全文获取类型
收费全文 | 1262篇 |
免费 | 76篇 |
国内免费 | 79篇 |
专业分类
耳鼻咽喉 | 15篇 |
儿科学 | 9篇 |
妇产科学 | 35篇 |
基础医学 | 225篇 |
口腔科学 | 37篇 |
临床医学 | 101篇 |
内科学 | 157篇 |
皮肤病学 | 11篇 |
神经病学 | 65篇 |
特种医学 | 32篇 |
外科学 | 239篇 |
综合类 | 211篇 |
预防医学 | 37篇 |
眼科学 | 19篇 |
药学 | 101篇 |
中国医学 | 1篇 |
肿瘤学 | 122篇 |
出版年
2023年 | 2篇 |
2022年 | 3篇 |
2021年 | 4篇 |
2020年 | 4篇 |
2019年 | 6篇 |
2018年 | 10篇 |
2017年 | 10篇 |
2016年 | 24篇 |
2015年 | 36篇 |
2014年 | 45篇 |
2013年 | 70篇 |
2012年 | 86篇 |
2011年 | 104篇 |
2010年 | 102篇 |
2009年 | 103篇 |
2008年 | 102篇 |
2007年 | 112篇 |
2006年 | 105篇 |
2005年 | 102篇 |
2004年 | 84篇 |
2003年 | 61篇 |
2002年 | 62篇 |
2001年 | 46篇 |
2000年 | 32篇 |
1999年 | 28篇 |
1998年 | 13篇 |
1997年 | 16篇 |
1996年 | 9篇 |
1995年 | 11篇 |
1994年 | 5篇 |
1993年 | 7篇 |
1992年 | 3篇 |
1991年 | 3篇 |
1989年 | 2篇 |
1988年 | 3篇 |
1986年 | 1篇 |
1985年 | 1篇 |
排序方式: 共有1417条查询结果,搜索用时 15 毫秒
1.
Ferry Verbaan Inger van Dam Yoshinubu Takakura Mitsuru Hashida Wim Hennink Gert Storm Christien Oussoren 《European journal of pharmaceutical sciences》2003,20(4-5):419-427
The objective of this study was to assess the in vivo fate of poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA)-based polyplexes after intravenous administration into mice. Circulation kinetics and tissue distribution in terms of plasmid localization and transfection efficiency were assessed. To gain more insight into the observed biodistribution and gene expression profile, the interaction of pDMAEMA-based polyplexes with blood components (erythrocytes and albumin) was investigated in vitro. In the case of i.v. injection of positively charged polyplexes at a dose of 30 microg DNA most of the radioactivity was found in the lungs and the liver 60 min after injection. In the case of pDMAEMA/DNA polyplexes with a negative charge, uptake occurred mainly by the liver. Administration of positively charged complexes at a 30 microg DNA dose resulted in reporter gene expression primarily in the lungs. Injection of negatively charged complexes and naked plasmid did not result in luciferase expression in any of the organs examined. In vitro turbidity experiments showed the induction of a charge dependent aggregation process upon addition of albumin to the polyplexes pointing out to the involvement of aggregate formation in the dominant lung uptake of the positively charged polyplexes. Also, incubations of polyplexes after pre-incubation with a physiological concentration of albumin with washed erythrocytes confirmed that polyplexes induce the formation of extremely large structures. This paper underlines the need for the design of systems with reduced interaction with blood components to promote the delivery of DNA to target tissues outside the lungs. 相似文献
2.
3.
人骨形态发生蛋白-2基因转染脂肪源性间充质干细胞的成骨研究 总被引:1,自引:0,他引:1
目的 探讨人骨形态发生蛋白-2(hBMP-2)基因修饰的人脂肪源性间充质干细胞(ADSCs)的成骨能力及基因转染对ADSCs成骨分化的生物学调控。方法 流式细胞技术鉴定从人脂肪组织中提取的细胞为间充质来源的干细胞。将ADSCs转染hBMP+2基因,免疫沉淀+Western blotting法和酶联免疫吸附试验(ELISA)检测hBMP-2表达,确定hBMP-2表达量及表达稳定性,碱性磷酸酶(ALP)染色、ALP定量测定、钙结节染色及RT—PCR分析hBMP-2基因转染对ADSCs向成骨细胞转化的调控,并将转基因细胞注入裸鼠股部肌内,通过X线及组织学检查观察体内成骨情况。结果 流式细胞术证实从脂肪中提取的细胞为间充质来源的干细胞。转基因后免疫沉淀+Western blotting法和ELISA法显示转染hBMP-2的ADSCs具有较高且稳定的hBMP-2的表达,转染21d后表达量无明显降低。ALP染色、ALP定量测定、钙结节染色及RT—PCR发现转基因ADSCs向成骨细胞方向发生分化。裸鼠肌内注射转基因细胞后2周即显示有异位骨形成,4周明显增多,对照组无骨组织形成。结论 转染hBMP-2基因的ADSCs在持续稳定高表达目的蛋白的同时,自身向成骨细胞发生分化,并在裸鼠体内形成异位骨。因此,ADSCs有望作为新的基因工程种子细胞。 相似文献
4.
高转移小鼠肺癌株细胞导入人基因组DNA后的转移表型抑制 总被引:2,自引:0,他引:2
目的 探讨从正常人细胞基因组DNA中寻找、分离并鉴定肿瘤转移抑制基因或相应DNA序列的新途径。方法 采用体内转移灶体外再培养再硬琼脂集落克隆化筛选,获体内90%以上高转移率小鼠肺腺癌LM2细胞株;使用磷酸钙共沉淀DNA转染技术,将正常细胞人基因组DNA随机片段,导入高转移小鼠肺癌细胞株LM2后,使用有限稀释法克隆化其中形态扁平的回复突变株。为证实细胞确实含有外源人DNA序列,设计人特异性Alu引物 相似文献
5.
用玻璃纤维处理BALB/C3T3细胞,使细胞发生转化,从转化细胞中抽提DNA用以转染NIH3T3细胞,结果被转染的细胞亦发生转化。用~(32)P标记的Ha-ras癌基因作探针,通过斑点杂交,征明玻璃纤维诱发的BALB/3T3转化细胞中,Ha—ras的原癌基因被激活。 相似文献
6.
脂质体转染AFP基因构建树突状细胞肝癌瘤苗及体外活性检测 总被引:5,自引:0,他引:5
目的:探讨以AFP为靶点,构建AFP-DC肝癌瘤苗及其抗肝癌免疫治疗的可能性。方法:应用脂质体将AFP基因转染体外培养的未成熟 BMDC,构建 AFP-DC肝癌瘤苗,以流式细胞术、Western blot、3H-TdR法和 MTT法等检测其免疫活性。结果:AFP-DC瘤苗不仅能产生和分泌AFP,而且能上调自身的B7分子和MHC分子,明显刺激T细胞增殖及提高CTL的杀伤作用。结论:提示肝癌相关基因AFP可作为抗肝癌基因治疗的切入点。 相似文献
7.
8.
P. M. T. Deen Søren Nielsen René J. M. Bindels Carel H. van Os 《Pflügers Archiv : European journal of physiology》1997,433(6):780-787
Aquaporin-1 is present in the apical and basolateral membranes in proximal tubules and descending limbs of Henlé’s loop.
In order to be able to study the routing of Aquaporin-1 and the regulation of Aquaporin-1-mediated transcellular water flow,
we stably transfected LLC-PK1 and MDCK-HRS cell lines with an Aquaporin-1 expression construct. LLC-PK1 clone 7 and MDCK clone K integrated two and one copies, respectively, which was reflected in the amount of Aquaporin-1 mRNA expressed in both clones. The Aquaporin-1 protein levels, however, were similar. In both clones, immuno-electronmicroscopy
showed extensive labelling of Aquaporin-1 on the basolateral plasma membrane, endosomal vesicles and the apical plasma membrane,
including the microvilli. To measure transcellular water permeation, a simple method was applied using phenol-red as a cell-impermeant
marker of concentration. In contrast to the native cell lines, both clones revealed a high transcellular osmotic water permeability,
which could not be influenced by forskolin add/3-isobutyl-1-methylxanthine (IBMX) or the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA). After glutaraldehyde fixation, it was inhibitable by HgCl2. These results indicate that targeting of Aquaporin-1 to the apical and basolateral plasma membrane is independent of cell
type and show for the first time that water flow through a cultured epithelium can be blocked by mercurial compounds.
Received: 9 October 1996 / Received after revision: 3 January 1997 / Accepted: 8 January 1997 相似文献
9.
Jos Alejandro Lpez Jonathan H. Lebowitz Stephen M. Beverley Hans-Georg Rammensee Peter Overath 《European journal of immunology》1993,23(1):217-223
The question is addressed whether antigens of Leishmania, a parasite residing in the endosomal compartment of macrophages, can be presented in the context of major histocompatibility complex class I molecules. We used E. coli β-galactosidase as a model antigen which can be expressed in high levels in L. mexicana promastigotes (L. mexicana-gal). Infection of BALB/c mice with L. mexicanagal induces β-galactosidase-specific cytotoxic T cells (CTL), which can be isolated using a β-galactosidase-expressing mastocytoma line as an antigen-presenting cell. These CTL recognize epitopes of β-galactosidase in the context of H-2Kd; however, they do not recognize L. mexicanagal-infected macrophages even after killing of the intracellular amastigotes by drug treatment or macrophage activation by lymphokines, although class I-peptide interaction and the presentation of endogenously produced antigens is normal. It is concluded that parasite antigens can induce a CTL response in vivo but that these CTL cannot recognize infected macrophages because the relevant epitopes cannot gain access to class I molecules. The effect of priming in vivo may be explained by the well-known but ill-understood phenomenon of cross-priming. 相似文献
10.
血管内皮生长因子反义基因转染对高转移人巨细胞肺癌细胞系生 … 总被引:1,自引:1,他引:1
目的 探讨反义血管内皮生长因子(VEGF)基因转染在抑制恶性肿瘤生长和转移的抗肿瘤血管基因治疗中的意义。方法 利用基因重组技术构建正义和反义VEGF121 cDNA真核表达载体,用脂质体法转染高转移性人巨细胞肺癌细胞(PG),经Northem杂交和Western印迹免疫化学检测VEGF mRNA和蛋白质的表达水平,并对转染前后细胞进行体外生长和裸鼠体内生长转移等多项生物学行为实验,结果 转染反义转 相似文献