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1.
M Orita  Y Suzuki  T Sekiya  K Hayashi 《Genomics》1989,5(4):874-879
We report a rapid and sensitive method for the detection of base changes in given sequences of genomic DNA. This technique is based on the facts that specific regions of genomic sequences can be efficently labeled and amplified simultaneously by using labeled substrates in the polymerase chain reaction and that in nondenaturing polyacrylamide gels, the electrophoretic mobility of single-stranded nucleic acid depends not only on its size but also on its sequence. The process does not involve restriction enzyme digestion, blotting, or hybridization to probes. We found that most single base changes in up to 200-base fragments could be detected as mobility shifts. RAS oncogene activation was detected by this technique. We also show that the interspersed repetitive sequences of human, Alu repeats are highly polymorphic.  相似文献
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BMP-6 enhances chondrogenesis in a subpopulation of human marrow stromal cells   总被引:42,自引:0,他引:42  
Marrow stromal cells (MSCs) can differentiate into several mesenchymal lineages. MSCs were recently shown to form cartilage in micromass cultures with serum-free medium containing TGF-beta and dexamethasone. Here we found that addition of BMP-6 increased the weight of the pellets about 10-fold and they stained more extensively for proteoglycans. mRNAs for type II procollagen and type X collagen were detected at 1 week and the levels were increased at 3 weeks. We also compared two subpopulation of cultures of MSCs: Small and rapidly self-renewing cells (RS cells) and the large, more mature and slowly replicating cells (mMSCs). The cartilage pellets prepared from cultures enriched for RS cells were about 2.5-fold larger, stained more extensively for proteoglycans, and had levels of mRNA for type II procollagen that were 1.6-fold higher. Also, RS cells retained more of their chondrogenic potential as the cells were passaged.  相似文献
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A new technique is described for amplifying individual alleles in a mixture of two or more alleles by the polymerase chain reaction (PCR) to determine their nucleotide sequence. This technique involves amplifying and separating target sequences by the PCR-mediated single-strand conformation polymorphism (PCR-SSCP) method, isolating each polymorphic DNA strand, and amplifying it by a second-stage PCR for its sequence determination. By this technique, the sequence of a minor constituent (approximately 3%) can be determined accurately.  相似文献
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CCR4 is now known to be selectively expressed in Th2 cells. Since the bronchial epithelium is recognized as an important source of mediators fundamental to the manifestation of respiratory allergic inflammation, we studied the expression of two functional ligands for CCR4, i.e., macrophage-derived chemokine (MDC) and thymus- and activation-regulated chemokine (TARC), in bronchial epithelial cells. The bronchial epithelium of asthmatics and normal subjects expressed TARC protein, and the asthmatics showed more intense expression than the normal subjects. On the other hand, MDC expression was only weakly detected in the asthmatics, but the intensity was not significantly different from that of normal subjects. Combination of TNF-alpha and IL-4 induced expression of TARC protein and mRNA in bronchial epithelial A549 cells, which was slightly up-regulated by IFN-gamma. The enhancement by IFN-gamma was more pronounced in bronchial epithelial BEAS-2B cells, and a maximum production occurred with combination of TNF-alpha, IL-4, and IFN-gamma. On the other hand, MDC was essentially not expressed in any of the cultures. Furthermore, expressions of TARC protein and mRNA were almost completely inhibited by glucocorticoids. These results indicate that the airway epithelium represents an important source of TARC, which potentially plays a role via a paracrine mechanism in the development of allergic respiratory diseases. Furthermore, the beneficial effect of inhaled glucocorticoids on asthma may be at least in part due to their direct inhibitory effect on TARC generation by the bronchial epithelium.  相似文献
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Mesenchymal stem cells (MSCs) are increasingly being reported as occurring in a variety of tissues. Although MSCs from human bone marrow are relatively easy to harvest, the isolation of rodent MSCs is more difficult, thereby limiting the number of experiments in vivo. To determine a suitable cell source, we isolated rat MSCs from bone marrow, synovium, periosteum, adipose, and muscle and compared their properties for yield, expansion, and multipotentiality. After two passages, the cells in each population were CD11b (−), CD45 (−), and CD90 (+). The colony number per nucleated cells derived from synovium was 100-fold higher than that for cells derived from bone marrow. With regard to expansion potential, synovium-derived cells were the highest in colony-forming efficiency, fold increase, and growth kinetics. An in vitro chondrogenesis assay demonstrated that the pellets derived from synovium were heavier, because of their greater production of cartilage matrix, than those from other tissues, indicating their superiority in chondrogenesis. Synovium-derived cells retained their chondrogenic potential after a few passages. The Oil Red-O positive colony-rate assay demonstrated higher adipogenic potential in synovium- and adipose-derived cells. Alkaline phosphatase activity was greater in periosteum- and muscle-derived cells during calcification. The yield and proliferation potential of rat MSCs from solid tissues was much better than those from bone marrow. In particular, synovium-derived cells had the greatest potential for both proliferation and chondrogenesis, indicating their usefulness for cartilage study in a rat model. This study was supported in part by grants from the Japan Latest Osteoarthritis Society and from the Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone in Tokyo Medical and Dental University (to T.M.), and by the Japan Society for the Promotion of Science (grant no. 18591657 to I.S.). Recombinant human bone morphogenetic protein-2 was kindly provided by Astellas Pharma.  相似文献
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