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91.
目的:克隆人异质性胞核核糖核蛋白I(hnRNP I)基因并构建原核表达载体,用纯化的重组蛋白进行系统性硬化症(SSc)体外诊断应用研究.方法:从体外培养的HeLa细胞中提取总RNA,用逆转录-聚合酶链反应(RT-PCR)扩增hnRNP I基因,构建原核表达载体pET-30a-hnRNP I,在原核表达系统E.coli BL21(DE3)中表达重组蛋白.重组蛋白纯化后作为抗原对包括系统性硬化症(SSc)、系统性红斑狼疮(SLE)、干燥综合征(SS)、混合型结缔组织病(MCTD)、未分化型结缔组织病(UCTD)、类风湿性关节炎(RA)、正常对照(control)在内的血清相应抗体进行ELISA检测.结果:成功构建了原核表达载体pET-30a-hnRNP I,在IPTG诱导下在E.coli BL21(DE3)中高效表达相对分子质量(M_r)为59 600的重组hnRNP I蛋白,重组蛋白可以可溶蛋白和包涵体蛋白两种形式存在.hnRNP I蛋白抗体在SSc中阳性率(48.72%)明显高于其他各组(P<0.05).结论:利用构建的原核表达载体成功表达出hnRNP I蛋白,此重组蛋白可用于SSc的临床诊断检测. UCTD) 类风湿性关节炎(RA)、正常对照(control)在内的血清相应抗体进行ELISA检测.结果:成功构建了原核表达载体pET-30a-hnRNP I,在IPTG诱导下在E.coli BL21(DE3)中高效表达相对分子质量(Mr)为59 600的重组hnRNP I蛋白,重组蛋白可以可溶蛋白和包涵体蛋白两种形式存在.hnRNP I蛋白抗体在SSc中阳性率(48.72%)明显高于其他各组(P<0.05).结论:利用构建的原核表达载体成功表达出hnRNP I蛋白,此重组蛋白可用于SSc的临床诊断检测. UCTD) 类风湿性关节炎(RA)、正常对照(control) 相似文献
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BACKGROUND: Overexpression of α-synuclein can induce cell apoptosis. RNA interference (RNAi) may block specific gene function and cause gene silencing. OBJECTIVE: To construct a specific and effective RNAi plasmid for the α-synuclein gene and investigate if RNAi can block apoptosis in HEK293 cells, induced by overexpression of wild-type α-synuclein.
DESIGN, TIME AND SETTING: A contrast experiment based on genetically engineered cytobiology was performed at the State Key Lab of Medical Genetics of China, Xiangya Medical College of Central South University, between October 2004 and October 2008.
MATERIALS: HEK293 cells and pBSHH1 plasmid were provided by the State Key Lab of Medical Genetics of China; OligDNA sequence by Sagon Bioengineering Company, Shanghai; Lipofectamine 2000 by Invitrogen, USA; α-synuclein monoclonal antibody, Hoechst 33258, and MTT by Sigma, USA; Horseradish peroxidase-coupled goat anti-rat IgG by KPL, USA; FACSan flow cytometry by BD, USA.
METHODS: Four target sites were used to construct hairpin RNA pBSHH1 vectors - pSYNi-1, pSYNi-2, pSYNi-3 and pSYNi-4 - which were cloned in the pBSHH1 plasmid. HEK293 cells were transfected using Lipofectamine 2000. In addition, a non-transfect group and a negative plasmid transfect group were established. The cultured HEK293 cells were processed as follows: transfection of blank plasmid (blank control group), transfection of α-synuclein-pEGFP and RNAi negative vector (negative control group), and transfection of α-synuclein-pEGFP and pSYNi-1 (transfection group). Cells in all groups were transfected with Lipofectamine 2000 for 48 hours.
MAIN OUTCOME MEASURES: Expression of α-synuclein mRNA and protein were detected by RT-PCR and Western blot. Cell morphology was observed under an inverted fluorescence microscope; cell viability was measured using MTT method; and cell apoptosis was determined with Annexin V-PE flow cytometry.
RESULTS: α-synuclein mRNA and protein expressions were significantly decreased in the pSYNi-1 group when compared with the non-transfect and negative plasmid transfect groups (P 〈 0.05). The expressions were partially decreased in the pSYNi-2 group, but there was no significant difference in the pSYNi-3 and pSYNi-4 groups. Hoechst staining indicated that cell nuclei were enlarged in the negative control group, coloring was not uniform, and chromatin was accumulated and appeared spot-like. The nucleus coloring was uniform in the transfection group compared to negative control group. Cell viability in the negative control group was significantly lower than blank control group with cell apoptosis being significantly increased (P 〈 0.05). In comparison with negative control group, cell viability was significantly increased in the transfection group and cell apoptosis was significantly decreased (P 〈 0.05).
CONCLUSION: pSYNi-1 can inhibit α-synuclein gene expression and block apoptosis of HEK293 cells induced by overexpression of wild-type α-synuclein. 相似文献
DESIGN, TIME AND SETTING: A contrast experiment based on genetically engineered cytobiology was performed at the State Key Lab of Medical Genetics of China, Xiangya Medical College of Central South University, between October 2004 and October 2008.
MATERIALS: HEK293 cells and pBSHH1 plasmid were provided by the State Key Lab of Medical Genetics of China; OligDNA sequence by Sagon Bioengineering Company, Shanghai; Lipofectamine 2000 by Invitrogen, USA; α-synuclein monoclonal antibody, Hoechst 33258, and MTT by Sigma, USA; Horseradish peroxidase-coupled goat anti-rat IgG by KPL, USA; FACSan flow cytometry by BD, USA.
METHODS: Four target sites were used to construct hairpin RNA pBSHH1 vectors - pSYNi-1, pSYNi-2, pSYNi-3 and pSYNi-4 - which were cloned in the pBSHH1 plasmid. HEK293 cells were transfected using Lipofectamine 2000. In addition, a non-transfect group and a negative plasmid transfect group were established. The cultured HEK293 cells were processed as follows: transfection of blank plasmid (blank control group), transfection of α-synuclein-pEGFP and RNAi negative vector (negative control group), and transfection of α-synuclein-pEGFP and pSYNi-1 (transfection group). Cells in all groups were transfected with Lipofectamine 2000 for 48 hours.
MAIN OUTCOME MEASURES: Expression of α-synuclein mRNA and protein were detected by RT-PCR and Western blot. Cell morphology was observed under an inverted fluorescence microscope; cell viability was measured using MTT method; and cell apoptosis was determined with Annexin V-PE flow cytometry.
RESULTS: α-synuclein mRNA and protein expressions were significantly decreased in the pSYNi-1 group when compared with the non-transfect and negative plasmid transfect groups (P 〈 0.05). The expressions were partially decreased in the pSYNi-2 group, but there was no significant difference in the pSYNi-3 and pSYNi-4 groups. Hoechst staining indicated that cell nuclei were enlarged in the negative control group, coloring was not uniform, and chromatin was accumulated and appeared spot-like. The nucleus coloring was uniform in the transfection group compared to negative control group. Cell viability in the negative control group was significantly lower than blank control group with cell apoptosis being significantly increased (P 〈 0.05). In comparison with negative control group, cell viability was significantly increased in the transfection group and cell apoptosis was significantly decreased (P 〈 0.05).
CONCLUSION: pSYNi-1 can inhibit α-synuclein gene expression and block apoptosis of HEK293 cells induced by overexpression of wild-type α-synuclein. 相似文献
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《实用口腔医学杂志》2020,(5)
目的探讨丹参多酚对缺氧复氧诱导的心肌细胞凋亡及聚腺苷二磷酸核糖聚合酶-1(PARP-1)表达的影响。方法对心肌细胞H9C2进行缺氧复氧处理构建体外心肌细胞凋亡模型,并分为模型组与丹参多酚组,其中丹参多酚组用丹参多酚进行预处理,模型组不做处理,另设对照组细胞不进行缺氧复氧处理。四甲基偶氮唑盐(MTT)法检测细胞增殖,流式细胞术检测细胞凋亡,丙二醛(MDA)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)试剂盒检测各个活性物质含量变化,蛋白质印迹法检测PARP-1的表达。结果丹参多酚组细胞存活率、细胞凋亡率与对照组差异无统计学意义,并且显著高于模型组(P<0.001),SOD的表达明显高于模型组(P<0.05),LDH、MDA的表达明显低于模型组(P<0.05),模型组PARP-1蛋白相对表量明显高于对照组,差异具有统计学意义(P<0.001)。丹参多酚组PARP-1表达显著低于模型组,差异具有统计学意义(P<0.001)。结论丹参多酚能够抑制缺氧复氧诱导的心肌细胞凋亡,这种调控作用可能与其对PARP-1表达的抑制作用有关。 相似文献
97.
目的通过RT-PCR方法对2009年宁波市狂犬病病毒分离株(NB0901)的N基因扩增并测序,了解宁波地区狂犬病病毒的流行特点。方法运用RT-PCR方法和序列测定方法获得NB0901株的N基因序列,并在核苷酸和氨基酸水平与疫苗株(3aG和CNT-1)进行同源性比较,同时进行遗传进化分析。结果狂犬病病毒NB0901株与疫苗株的核苷酸和氨基酸序列同源性分别在86.6%~90.1%和95.5%~98.8%之间。NB0901株N蛋白的抗原表位与疫苗株相比后发现在NⅠ和NⅡ抗原表位存在突变。而在Th细胞表位上,NB0901株与CNT-1株完全一致,与3aG株相比变异较大。进化结果表明NB0901株属于狂犬病病毒Ⅰ群中的A亚型。结论狂犬病病毒NB0901株和当前疫苗株虽同属于Ⅰ群,但是与疫苗株相比存在差异,从氨基酸序列水平上分析,CNT-1株较3aG株更能有效保护流行毒株感染。 相似文献
98.
目的 探讨康复训练对大鼠脑出血后神经细胞再生的影响.方法 将75只SD大鼠分为康复组、制动组和假手术组,每组25只.康复组和制动组应用胶原酶Ⅶ注射入苍白球诱导脑出血模型,假手术组用生理盐水替代胶原酶.康复组每天予以抓握、平衡、旋转等训练,制动组置于网状笼内固定.各组分成脑出血后第1,4,7,14,28天共5个时间点,每... 相似文献
99.
目的了解云南省狂犬病毒的流行情况以及街毒株N、G基因结构特征,为有效控制狂犬病疫情提供初步科学依据。方法对云南省2006-2010年10株狂犬病街毒株N、G基因进行全基因克隆、测序,并与已知国内外代表毒株核苷酸及推导氨基酸进行序列比对及系统发育分析。结果序列分析表明所研究的10个云南毒株与Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ型代表毒株N、G基因核苷酸序列同源性分别为72.1%~78.5%、58.4%~71.0%,与基因Ⅰ型毒株序列同源性为85.2%~90.1%、82.7~99.6%,云南毒株之间核苷酸序列同源性为89.3%~99.2%、86.0~99.6%。云南10个毒株均为基因Ⅰ型和血清1型毒株,分为2个进化分支,除YNTC06与GXN119和HM88单独属于分支Ⅲ外,其余云南毒株均分布在分支Ⅰ上,且进一步分为3个小亚分支。遗传进化分析表明,各毒株氨基酸位点均存在不同程度的变异,其中YNTC06在360、369、375等多个NP抗原位点存在变异;云南毒株在GP330、333位毒力决定位点未发生变异,均为强毒株。结论云南狂犬病毒属于基因Ⅰ型,存在2个分支;云南狂犬病毒NP和GP存在特异性氨基酸变异位点,其基因结构及变异、亚组群划分与地理位置无相关性。 相似文献
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Smoothened(Smo)基因在结肠腺癌Lovo细胞的表达及其作用 总被引:1,自引:0,他引:1
目的探讨Smoothened(Smo)基因在结肠腺癌Lovo细胞的表达水平及在细胞增殖与凋亡过程中的作用。方法应用Westernblot及半定量RT—PCR检测结肠腺癌Lovo细胞内Smo基因的表达水平,再应用RNAi技术降解Lovo细胞内的SmomRNA表达,然后应用MTT法、流式细胞术检测SmomRNA被降解后Lovo细胞增殖水平与凋亡水平的变化。结果结肠腺癌Lovo细胞内有Smo蛋白及SmomRNA的高表达,SmomRNA被降解后,Lovo细胞的增殖水平明显抑制(P〈0.05),凋亡率明显升高(P〈0.001)。结论结肠腺癌Lovo细胞的发生与Smo基因的高表达有关,Smo基因参与结直肠癌的增殖与凋亡过程。 相似文献