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1.
目的:研究H1N1流感病毒血凝素HA对人胚肺成纤维细胞的损伤作用并初步探讨其机理。方法:合成2009 H1N1的HA基因全长,分别将HA的PCR产物和pEGFP-N1质粒经Hind Ⅲ和EcoRI酶切电泳、纯化、连接并转化大肠杆菌DH5α,构建真核表达质粒pEGFP-N1/HA。质粒转染293细胞,检测其转染效率,并收获细胞总蛋白进行Western blotting检测。将阳性质粒转染MRC-5细胞,CCK-8检测HA的表达对细胞增殖活性,线粒体膜电位检测试剂盒检测其对细胞的早期凋亡的影响,ATP检测试剂盒检测HA的表达对ATP水平的改变。结果:PCR电泳检测获得分子量为1700 bp左右的目的条带,菌落PCR鉴定HA片段成功插入pEGFP-N1载体。荧光显微镜下检测pEGFP-N1/HA转染293细胞有明显荧光,Western blotting结果显示pEGFP-N1/HA转染细胞有单一的目的蛋白表达。HA的表达可明显抑制MRC-5细胞活力,HA的高表达降低MRC-5细胞的线粒体膜电位及ATP水平。结论:H1N1流感病毒HA对人肺细胞的损伤作用可能与影响肺细胞的线粒体功能有关。  相似文献   
2.
目的:肝脏是维持人体发挥功能的重要器官,同时肝脏再生能力十分强大。本文通过部分肝切除术后小鼠肝再生模型,观察肝再生过程中氧化应激及线粒体代谢变化规律,以期为将来的调控肝再生提供新的干预靶点。方法:选择雄性健康体重均匀的Balb/c小鼠,采用经典70%肝切除模型,随机分为假手术对照组(Sham组)以及70%肝切除组(70%PH组)。肝切除术后6 h、1d、2 d、3 d、5 d、7 d不同时间点取肝组织,制备冰冻切片检测活性氧(ROS)水平,Western blot分别检测细胞增殖相关蛋白PCNA、Cyclin D1;氧化应激相关蛋白SOD1、SOD2、CAT、GPX1;以及线粒体代谢相关蛋白PGC-1α、Nrf1、TFAM、Drp1、Fis1、Mfn1、Mfn2、OPA1的表达并分析其变化规律。结果:70%肝切除术后小鼠肝脏增长迅速,细胞增殖关键蛋白PCNA和Cyclin D1表达显著增加;在此过程中细胞ROS水平呈现先升高后降低的变化,细胞主要抗氧化酶SOD1、SOD2、CAT、Gpx1与ROS相一致出现先升高后降低的变化。线粒体生物合成调控因子PGC-1α、Nrf1、TFAM呈现先降低后升高的趋势,而线粒体分裂蛋白Drp1和Fis1呈现先降低后显著升高的趋势,线粒体融合相关蛋白Mfn1、Mfn2和OPA1总体为先降低后恢复至正常水平。结论:在小鼠70%肝切除再生过程中,存在着明显的氧化应激,线粒体生物合成增加,线粒体分裂/融合平衡偏向分裂,并且这些变化呈现具有一定的时间变化规律,这些变化及规律很可能作为将来调控肝再生的重要的潜在干预靶点。  相似文献   
3.

Background

The epidermis forms a critical barrier that is maintained by orchestrated programs of proliferation, differentiation, and cell death. Gene mutations that disturb this turnover process may cause skin diseases. Human GASDERMIN A (GSDMA) is frequently silenced in gastric cancer cell lines and its overexpression has been reported to induce apoptosis. GSDMA has also been linked with airway hyperresponsiveness in genetic association studies. The function of GSDMA in the skin was deduced by dominant mutations in mouse gasdermin A3 (Gsdma3), which caused skin inflammation and hair loss. However, the mechanism for the autosomal dominance of Gsdma3 mutations and the mode of Gsdma3’s action remain unanswered.

Results

We demonstrated a novel function of Gsdma3 in modulating mitochondrial oxidative stress. We showed that Gsdma3 is regulated by intramolecular fold-back inhibition, which is disrupted by dominant mutations in the C-terminal domain. The unmasked N-terminal domain of Gsdma3 associates with Hsp90 and is delivered to mitochondrial via mitochondrial importer receptor Tom70, where it interacts with the mitochondrial chaperone Trap1 and causes increased production of mitochondrial reactive oxygen species (ROS), dissipation of mitochondrial membrane potential, and mitochondrial permeability transition (MPT). Overexpression of the C-terminal domain of Gsdma3 as well as pharmacological interventions of mitochondrial translocation, ROS production, and MPT pore opening alleviate the cell death induced by Gsdma3 mutants.

Conclusions

Our results indicate that the genetic mutations in the C-terminal domain of Gsdma3 are gain-of-function mutations which unmask the N-terminal functional domain of Gsdma3. Gsdma3 regulates mitochondrial oxidative stress through mitochondrial targeting. Since mitochondrial ROS has been shown to promote epidermal differentiation, we hypothesize that Gsdma3 regulates context-dependent response of keratinocytes to differentiation and cell death signals by impinging on mitochondria.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0152-0) contains supplementary material, which is available to authorized users.  相似文献   
4.
Cancer cells are characterized by elevated levels of reactive oxygen species, which are produced mainly by the mitochondria. The dismutase SOD2 localizes in the matrix and is a major antioxidant. The activity of SOD2 is regulated by the deacetylase SIRT3. Recent studies indicated that SIRT3 is decreased in 87% of breast cancers, implying that the activity of SOD2 is compromised. The resulting elevation in reactive oxygen species was shown to be essential for the metabolic reprograming toward glycolysis. Here, we show that SOD2 itself is down-regulated in breast cancer cell lines. Further, activation of oncogenes, such as Ras, promotes the rapid down-regulation of SOD2. Because in the absence of SOD2, superoxide levels are elevated in the matrix, we reasoned that mechanisms must exist to retain low levels of superoxide in other cellular compartments especially in the intermembrane space of the mitochondrial to avoid irreversible damage. The dismutase SOD1 also acts as an antioxidant, but it localizes to the cytoplasm and the intermembrane space of the mitochondria. We report here that loss of SOD2 correlates with the overexpression of SOD1. Further, we show that mitochondrial SOD1 is the main dismutase activity in breast cancer cells but not in non-transformed cells. In addition, we show that the SOD1 inhibitor LCS-1 leads to a drastic fragmentation and swelling of the matrix, suggesting that in the absence of SOD2, SOD1 is required to maintain the integrity of the organelle. We propose that by analogy to the cadherin switch during epithelial-mesenchymal transition, cancer cells also undergo a SOD switch during transformation.  相似文献   
5.
Mutations in the human mitochondrial polymerase (polymerase-γ (Pol-γ)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. To correlate biochemically quantifiable defects resulting from point mutations in Pol-γ with their physiological consequences, we created “humanized” yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol-γ. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also examined the effects of four disease-related point mutations (S305R, H932Y, Y951N, and Y955C) and an exonuclease-deficient mutant (D198A/E200A). In haploid cells, each mutant results in rapid mtDNA depletion, increased mutation frequency, and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild-type Pol-γ suppresses mutation-associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content, and depolarized mitochondrial membranes. The severity of the Pol-γ mutant phenotype in heterozygous diploid humanized yeast correlates with the approximate age of disease onset and the severity of symptoms observed in humans.  相似文献   
6.
Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca2+ uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca2+ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca2+ uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.  相似文献   
7.
Mitochondria frequently change their shape through fission and fusion in response to physiological stimuli as well as pathological insults. Disrupted mitochondrial morphology has been observed in cholestatic liver disease. However, the role of mitochondrial shape change in cholestasis is not defined. In this study, using in vitro and in vivo models of bile acid-induced liver injury, we investigated the contribution of mitochondrial morphology to the pathogenesis of cholestatic liver disease. We found that the toxic bile salt glycochenodeoxycholate (GCDC) rapidly fragmented mitochondria, both in primary mouse hepatocytes and in the bile transporter-expressing hepatic cell line McNtcp.24, leading to a significant increase in cell death. GCDC-induced mitochondrial fragmentation was associated with an increase in reactive oxygen species (ROS) levels. We found that preventing mitochondrial fragmentation in GCDC by inhibiting mitochondrial fission significantly decreased not only ROS levels but also cell death. We also induced cholestasis in mouse livers via common bile duct ligation. Using a transgenic mouse model inducibly expressing a dominant-negative fission mutant specifically in the liver, we demonstrated that decreasing mitochondrial fission substantially diminished ROS levels, liver injury, and fibrosis under cholestatic conditions. Taken together, our results provide new evidence that controlling mitochondrial fission is an effective strategy for ameliorating cholestatic liver injury.  相似文献   
8.
The respiratory response to heat shock in Neurospora crassa   总被引:2,自引:0,他引:2  
A sharp decrease in oxygen uptake occurred in Neurospora crassa cells that were transferred from 30 degrees C to 45 degrees C, and the respiration that resumed later at 45 degrees C was cyanide-insensitive. Energization of mitochondria, measured in vivo with fluorescence microscopy and a carbocyanine dye, also declined sharply in cells at 45 degrees C. Electron microscopy showed no changes in mitochondrial complexity; however, the cytoplasm of heat-shocked cells was deficient in glycogen granules.  相似文献   
9.
Mercuric ion, a well-known nephrotoxin, promotes oxidative tissue damage to kidney cells. One principal toxic action of Hg(II) is the disruption of mitochondrial functions, although the exact significance of this effect with regard to Hg(II) toxicity is poorly understood. In studies of the effects of Hg(II) on superoxide (O) and hydrogen peroxide (H2O2) production by rat kidney mitochondria, Hg(II) (1–6 μM), in the presence of antimycin A, caused a concentration-dependent increase (up to fivefold) in mitochondrial H2O2 production but an apparent decrease in mitochondrial O production. Hg(II) also inhibited O-dependent cytochrome c reduction (IC50 ≈?2–3 μM) when O was produced from xanthine oxidase. In contrast, Hg(I) did not react with O in either system, suggesting little involvement of Hg(I) in the apparent dismutation of O by Hg(II). Hg(II) also inhibited the reactions of KO2 (i.e., O) with hemin or horseradish peroxidase dissolved in dimethyl sulfoxide (DMSO). Finally, a combination of Hg(II) and KO2 in DMSO resulted in a stable UV absorbance spectrum [currently assigned Hg(II)-peroxide] distinct from either Hg(II) or KO2. These results suggest that Hg(II), despite possessing little redox activity, enhances the rate of O dismutation, leading to increased production of H2O2 by renal mitochondria. This property of Hg(II) may contribute to the oxidative tissue-damaging properties of mercury compounds.  相似文献   
10.
This paper reports an investigation on the effects of the hydrophobic, bifunctional SH group reagent phenylarsine oxide (PhAsO) on mitochondrial membrane permeability. We show that PhAsO is a potent inducer of the mitochondrial permeability transition in a process which is sensitive to both the oxygen radical scavanger BHT and to cyclosporin A. The PhAsO-induced permeability transition is stimulated by Ca2+ but takes place also in the presence of EGTA in a process that maintains its sensitivity to BHT and cyclosporin A. Our findings suggest that, at variance from other known inducers of the permeability transition, PhAsO reacts directly with functional SH groups that are inaccessible to hydrophilic reagents in the absence of Ca2+.  相似文献   
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