全文获取类型
收费全文 | 4933篇 |
免费 | 80篇 |
国内免费 | 53篇 |
出版年
2024年 | 2篇 |
2023年 | 24篇 |
2022年 | 24篇 |
2021年 | 45篇 |
2020年 | 30篇 |
2019年 | 37篇 |
2018年 | 43篇 |
2017年 | 34篇 |
2016年 | 227篇 |
2015年 | 643篇 |
2014年 | 1027篇 |
2013年 | 665篇 |
2012年 | 680篇 |
2011年 | 384篇 |
2010年 | 205篇 |
2009年 | 127篇 |
2008年 | 97篇 |
2007年 | 111篇 |
2006年 | 87篇 |
2005年 | 83篇 |
2004年 | 95篇 |
2003年 | 57篇 |
2002年 | 59篇 |
2001年 | 26篇 |
2000年 | 28篇 |
1999年 | 24篇 |
1998年 | 27篇 |
1997年 | 25篇 |
1996年 | 18篇 |
1995年 | 18篇 |
1994年 | 22篇 |
1993年 | 14篇 |
1992年 | 13篇 |
1991年 | 11篇 |
1990年 | 4篇 |
1989年 | 4篇 |
1988年 | 7篇 |
1985年 | 2篇 |
1984年 | 7篇 |
1983年 | 2篇 |
1982年 | 6篇 |
1981年 | 3篇 |
1980年 | 2篇 |
1979年 | 1篇 |
1978年 | 2篇 |
1977年 | 2篇 |
1976年 | 2篇 |
1975年 | 2篇 |
1974年 | 4篇 |
1973年 | 3篇 |
排序方式: 共有5066条查询结果,搜索用时 15 毫秒
981.
Lynne A. Fieber Stephen L. Carlson Andrew T. Kempsell Justin B. Greer Michael C. Schmale 《Journal of visualized experiments : JoVE》2013,(77)
The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum. 相似文献
982.
983.
Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes. 相似文献
984.
Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices. 相似文献
985.
Daniel Goldreich Michael Wong Ryan M. Peters Ingrid M. Kanics 《Journal of visualized experiments : JoVE》2009,(28)
Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering controlled spatially structured stimuli to the skin. Consequently, investigators often apply tactile stimuli manually. Manual stimulus application is time consuming, requires great care and concentration on the part of the investigator, and leaves many stimulus parameters uncontrolled. We describe here a computer-controlled tactile stimulus system, the Tactile Automated Passive-finger Stimulator (TAPS), that applies spatially structured stimuli to the skin, controlling for onset velocity, contact force, and contact duration. TAPS is a versatile, programmable system, capable of efficiently conducting a variety of psychophysical procedures. We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.Corresponding Author: Daniel Goldreich 相似文献
986.
Mark L. Ormiston Mark R. Toshner Fedir N. Kiskin Christopher J. Z. Huang Emily Groves Nicholas W. Morrell Amer A. Rana 《Journal of visualized experiments : JoVE》2015,(106)
Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression. 相似文献
987.
Pierfrancesco Pagella Shayee Miran Tim Mitsiadis 《Journal of visualized experiments : JoVE》2015,(102)
Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment. 相似文献
988.
Martha M. Robinson Jonathan M. Martin Harold L. Atwood Robin L. Cooper 《Journal of visualized experiments : JoVE》2011,(47)
This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors. 相似文献
989.
Rubén Martín Marita Hernández Elvira Ibeas Lucia Fuentes Veronica Salicio Mercedes Arnés María Luisa Nieto 《Journal of neurochemistry》2009,111(4):988-999
Human group IIA secreted phospholipase A2 (sPLA2 -IIA) has been characterized in numerous inflammatory and neoplastic conditions. sPLA2 -IIA can either promote or inhibit cell growth depending on the cellular type and the specific injury. We have previously demonstrated that exogenous sPLA2 -IIA, by engagement to a membrane structure, induces proliferation and activation of mitogen-activated protein kinases cascade in human astrocytoma cells. In this study, we used human astrocytoma 1321N1 cells to investigate the key molecules mediating sPLA2 -IIA-induced cell proliferation. We found that sPLA2 -IIA promoted reactive oxygen species (ROS) accumulation, which was abrogated in the presence of allopurinol and DPI, but not by rotenone, discarding mitochondria as a ROS source. In addition, sPLA2 -IIA triggered Ras and Raf-1 activation, with kinetics that paralleled ERK phosphorylation, and co-immunoprecipitation assays indicated an association between Ras, Raf-1 and ERK. Additionally, Akt, p70 ribosomal protein S6 kinase, and S6 ribosomal protein were also phosphorylated upon sPLA2 -IIA treatment, effect that was abrogated by N -acetylcysteine or LY294002 treatment indicating that ROS and phosphatidylinositol 3 kinase are upstream signaling regulators. As the inhibitors N -acetylcysteine, PD98059, LY294002 or rapamycin blocked sPLA2 -IIA-induced proliferation without activation of the apoptotic program, we suggest that inhibition of these intracellular signal transduction elements may represent a mechanism of growth arrest. Our results reveal new potential targets for therapeutic intervention in neuroinflammatory disorders and brain cancer in particular. 相似文献
990.
Yasuhiko Murata Takuma Hashimoto Yusuke Urushihara Soichiro Shiga Kazuya Takeda Keiichi Jingu Yoshio Hosoi 《Biochemical and biophysical research communications》2018,495(4):2566-2572