while preserving cell viability.MethodsVarying amounts of diverse cationic lipids were used to obtain the reagent-to-DNA mass ratio showing highest luciferase activity. Transfection efficiency (flow cytometry) was tested on plasmid-green fluorescent protein-transfected MSCs at increasing DNA mass.ResultsLipofectamine LTX 5 μL and Plus reagent 4 μL with 2 μg of DNA yielded 42.3 ± 4.7% transfection efficiency, while preserving cell viability. Using these transfection conditions, we transfected MSCs with a plasmid encoding human vascular endothelial growth factor (VEGF) and found high VEGF protein concentrations in the culture supernatant from day 2 (1968 ± 324 pg/mL per μg DNA) through at least day 12 (888 ± 386 pg/mL per μg DNA) after transfection.ConclusionsPlasmid-mediated transfection of ovine MSCs to over-express paracrine heart-regenerative growth factors is feasible and efficient and overcomes the risks and limitations associated with the use of viral vectors. 相似文献
995.
Pedro Casado Maria P Alcolea Francesco Iorio Juan-Carlos Rodríguez-Prados Bart Vanhaesebroeck Julio Saez-Rodriguez Simon Joel Pedro R Cutillas 《Genome biology》2013,14(4):R37
Background
Tumor classification based on their predicted responses to kinase inhibitors is a major goal for advancing targeted personalized therapies. Here, we used a phosphoproteomic approach to investigate biological heterogeneity across hematological cancer cell lines including acute myeloid leukemia, lymphoma, and multiple myeloma.Results
Mass spectrometry was used to quantify 2,000 phosphorylation sites across three acute myeloid leukemia, three lymphoma, and three multiple myeloma cell lines in six biological replicates. The intensities of the phosphorylation sites grouped these cancer cell lines according to their tumor type. In addition, a phosphoproteomic analysis of seven acute myeloid leukemia cell lines revealed a battery of phosphorylation sites whose combined intensities correlated with the growth-inhibitory responses to three kinase inhibitors with remarkable correlation coefficients and fold changes (> 100 between the most resistant and sensitive cells). Modeling based on regression analysis indicated that a subset of phosphorylation sites could be used to predict response to the tested drugs. Quantitative analysis of phosphorylation motifs indicated that resistant and sensitive cells differed in their patterns of kinase activities, but, interestingly, phosphorylations correlating with responses were not on members of the pathway being targeted; instead, these mainly were on parallel kinase pathways.Conclusion
This study reveals that the information on kinase activation encoded in phosphoproteomics data correlates remarkably well with the phenotypic responses of cancer cells to compounds that target kinase signaling and could be useful for the identification of novel markers of resistance or sensitivity to drugs that target the signaling network. 相似文献996.
Christopher I Keeling Macaire MS Yuen Nancy Y Liao T Roderick Docking Simon K Chan Greg A Taylor Diana L Palmquist Shaun D Jackman Anh Nguyen Maria Li Hannah Henderson Jasmine K Janes Yongjun Zhao Pawan Pandoh Richard Moore Felix AH Sperling Dezene P W Huber Inanc Birol Steven JM Jones Joerg Bohlmann 《Genome biology》2013,14(3):R27
997.
Susana Freitas Anna Vavakou Marine Arakelyan Sergei V. Drovetski Jelka Crnobrnja-isailović Artem A. Kidov 《分类学与生物多样性》2013,11(2):184-197
Darevskia praticola differs from the other species of the genus in having a large but disjunct distribution, covering the Balkan and the Caucasus regions. Furthermore, most Darevskia species occupy saxicolous habitats, whereas D. praticola inhabits meadows and forest environments. Here we determine the phylogeographic and phylogenetic relationships of Darevskia praticola sensu lato and evaluate the current, morphology-based taxonomy. We sequenced two mtDNA genes (Cyt-b and ND4) and two nuclear loci (MC1R and RELN) for samples collected across the species range. Because our sequences amplified with the Cyt-b primers appear to represent a nuclear pseudogene we excluded this marker from the final analysis. Our results support monophyly of D. praticola and show its division into three clades. The first divergence, dated to the Late Pliocene, is between the Balkans and the Caucasus. The Caucasus lineage is further subdivided in a western Greater Caucasus and a Transcaucasia clade, likely due to subsequent differentiation during the Pleistocene. Our findings do not support the current taxonomic arrangement within D. praticola. The main geographic divergence likely happened due to a vicariance event associated with Plio-Pleistocene climatic and vegetation oscillations. 相似文献
998.
Cecilia Marini Barbara Salani Michela Massollo Adriana Amaro Alessia Isabella Esposito Anna Maria Orengo Selene Capitanio Laura Emionite Mattia Riondato Gianluca Bottoni Cinzia Massara Simona Boccardo Marina Fabbi Cristina Campi Silvia Ravera Giovanna Angelini Silvia Morbelli Michele Cilli Renzo Cordera Mauro Truini Davide Maggi Ulrich Pfeffer Gianmario Sambuceti 《Cell cycle (Georgetown, Tex.)》2013,12(22):3490-3499
Emerging evidence suggests that metformin, a widely used anti-diabetic drug, may be useful in the prevention and treatment of different cancers. In the present study, we demonstrate that metformin directly inhibits the enzymatic function of hexokinase (HK) I and II in a cell line of triple-negative breast cancer (MDA-MB-231). The inhibition is selective for these isoforms, as documented by experiments with purified HK I and II as well as with cell lysates. Measurements of 18F-fluoro-deoxyglycose uptake document that it is dose- and time-dependent and powerful enough to virtually abolish glucose consumption despite unchanged availability of membrane glucose transporters. The profound energetic imbalance activates phosphorylation and is subsequently followed by cell death. More importantly, the “in vivo” relevance of this effect is confirmed by studies of orthotopic xenografts of MDA-MB-231 cells in athymic (nu/nu) mice. Administration of high drug doses after tumor development caused an evident tumor necrosis in a time as short as 48 h. On the other hand, 1 mo metformin treatment markedly reduced cancer glucose consumption and growth. Taken together, our results strongly suggest that HK inhibition contributes to metformin therapeutic and preventive potential in breast cancer. 相似文献
999.
Didac Carmona-Gutierrez Ali Alavian-Ghavanini Lukas Habernig Maria Anna Bauer Astrid Hammer Christine Rossmann Andreas S. Zimmermann Christoph Ruckenstuhl Sabrina Büttner Tobias Eisenberg Wolfgang Sattler Ernst Malle Frank Madeo 《Cell cycle (Georgetown, Tex.)》2013,12(11):1704-1712
Following microbial pathogen invasion, the human immune system of activated phagocytes generates and releases the potent oxidant hypochlorous acid (HOCl), which contributes to the killing of menacing microorganisms. Though tightly controlled, HOCl generation by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils/monocytes may occur in excess and lead to tissue damage. It is thus of marked importance to delineate the molecular pathways underlying HOCl cytotoxicity in both microbial and human cells. Here, we show that HOCl induces the generation of reactive oxygen species (ROS), apoptotic cell death and the formation of specific HOCl-modified epitopes in the budding yeast Saccharomyces cerevisiae. Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect. The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification. By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes. 相似文献
1000.
Tremendous efforts have been made over the past few decades to discover novel cancer biomarkers for use in clinical practice. However, a striking discrepancy exists between the effort directed toward biomarker discovery and the number of markers that make it into clinical practice. One of the confounding issues in translating a novel discovery into clinical practice is that quite often the scientists working on biomarker discovery have limited knowledge of the analytical, diagnostic, and regulatory requirements for a clinical assay. This review provides an introduction to such considerations with the aim of generating more extensive discussion for study design, assay performance, and regulatory approval in the process of translating new proteomic biomarkers from discovery into cancer diagnostics. We first describe the analytical requirements for a robust clinical biomarker assay, including concepts of precision, trueness, specificity and analytical interference, and carryover. We next introduce the clinical considerations of diagnostic accuracy, receiver operating characteristic analysis, positive and negative predictive values, and clinical utility. We finish the review by describing components of the FDA approval process for protein-based biomarkers, including classification of biomarker assays as medical devices, analytical and clinical performance requirements, and the approval process workflow. While we recognize that the road from biomarker discovery, validation, and regulatory approval to the translation into the clinical setting could be long and difficult, the reward for patients, clinicians and scientists could be rather significant. 相似文献