全文获取类型
收费全文 | 15571篇 |
免费 | 1343篇 |
国内免费 | 1905篇 |
专业分类
18819篇 |
出版年
2024年 | 52篇 |
2023年 | 281篇 |
2022年 | 587篇 |
2021年 | 905篇 |
2020年 | 701篇 |
2019年 | 813篇 |
2018年 | 755篇 |
2017年 | 553篇 |
2016年 | 724篇 |
2015年 | 1035篇 |
2014年 | 1249篇 |
2013年 | 1278篇 |
2012年 | 1558篇 |
2011年 | 1434篇 |
2010年 | 905篇 |
2009年 | 727篇 |
2008年 | 807篇 |
2007年 | 743篇 |
2006年 | 604篇 |
2005年 | 527篇 |
2004年 | 385篇 |
2003年 | 303篇 |
2002年 | 279篇 |
2001年 | 191篇 |
2000年 | 180篇 |
1999年 | 174篇 |
1998年 | 120篇 |
1997年 | 114篇 |
1996年 | 117篇 |
1995年 | 96篇 |
1994年 | 89篇 |
1993年 | 66篇 |
1992年 | 85篇 |
1991年 | 55篇 |
1990年 | 57篇 |
1989年 | 50篇 |
1988年 | 35篇 |
1987年 | 27篇 |
1986年 | 28篇 |
1985年 | 29篇 |
1984年 | 15篇 |
1983年 | 14篇 |
1982年 | 13篇 |
1981年 | 5篇 |
1980年 | 4篇 |
1979年 | 10篇 |
1978年 | 4篇 |
1976年 | 9篇 |
1975年 | 8篇 |
1971年 | 3篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
71.
【目的】阿尔茨海默症治疗药物石杉碱甲(Huperzine A,Hup A)的生物合成途径起始于赖氨酸脱羧酶(Lysine decarboxylase,LDC)。本研究克隆及表达了来源于产Hup A的植物内生真菌的LDC基因,并研究了其功能。【方法】采用RT-PCR扩增法,从一株产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14获得LDC基因,构建表达质粒p ET-22b-LDC与p ET-32a-LDC,转化感受态细胞E.coli BL21,加入IPTG至终浓度为1×10~(–3) mol/L,于24°C、200 r/min培养8 h,诱导表达LDC蛋白质;通过Ni~(2+)金属亲和层析纯化重组LDC并建立酶促反应体系,利用TLC检测了LDC催化活性。利用生物信息学软件分析了LDC的理化性质及蛋白质的空间结构。【结果】成功克隆并异源表达出重组蛋白LDC与Trx-LDC,经SDS-PAGE电泳鉴定分子量分别为24.4 k Da和42.7 k Da,与预计大小相符。TLC结果表明LDC与Trx-LDC均具有赖氨酸脱羧酶活性。【结论】本研究从产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14中成功克隆到LDC基因并进行了异源表达,检测到了其催化活性,为丰富LDC分子信息及阐明内生真菌中Hup A生物合成机制提供参考数据。 相似文献
72.
73.
Replicating rather than nonreplicating adenovirus-human immunodeficiency virus recombinant vaccines are better at eliciting potent cellular immunity and priming high-titer antibodies 总被引:3,自引:0,他引:3 下载免费PDF全文
Peng B Wang LR Gómez-Román VR Davis-Warren A Montefiori DC Kalyanaraman VS Venzon D Zhao J Kan E Rowell TJ Murthy KK Srivastava I Barnett SW Robert-Guroff M 《Journal of virology》2005,79(16):10200-10209
A major challenge in combating the human immunodeficiency virus (HIV) epidemic is the development of vaccines capable of inducing potent, persistent cellular immunity and broadly reactive neutralizing antibody responses to HIV type 1 (HIV-1). We report here the results of a preclinical trial using the chimpanzee model to investigate a combination vaccine strategy involving sequential priming immunizations with different serotypes of adenovirus (Ad)/HIV-1(MN)env/rev recombinants and boosting with an HIV envelope subunit protein, oligomeric HIV(SF162) gp140deltaV2. The immunogenicities of replicating and nonreplicating Ad/HIV-1(MN)env/rev recombinants were compared. Replicating Ad/HIV recombinants were better at eliciting HIV-specific cellular immune responses and better at priming humoral immunity against HIV than nonreplicating Ad-HIV recombinants carrying the same gene insert. Enhanced cellular immunity was manifested by a greater frequency of HIV envelope-specific gamma interferon-secreting peripheral blood lymphocytes and better priming of T-cell proliferative responses. Enhanced humoral immunity was seen in higher anti-envelope binding and neutralizing antibody titers and better induction of antibody-dependent cellular cytotoxicity. More animals primed with replicating Ad recombinants mounted neutralizing antibodies against heterologous R5 viruses after one or two booster immunizations with the mismatched oligomeric HIV-1(SF162) gp140deltaV2 protein. These results support continued development of the replicating Ad-HIV recombinant vaccine approach and suggest that the use of replicating vectors for other vaccines may prove fruitful. 相似文献
74.
75.
76.
Tao Fang Wang Heng Wang Ai Fen Peng Qing Feng Luo Zhi Li Liu Rong Ping Zhou Song Gao Yang Zhou Wen Zhao Chen 《Biochemical and biophysical research communications》2013
FASN plays an important role in the malignant phenotype of various tumors. Our previous studies show that inhibition FASN could induce apoptosis and inhibit proliferation in human osteosarcoma (OS) cell in vivo and vitro. The aim in this study was to investigate the effect of inhibition FASN on the activity of HER2/PI3K/AKT axis and invasion and migration of OS cell. The expression of FASN, HER2 and p-HER2(Y1248) proteins was detected by immunohistochemistry in OS tissues from 24 patients with pulmonary metastatic disease, and the relationship between FASN and p-HER2 as well as HER2 was investigated. The results showed that there was a positive correlation between FASN and HER2 as well as p-HER2 protein expression. The U-2 OS cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid. FASN mRNA was measured by RT-PCR. Western blot assays was performed to examine the protein expression of FASN, HER2, p-HER2(Y1248), PI3K, Akt and p-Akt (Ser473). Migration and invasion of cells were investigated by wound healing and transwell invasion assays. The results showed that the activity of HER2/PI3K/AKT signaling pathway was suppressed by inhibiting FASN. Meanwhile, the U-2OS cells migration and invasion were also impaired by inhibiting the activity of FASN/HER2/PI3K/AKT. Our results indicated that inhibition of FASN suppresses OS cell invasion and migration via down-regulation of the “HER2/PI3K/AKT” axis in vitro. FASN blocker may be a new therapeutic strategy in OS management. 相似文献
77.
78.
William K. K. Wu Minyi He Liang Zhao Xuegang Sun Hui Li Yong Jiang Yungao Yang Kang Peng 《Cell biochemistry and function》2012,30(4):271-278
Triptolide is a diterpenoid triepoxide derived from the traditional Chinese medical herb Tripterygium wilfordii. In the present study, we demonstrated that this phytochemical attenuated colon cancer growth in vitro and in vivo. Using a proteomic approach, we found that 14‐3‐3 epsilon, a cell cycle‐ and apoptosis‐related protein, was altered in colon cancer cells treated with triptolide. In this regard, triptolide induced cleavage and perinuclear translocation of 14‐3‐3 epsilon. Taken together, our findings suggest that triptolide may merit investigation as a potential therapeutic agent for colon cancer, and its anticancer action may be associated with alteration of 14‐3‐3 epsilon. Copyright © 2012 John Wiley & Sons, Ltd. 相似文献
79.
80.
He WQ Qiao YN Zhang CH Peng YJ Chen C Wang P Gao YQ Chen C Chen X Tao T Su XH Li CJ Kamm KE Stull JT Zhu MS 《American journal of physiology. Heart and circulatory physiology》2011,301(2):H584-H591
Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) but Ca(2+)-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCK(SMKO)) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCK(SMKO) mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCK(SMKO) mice may be a useful model of vascular failure and hypotension. 相似文献