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61.
目的 :研究膜结合型肿瘤坏死因子α(TNFα)在基因转染中的特点及其在肿瘤基因治疗中的作用等特性。方法 :用反转录病毒为载体 ,将含有突变的膜结合型TNFα的质粒 ,用lipofectin法转入包装细胞株crip细胞。又用收集到的病毒上清感染人乳腺癌细胞MDA2 31,采用G418筛选 ,DNA印迹法和聚合酶链反应方法鉴定该基因转染的结果。结果 :经上述方法鉴定表明该外源基因已成功转染人乳腺癌细胞中 ,为进一步研究膜结合型TNFα的功能奠定了基础。结论 :经初步研究表明 ,含有膜结合型肿瘤坏死因子的质粒可以通过lipofectin法成功导入crip细胞 ,且该病毒可成功感染人乳腺癌细胞并整合 相似文献
62.
Legendre Jean-Yves Trzeciak Arnold Bur Daniel Deuschle Ulrich Supersaxo Andreas 《Pharmaceutical research》1997,14(5):619-624
Purpose. This study investigates the structure/activity relationship of a series of N-acyl-peptides (lipopeptides) for the transfection of mammalian cells.
Methods. Lipopeptides comprising 1 to 3 basic amino-acids and a single fatty acid chain were synthesized. Transfecting complexes between lipopeptide, plasmid DNA and dioleoyl phosphatidylethanolamine were prepared and applied on cells in culture. Transfection efficiency was evaluated by measuring -galactosidase activity 48 h post-transfection. Lipopeptide-DNA binding was also investigated by physical means and molecular modelling.
Results. Besides the length of the fatty acid chain, the nature of the basic amino-acid and the C-terminal group were crucial parameters for high transfection efficiency. The N-acyl-(diaminobutyric acid)n derivatives were the most potent transfecting agents among those tested and induced a -galactosidase activity 2 to 20 times higher than the N-acyl-lysine, -ornithine or -diaminopropionic acid derivatives. Furthermore, a hydrazide C-terminal modification greatly enhanced transfection efficiency for all compounds tested. The reason why , -diaminobutyric acid hydrazide-based lipopeptides were the most potent in transfection is not fully understood but could be related to their high DNA binding.
Conclusions. Poly- or oligo-diaminobutyric acid containing or not a hydrazide C-terminus could advantageously be used in peptide-based gene delivery systems. 相似文献
63.
采用脂质体转染法将人中性粒细胞防御素( H N P1 )的 c D N A 重组真核表达质粒p Babe Neo H N P1导入无血清培养的人和兔的气管粘膜上皮细胞,利用逆转录聚合酶链反应( R T P C R)法,在核酸水平检测 H N P1 在气管上皮细胞的表达。结果:在人和兔的转染上皮细胞中均可检测到 H N P1m R N A 的表达,而在未转染的上皮细胞中 R T P C R检测结果呈阴性。这一结果与作者先前用免疫组化法在蛋白质水平上检测的结果一致,并证明p Babe Neo H N P1 转染至气管粘膜上皮细胞后能得到有效表达。 相似文献
64.
Petrus J. Pauwels Thierry Wurch Christiane Palmier Francis C. Colpaert 《Naunyn-Schmiedeberg's archives of pharmacology》1996,354(2):136-144
5-Hydroxytryptamine (serotonin, 5-HT), essentially known as a neurotransmitter and vasoactive agent, also functions as a mitogen in various cell types through several different second messenger systems. Stimulation of cloned human 5-HT1D receptor sites by sumatriptan in stably transfected rat C6-glial/5-HT1D cells promotes cell growth (Pauwels et al. (1996) Naunyn-Schmiedeberg's Arch Pharmacol 353:144–156). In the present study, the pharmacology of this growth response was investigated using a broad series of 5-HT receptor ligands. The data were compared with the responses obtained by measuring inhibition of forskolin-stimulated cAMP formation. 5-HT (EC50: 25 nM) promoted cell growth of C6-glial/5-HT1D cells, and this in contrast to the absence of any measurable effect in pcDNA3-plasmid transfected and non-transfected C6-glial cells. The 5-HT effect could be mimicked by the following compounds (EC50 in nM): zolmitriptan (0.41), 2-methyl-4-(5-methyl[1,2,4] oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR 127,935; 0.86), naratriptan (0.92), metergoline (1.9), sumatriptan (2.9), (N,N-dimethyl-2-[5-(1,2,4-triazol-1-ylmethyl)-1H-indol-3-y)]ethylamine (MK-462; 3.0), and R(+)-8-hydroxy-2-(di-n-propylamino)tetralin (R(+)-8-OH-DPAT; 30.7). These EC50-values correspond to the compounds binding affinities at the human 5-HT1D receptor site and, with the exception of GR 127,935 and metergoline, also to the EC50-values found by measuring over 5 min inhibition of forskolin (100 M)-stimulated cAMP formation. Prolonged exposure of GR 127,935 (3 h) and metergoline (30 min) to cells yielded EC50 values in the cAMP assay more close to those measured in the mitogenic response. The growth response to sumatriptan, 5-HT, GR 127,935 and metergoline was blocked by the apparently silent antagonists methiothepin, ritanserin and ketanserin with potencies similar to blockade of inhibition of stimulated CAMP formation. The 8-OH-DPAT effect also is likely mediated by 5-HT1D receptors; stereoselectivity was found with its enantiomers at this receptor site and the effect was blocked by ketanserin (1 M) but not by spiperone (1 M). Micromolar concentrations of the 5-HT1B receptor agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo[3, 2-b]pyril-5-one (CP 93,129) and of the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) induced cell growth with a potency that accorded with the affinity of these compounds for the human 5-HT1D receptor site. These effects were sensitive to ketanserin (1 M) antagonism, but not to blockade by -adrenergic blockers and the 5-HT2 receptor antagonist 2-anilino-N-[2-(3-chlorophenoxy)-propyl] acetamidine hydroiodide (BW 501-C-67). The findings suggest that 5-HT1A, 5-HT1B and 5-HT2 receptors are not implicated in 5-HT-stimulated C6-glial/5-HT1D cell growth. In conclusion, human 5-HT1D receptors are involved in the growth of C6-glial/5-HT1D cells. This cellular response is highly sensitive to the intrinsic activity of compounds at 5-HT1D receptors. 相似文献
65.
Anticancer Drug Resistance of HeLa Cells Transfected With Rat Glutathione S-transferase pi Gene 总被引:3,自引:0,他引:3
Cao W Zuo J Meng Y Wei Q Shi ZH Ju LM Fang FD 《Biomedical and environmental sciences : BES》2003,16(2):157-162
Objective To establish a cytologic expressing system of rat glutathione S-transferase pi (GST-pi) cDNA for detecting the resistance of HeLa cells to anticancer drugs. Methods The assessment was made with various anticancer drugs (adriamycin, mitomycin, cisplatinum and vincristine) that showed different cytotoxicities in transfectant HeLa cells with pSV-GT containing rat GST-pi cDNA (HeLa/pSV-GT) or control pSV-neo (HeLa/pSV-neo). Expression levels of GST-pi mRNA in HeLa/pSV-GT and HeLa/pSV-neo were measured by in situ hybridization using Digoxin-labelled cDNA probe. Results HeLa/pSV-GT expressed significantly high degree of GST-pi mRNA, whereas both HeLa/pSV-neo and HeLa cells had very low expression. Cytotoxicities of HeLa/pSV-GT and HeLa/pSV-neo with 4 anticancer drugs were measured by MTT assay. Drug concentrations for yielding 50% inhibition (IC50) in HeLa/pSV-GT by adriamycin, mitomycin and cisplatinum were 70.13μg/mL, 10.95μg/mL and 16.52μg/mE respectively. In contrast, IC50 in HeLa/pSV-neo was 10.34μg/mL, 7.48μg/mL and 13.70μg/mE respectively. The cytotoxicities of vincristine on both HeLa/pSV-GT and HeLa/pSV-neo were not significantly different. Conclusions Our findings suggest that HeLa/pSV-GT containing rat GST-pi cDNA is resistant to some anticancer drugs due to overexpression of GST-pi. Also, HeLa/pSV-GT cell line could serve as a useful cytogenetic model for further research. 相似文献
66.
脂质-鱼精蛋白-DNA复合物的构建及其对细胞的体外转染 总被引:4,自引:1,他引:4
目的研究新型非病毒载体脂质-聚阳离子-DNA(LPD)复合物的制备方法及其对体外细胞的转染率。方法用薄膜-挤压法制备空白阳离子脂质体,与鱼精蛋白-DNA复合物在室温孵育后,得到LPD;用透射电镜观察其形态,用激光粒度仪测定其粒径和zeta电位;LPD与DNA酶I溶液在37 ℃下孵育不同时间后,用琼脂糖凝胶电泳观察其降解情况;用荧光法测定其包封率;用X-gal染色法考察了LPD对张氏(Chang)肝细胞,HepG2肝癌细胞和SMMC-7721肝癌细胞的转染率。结果LPD的形态近似于球体,平均粒径为143.5 nm,平均zeta电位为+32.6 mV;37 ℃下核酸酶作用2 h后,LPD中的DNA几乎无降解;平均包封率为93.42%;LPD对张氏(Chang)肝细胞、HepG2肝癌细胞和SMMC-7721肝癌细胞的转染率分别为(69±6)%,(43±7)%和(96.2±1.8)%。结论LPD是一种制备工艺简单、体外稳定性好、转染率高,具有应用潜力的非病毒载体系统。 相似文献
67.
68.
[目的]探讨新基因MADP-1对雄激素非依赖性前列腺癌细胞系DU145增殖的影响,了解其功能.[方法]采用脂质体和绿色荧光蛋白基因标记的质粒载体pIRES-hrGFP-1α,将MADP-1基因导入前列腺癌细胞系DU145中.应用荧光显微镜、流式细胞仪分析和分选术及细胞生长曲线动态观察MADP-1基因对DU145细胞系的影响.[结果]转染细胞在荧光显微镜下显示绿色荧光.转染MADP-1基因组细胞生长加速,与转染空载体组及对照组相比,有显著差异(P<0.05).[结论]新基因MADP-1在体外具有促进前列腺癌细胞系DU145增殖的作用. 相似文献
69.
70.
目的 了解自杀基因胞嘧啶脱氨酶基因 (cytosinedeaminase ,CD)及其前药 5 氟胞嘧啶 (5 fluorucytosine,5 FC)和bcl Xs基因转移联合作用对卵巢癌细胞株生长的影响。方法 以复制缺陷型腺病毒为载体将CD基因和bcl Xs基因体外转染大鼠卵巢癌细胞株NUTU 19细胞 ,加入含 5 FC的培养基。MTT法检测培养细胞吸光度值 ,计算细胞存活率。结果 单一bcl Xs基因转移及单一CD /5 FC系统作用对NUTU 19细胞的生长抑制作用均随病毒滴度的增加而增大 ;将两者联合作用于NU TU 19细胞 ,其生长抑制率比两者单独使用时的生长抑制率之和更高 ,表现为协同效应 (P <0 .0 0 0 1)。结论 CD /5 FC系统与bcl Xs基因转移联合使用对NUTU 19细胞的生长抑制具有协同作用。 相似文献