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21.
TCRVβ7.1基因修饰T细胞对乳腺癌细胞杀伤作用的研究 总被引:1,自引:1,他引:1
目的:观察TCPVβ7.1基因转染前后正常人外周血淋巴细胞对乳腺癌细胞株杀伤活性的影响。方法:脂质体包裹PdWA3.1vβ7.1后转染健康人PBMC,流式细胞仪检测PdWA3.1Vβ7.1基因表达,改良MIT法检测TCRVβ7.1基因转染前后正常人外周血淋巴细胞对乳腺癌细胞株杀伤活性。结果:TCRVβ7.1基因转染可显著增加正常人PBMC该基因表达,转染前后正常人外周血淋巴细胞对乳腺癌细胞株杀伤活性有显著性差异。结论:用TCR基因修饰可明显提高正常人PBMC对乳腺癌细胞杀伤作用。 相似文献
22.
Summary To study the osteogenic potential of cultured bone marrow stromal cells (BMSCs) transfected with transforming growth factor
β1 (TGF-β1) genein vitro, cultured BMSCs were transfected with the complexes of pcDNA3-TGF-β1 and Lipofectamine Reagentin vitro. The cell proliferation was detected by MTT method and the morphological features of transfected BMSCs was observed. ALP
stains and PNP method were used to measure ALP activity. In addition, the collagen type I propeptides and mineralized matrixes
were examined by immunohistochemical staining and tetracycline fluorescence labeling respectively. The morphological and biological
characters of the transfected BMSCs were similar to those of osteoblasts and the cell proliferation was promoted. The cell
layer displayed strong positive reaction for ALP stains and immunohistochemical staining. ALP activity and collagen type I
expression increased remarkably after transfection. Mineralized matrixes formed earlier and more in transfected BMSCs as compared
with control group. It is concluded that transfecting with TGF-β1 gene could promote the osteogenic potential of cultured BMSCs. 相似文献
23.
目的研究HBV T1862变异的生物学意义。方法采用分子生物学方法,构建HBV前C/C基因EB病毒真核表达载体,利用体外定点突变技术诱导前C/C基因T1862变异,经PCR-RFLP初筛并经测序终鉴定出阳性克隆后,以脂质体介导方法将突变前后的重组质粒转染Cos7细胞, 以ELISA 检测HBeAg的表达量。结果未突变的重组质粒可稳定表达HBeAg,突变后的重组质粒未能检测到HBeAg表达。结论 HBV前C/C基因1862点突变的真核表达载体的构建,为体外研究该点突变引起HBV的一系列生物学改变奠定基础。 相似文献
24.
目的:构建白念珠菌钙调蛋白基因(CMD1)缺陷HS3酵母菌体,为进一步探讨钙调蛋白基因突变对真菌生长周期及致病性的影响奠定基础。方法:首先将含cmd1::TRP1置换序列的质粒I转化二倍体酵母菌株YPH501(his3trplural),经Soutthern印迹法筛选出含TRP1序列的菌株。其次,将含CMD1序列的质粒Ⅱ转化以上TRP1阳性菌株,进行减数分裂后选择得到TRP1阳性酵母菌株单倍体。最后,将含trp1:HIS3置换序列质粒Ⅲ转化上述TRP1阳性单倍体菌株,用不含His培养基培养得到钙调蛋白基因缺陷HIS3酵母菌株。结果:经Southern印迹法证实cmd1:TRP1基因置换克隆;减数分裂后选择得到了TRP1阳性酵母菌株单倍体;质粒Ⅲ转化单倍体后经不含His培养基培养得到CMD1缺陷HIS3菌株,接种于不含Trp倍养基上未见有菌落生长,说明质粒Ⅲ转化单倍体后已将his3TRP1转换成HIS3trp1。结论:成功构建了钙调蛋白基因缺陷HIS3酵母菌株,基因型为cmd1trp1HIS3。 相似文献
25.
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
26.
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
27.
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
28.
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
29.
30.
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