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排序方式: 共有1781条查询结果,搜索用时 15 毫秒
51.
Wolfgang Pfützner 《Journal der Deutschen Dermatologischen Gesellschaft》2010,8(8):582-590
The success of gene therapy mainly depends on the gene vector (GV) responsible for the efficient transport of genetic information. The qualities of a GV have a profound influence on the method of application, the efficiency of gene transfer in the target tissue, the amount and persistence of gene expression and the potential side effects and safety risks. Clinical gene therapy studies over the past 20 years have contributed to the development and testing of different GV systems, some of which also show great potential for the treatment of skin diseases. In this review the structures, methods of application, characteristics, clinical uses and possibilities for optimization of these GV will be discussed with regard to their cutaneous applications. 相似文献
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53.
Hepatocyte growth factor gene transfer with naked plasmid DNA ameliorates dimethylnitrosamine-induced liver fibrosis in rats 总被引:10,自引:0,他引:10
Hironari Kanemura Yuji Iimuro Masaharu Takeuchi Takahiro Ueki Tadamichi Hirano Kiyoshi Horiguchi Yasukane Asano Jiro Fujimoto 《Hepatology research》2008,38(9):930-939
Aim: Hepatocyte growth factor (HGF) has various biological properties, including antifibrogenic activity. In the present study, we tested the efficacy of HGF gene therapy using naked plasmid DNA in dimethylnitrosamine (DMN)-induced liver fibrosis in a rat model. Methods: Naked plasmid DNA encoding human HGF was injected once, together with a hypertonic solution, into the hepatic artery after DMN treatment on three consecutive days per week for 3 weeks. Naked plasmid DNA encoding beta-galactosidase was injected similarly in the DMN-treated control rats. DMN treatment was continued once weekly after gene transfer for additional 3 weeks. Results: The human HGF protein expression was detected in livers transfected with human HGF naked plasmid DNA, gradually decreasing by day 21. The expression of the endogenous rat HGF protein was also upregulated after human HGF gene transfer. Phosphorylation of c-Met, a HGF receptor, was detected only in livers transfected with human HGF plasmid DNA. Fibrosis was attenuated significantly in livers transfected with the human HGF plasmid. Attenuation wasaccompanied by decreased expression of alpha-smooth muscle actin. Increased portal vein pressure after treatment with DMN was suppressed significantly by HGF gene transfer. The upregulated hepatic protein expression of transforming growth factor-beta (TGF-beta) in response to DMN was markedly attenuated by HGF gene transfer accompanied by the increased protein expression for matrix metalloproteinases (MMP)-3 and -13. Conclusion: The hepatic arterial injection of human naked plasmid HGF DNA was effective in suppressing liver fibrosis induced in rats by DMN. The mechanisms by which HGF expression attenuated liver fibrosis may include the suppression of hepatic TGF-beta expression and the induction of MMP expression. 相似文献
54.
Yuki Otani Hidefumi Mukai Yuki Fuchigami Fumiyoshi Yamashita Mitsuru Hashida 《Journal of drug targeting》2015,23(5):427-435
Background: Achieving long-term gene expression in kidney will be beneficial for gene therapy of renal and congenital diseases, genetic studies constructing animal disease models, and the functional analysis of disease-related genes.Purpose: The purpose of this study was to develop an in vivo long-term gene expression system in murine kidney using ?C31 integrase.Methods: Gene expression in cultured RENCA, TCMK-1, and HEK293 cells was assessed. The long-term in vivo gene expression system in the kidney was achieved by co-transfecting 5?µg of pORF-luc/attB as a donor plasmid and 20?µg of pCMV-luc as a helper plasmid into the right kidney of mice by electroporation. Luciferase expression levels were measured to determine longevity of the expression.Results: Significantly high luciferase expression levels were observed in cultured RENCA, TCMK-1, and HEK293 cells over 1 month compared with controls (non-integrase system). The luciferase cDNA sequence was integrated at a pseudo attP site termed mpsL1. In vivo luciferase expression levels in the integrase group were sustained and significantly higher than those in the control group over 2 months. Furthermore, ?C31 integrase-transfected cells had less genomic DNA damage caused by integrase expression.Discussion and conclusion: These results demonstrated that the ?C31 integrase system could produce long-term (2 months) in vivo gene expression in mouse kidney. 相似文献
55.
目的:在传统方案的基础上建立一种经济有效且易于操作的类转录激活效应子转录因子(TALE‐TFs)的构建和功能检测方案。方法采用基于PCR的Golden gate克隆法分别尝试构建类转录激活效应物(TALEs)的六聚体、五聚体、四聚体和三聚体,比较构建结果,选择最优效果方案进行TALE‐TFs的构建。采用片段置换反应(FSR)构建了含有 TALE‐TFs结合片段的RFP质粒pminCMV ,并与TALE‐TFs进行共转染,观察红色荧光验证TALE‐TFs的转录活性。结果 TALEs中所含的串联重复模块越少,获得的构建产物越多。共转染时,TALE‐TFs使得pminCMV成功启动表达。结论该研究为不同条件下实验方案的选择提供了依据,并利用含有TALE‐TF结合片段和红色荧光的质粒建立了快捷直观的转录活性验证方法。 相似文献
56.
《第三军医大学学报》2015,37(1)
目的 分析沙眼衣原体隐匿质粒编码蛋白的相互作用.方法 采用基因克隆的方法将沙眼衣原体质粒基因pgp1~4及pgp8分别克隆到pRAC和pRBR上,通过转录激活细菌双杂交系统分析蛋白相互作用.结果 Western blot结果显示Pgp1、Pgp3与大肠杆菌RpoA氨基末端功能区(α-NTD)融合蛋白及Pgp1、Pgp3、Pgp4与DNA结合蛋白λCI融合蛋白,能在大肠杆菌中表达.在报告菌株中,共表达与α-NTD融合的Pgp3蛋白(α-Pgp3)及与λCI融合的Pgp3蛋白(CI-Pgp3),导致报告基因产物β-半乳糖苷酶的活性升高.而余无明显变化.结论 pgp3编码的Pgp3能与自身相互作用. 相似文献
57.
58.
目的构建能够稳定表达绿色荧光蛋白(GFP)的白色假丝酵母菌菌株,以便对目的基因进行示踪。方法构建pACT1-GFP质粒,以白色假丝酵母菌CAI4菌株作为感受态进行转化培养后,分别观察绿色荧光蛋白在两相型下的表达情况。结果含有pACT1-GFP的白色假丝酵母菌菌株,99%在两相型下均有较高水平的荧光蛋白表达,且荧光强度没有明显差别。结论pACT1-GFP能够在白色假丝酵母菌体内稳定表达。 相似文献
59.
Neurotrophin-3 (NT-3) can promote the repair of central nervous system and retinal damage. In previous reports, NT-3 has been expressed by viral vectors. However, plasmid vectors have a safer profile compared with viral vectors in clinical studies. This study recombined amplified human retinal NT-3 with a eukaryotic expression plasmid containing green fluorescent protein (GFP) to construct an NT-3 expression plasmid, pEGFP-N1-NT-3. The transfection efficiency 48 hours after pEGFP-N1-NT-3 transfection to 293T cells was 50.06 ± 2.78%. Abundant NT-3-GFP was expressed in 293T cells as observed by fluorescence microscopy, suggesting the construct pEGFP-N1-NT-3 effectively expressed and secreted NT-3-GFP. Secretory vesicles containing NT-3-GFP were observed in a constant location in cells by laser scan confocal microscopy, indicating the expression and secretion processes of NT-3 in eukaryotic cells were in accordance with the physical synthesis processes of secreted proteins. Western blot assay showed that pro-NT-3-GFP had a molecular weight of 56 kDa, further confirming NT-3-GFP expression. At 48 hours after transfection, the concentration of NT-3 in culture medium was 22.3 ng/mL, suggesting NT-3 produced by pEGFP-N1-NT-3 was efficiently secreted. This study constructed a human retinal-derived NT-3 eukaryotic expression plasmid that efficiently expressed and secreted NT-3. 相似文献
60.
Zhonghua Liu Shengliang Li Zibin Liang Yan Zhao Yulin Zhang Yaqi Yang Minjuan Wang FengLi 《中国神经再生研究》2013,(33):3095-3106
There are several major pathological changes in Alzheimer's disease, including apoptosis of cho- linergic neurons, overactivity or overexpression of 13-site amyloid precursor protein cleaving enzyme 1 (BACE1) and inflammation. In this study, we synthesized a 19-nt oligonucleotide targeting BACE1, the key enzyme in amyloid beta protein (AI3) production, and introduced it into the pSilenCircle vector to construct a short hairpin (shRNA) expression plasmid against the BACE1 gene. We transfected this vector into C17.2 neural stem cells and primary neural stem cells, resulting in downregulation of the BACE1 gene, which in turn induced a considerable reduction in reducing AI3 protein production. We anticipate that this technique combining cell transplantation and gene ther- apy will open up novel therapeutic avenues for Alzheimer's disease, particularly because it can be used to simultaneously target several pathogenetic changes in the disease. 相似文献