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61.
62.
Lin-Li Lv Yuhan Cao Dan Liu Min Xu Hong Liu Ri-Ning Tang Kun-Ling Ma Bi-Cheng Liu 《International journal of biological sciences》2013,9(10):1021-1031
Recent studies indicate that microRNA (miRNA) is contained within exosome. Here we sought to optimize the methodologies for the isolation and quantification of urinary exosomal microRNA as a prelude to biomarker discovery studies. Exosomes were isolated through ultracentrifugation and characterized by immunoelectron microscopy. To determine the RNA was confined inside exosomes, the pellet was treated with RNase before RNA isolation. The minimum urine volume, storage conditions for exosomes and exosomal miRNA was evaluated. The presence of miRNAs in patients with various kidney diseases was validated with real-time PCR. The result shows that miRNAs extracted from the exosomal fraction were resistant to RNase digestion and with high quality confirmed by agarose electrophoresis. 16ml of urine was sufficient for miRNA isolation by absolute quantification with 4.15×105 copies/ul for miR-200c. Exosomes was stable at 4℃ 24h for shipping before stored at -80℃ and was stable in urine when stored at -80°C for 12months. Exosomal miRNA was detectable despite 5 repeat freeze-thaw cycles. The detection of miRNA by quantitative PCR showed high reproducibility (>94% for intra-assay and >76% for inter-assay), high sensitivity (positive call 100% for CKD patients), broad dynamic range (8-log wide) and good linearity for quantification (R2>0.99). miR-29c and miR-200c showed different expression in different types of kidney disease. In summary, the presence of urinary exosomal miRNA was confirmed for patients with a diversity of chronic kidney disease. The conditions of urine collection, storage and miRNA detection determined in this study may be useful for future biomarker discovery efforts. 相似文献
63.
《Cell Adhesion & Migration》2013,7(1):38-44
Stress is an important physiological regulator of brain function in young and adult mammals. The mechanisms underlying regulation of the consequences of stress, and in particular severe chronic stress, are thus important to investigate. These consequences most likely involve changes in synaptic function of brain areas being part of neural networks that regulate responses to stress. Cell adhesion molecules have been shown to regulate synaptic function in the adult and we were thus interested to investigate a regulatory mechanism that could influence expression of three adhesion molecules of the immunoglobulin superfamily (NCAM, L1 and CHL1) after exposure of early postnatal and adult mice to repeated stress. We hypothesized that reduction of adhesion molecule expression after chronic stress, as observed previously in vivo, could be due to gene silencing of the three molecules by DNA methylation. Although adhesion molecule expression was reduced after exposure of C57BL/6 mice to stress, thus validating our stress paradigm as imposing changes in adhesion molecule expression, we did not observe differences in methylation of CpG islands in the promoter regions of NCAM, L1 and CHL1, nor in the promoter region of the glucocorticoid receptor in the hippocampus, the expression of which at the protein level was also reduced after stress. We must therefore infer that severe stress in mice of the C57BL/6 strain downregulates adhesion molecule levels by mechanisms that do not relate to DNA methylation. 相似文献
64.
65.
Arianne van Koppen Marianne C. Verhaar Lennart G. Bongartz Jaap A. Joles 《Journal of visualized experiments : JoVE》2013,(77)
Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using ''gold standard'' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure>150 mmHg), proteinuria (> 50 mg/24 hr) and mild uremia (>10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (<10%). Follow-up until 12 weeks after SNX shows further progression of CKD. 相似文献
66.
摘要目的:观察无创呼吸机对不伴有呼吸衰竭的慢性阻塞性肺疾病急性加重期(AECOPD)患者症状、体征、血气分析和肺功能的影响。方法:80例AECOPD患者随机分为对照组和试验组,对照组给予常规治疗,如常规抗感染、化痰、平喘等治疗。试验组在常规治疗的基础上给予无创呼吸机正压通气(NIPPV)治疗。评价两组患者治疗前和治疗一周后临床症状体征、血气分析和肺功能的变化。结果:两组患者治疗后临床症状及体征评分较治疗前均呈下降趋势(P〈0.05),但试验组下降更为明显(P〈0.05),并且试验组症状改善时间明显缩短(P〈0.05);治疗后两组的pH,PO2和SaO2均较治疗前升高,PC02较治疗前下降(P〈0.05),和对照组相比,治疗后试验组的PO:和PC02改善更明显(P〈0.05),而pH和Sa02无显著差异(P〉0.05);治疗后两组的FEV1,FVC,FEV1实测值/预计值和FEVI%均较治疗前升高(P〈0.05),试验组升高更明显(P〈0.05)。结论:对不伴有呼吸衰竭的AECOPD患者早期应用NIPPV治疗能有效改善临床症状和体征,缩短症状改善时间,改善通气,缓解呼吸肌疲劳,预防呼吸衰竭的发生。 相似文献
67.
Barbara G Schneider M Blanca Piazuelo Liviu A Sicinschi Robertino Mera Dun-Fa Peng Juan Carlos Roa Judith Romero-Gallo Alberto G Delgado Thibaut de Sablet Luis E Bravo Keith T Wilson Wael El-Rifai Richard M Peek Jr Pelayo Correa 《Epigenetics》2013,8(11):1153-1161
DNA methylation changes are known to occur in gastric cancers and in premalignant lesions of the gastric mucosae. In order to examine variables associated with methylation levels, we quantitatively evaluated DNA methylation in tumors, non-tumor gastric mucosae, and in gastric biopsies at promoters of 5 genes with methylation alterations that discriminate gastric cancers from non-tumor epithelia (EN1, PCDH10, RSPO2, ZIC1, and ZNF610). Among Colombian subjects at high and low risk for gastric cancer, biopsies from subjects from the high-risk region had significantly higher levels of methylation at these 5 genes than samples from subjects in the low risk region (p ≤ 0.003). When results were stratified by Helicobacter pylori infection status, infection with a cagA positive, vacA s1m1 strain was significantly associated with highest methylation levels, compared with other strains (p = 0.024 to 0.001). More severe gastric inflammation and more advanced precancerous lesions were also associated with higher levels of DNA methylation (p ≤ 0.001). In a multivariate model, location of residence of the subject and the presence of cagA and vacA s1m1 in the H. pylori strain were independent variables associated with higher methylation in all 5 genes. High levels of mononuclear cell infiltration were significantly related to methylation in PCDH10, RSPO2, and ZIC1 genes. These results indicate that for these genes, levels of methylation in precancerous lesions are related to H. pylori virulence, geographic region and measures of chronic inflammation. These genes seem predisposed to sustain significant quantitative changes in DNA methylation at early stages of the gastric precancerous process. 相似文献
68.
《Epigenetics》2013,8(6):910-917
A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22–23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence of 11q-deletion, indicating that methylation and del(11q) independently silence these miRs. Finally, 5-azacytidine and trichostatin A exposure synergistically increased the expression of miR-34b/c in CLL cells, and transfection of miR-34b or miR-34c into HG3 CLL cells significantly increased apoptosis. Altogether, our novel data suggest that miR-34b/c is a candidate tumor suppressor that is epigenetically silenced in CLL. 相似文献
69.
《Epigenetics》2013,8(3):211-215
Ras-association domain family (RASSF) members are a family of genes containing an RA domain in either the C-terminus (RASSF1-RASSF6) or in the N-terminus (RASSF7-RASSF10). Members of this gene family are core members of the Salvador/Warts/Hippo (SWH) tumor suppressor network and have been shown to be involved in human tumorigenesis. Among the RASSF genes, RASSF1A is one of the most frequently methylated genes in a wide range of epithelial cancers, and we previously demonstrated that RASSF6 and RASSF10 genes are frequently epigenetically inactivated in acute leukemias, particularly in those of the B cell type. We here determined the methylation profiles of all members of the RASSF gene family as well as two recently identified (KIBRA, CRB3) upstream members of the SWH pathway in the leukemic B cells obtained from a well-characterized cohort of 95 patients with chronic lymphocytic leukemia (CLL). Among the RASSF genes, RASSF10 (50%) was the most frequently methylated gene, followed by RASSF6 (16%). The remaining RASSF genes were either unmethylated or showed a frequency of methylation < 10%. The upstream SWH member KIBRA was also frequently methylated in CLL (35%) in contrast to CRB3. Interestingly, the analysis of clinical-pathological parameters showed that KIBRA methylation was associated with unfavorable biological prognostic parameters, including unmutated IGHV genes (p = 0.007) and high CD38 expression (p < 0.05). 相似文献
70.