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41.
JIANG Xun ZENG Yao-ying HE Xian-hui XU Li-hui DI Jing-fang FENG Zheng ZHAO Jing-xian WANG Qing WANG Tong SHI Jian-bo 《园艺学报》2004,20(6):924-928
AIM: To investigate the effect of enhanced green fluorescence protein (EGFP) gene transfection on the cell cycle distribution of primary cultured human chondrocytes in order to establish a tracking method of cultured human nasoseptal chondrocytes. METHODS: pEGFP-N1 plasmid was amplified in E.coli, and purified by high purity kit. Primary cultured human chondrocytes,which were initially obtained from the nasoseptal cartilage, were cultured in vitro and transferred with pEGFP-N1 by means of electroporation with Amaxa nucleofector device. Transfering process and transient expression were evaluated by laser scanning confocal microscope (LSCM), the transfer efficiency and the cell cycle distribution were evaluated by flow cytometry. RESULTS: There was significant expression of EGFP at 24 h after transferring. The transfection efficiency of pEGFP-N1 into primary cultured human chondrocytes reached 35.37% at 48 h. It didn't affect the process of cell adherance and had no effect on the cell cycle distribution. CONCLUSION: Primary cultured human chondrocytes, which were transfected with pEGFP, are alive in vitro, and the transferring process doesn't affect the cell cycle distribution. These results suggest that pEGFP-N1 is an ideal transient expression vector for primary cultured human chondrocytes and it might be a well tracer in construction tissue engineered cartilage. 相似文献
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AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34+ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34+ and CD34- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34+ and CD34-,total MNC led to a significant increase in the expansion of CD34+ cells compared with CD34 enrichment (P<0.05). There was a trend toward decreased apoptosis in cultures when early passage was performed once the linear cord like structures appeared. There was no significant effect on apoptosis between with VEGF and without VEGF group (P>0.05). These differentiated EPCs were stained positive for CD34+, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34+ and AC133+cells accounted for 68.2%±6.3% (n=6) and 57.2%±9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34+ and CD34- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs. 相似文献
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AIM: To observe the change of insulin receptor in rabbit kidney with acute ischemic-reperfusion injury. METHODS: 15 Japanese white rabbits were allocated randomly into control group, ischemic-reperfusion group(IR group). IR group received clamping for 1 h followed by 2 h or 48 h of reperfusion. At 2 h or 48 h after reperfusion, glucose and insulin in serum were determined. Insulin receptor in renal tissue was analyzed by radioligand binging assay(BAD). RESULTS: The level of serum glucose increased after 2 h reperfusion in 2 groups, but in IR group the value increased much more higher than those in control groups(P<0.05). Plasma insulin of IR group was significantly higher than that in control after 2 h reperfusion(P<0.05). Scatchard analysis of data resulted in curvilinear profiles, indicating that there are two classes of receptors with different affinity or the presence of a single class of receptors with a negative cooperative hormone-receptor interaction. Data analyzed for a two-site model showed that the values of Bmax1(high affinity site), Bmax2(low affinity site) and Kd1, Kd2 were significantly lower than that of control (P<0.05) after 2 h perfusion. 48 h after IR there was no difference of Bmax1, Bmax2, and Kd1 between 2 groups,but Kd2 of IR group was higher than that of control (P<0.05). CONCLUSIONS: The results indicate that the effect of intrinsic insulin decreases in the progress of the renal ischemic-reperfusion. The resulting high serum glucose may aggravate renal injury in the progress of ischemic-reperfusion. 相似文献
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猪原始生殖嵴细胞(PGCs)建系因素的研究 总被引:4,自引:3,他引:4
从五指山猪(WSZP)近交系第8~13代培育群中,先后选用21头5~10月龄青年母猪,分别于授精后25~30d采集胎儿106个,进行原始生殖嵴(PGCs)细胞分离、培养等建系技术研究。以DMEM F10(1:1)为基础培养液,按添加或不添加生长因子,将培养液分为A、B、C3种,并以STO细胞作饲养层,在38℃、5.0%CO2和湿润的气相中进行培养建系。结果获得胚胎生殖嵴细胞(EG)细胞系6个细胞株,其中1个EG细胞株传至11代、2个传至5代、1个传至4代、2个传至3代冻存。并进行了AKP染色、体外分化、冷冻-解冻复苏和嵌合体制作等鉴定研究。研究发现:不同胚龄对EG细胞建系具有一定影响,不同培养液对EG细胞建系效果不同,STO细胞饲养层的质量是建株、传代、冷冻-解冻复苏的关键因素之一。EG细胞系的初步建立,为今后筛选进入种系的EG细胞系、实施体外基因操作提供了可能。 相似文献
47.
植物育种的物理学方法 总被引:10,自引:0,他引:10
介绍了草原生态恢复中选育优良牧草种子的一些物理学方法,包括电场、磁场、物理辐射、激光、离子束和交变应力等在植物的生物学效应、诱变育种和转基因技术中的应用. 相似文献
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构建表达eae和stx1/2B的融合基因,克隆eae基因的C端280个氨基酸残基(Int280)基因部分,以正确地阅读框定向插入到含有stx1/2B融合基因的质粒,构建重组质粒,将其转化于BL21(DE3),用IPTG进行诱导表达,经SDS—PAGE电泳检测,该融合蛋白获得了高效表达。薄层扫描分析表明:目的蛋白表达量占菌体总蛋白含量的50.67%。由于该融合蛋白由eae、stx1B、stx2B等三部分抗原组成,可刺激机体产生针对紧密素和StxB的抗体,在EHEC O157亚单位疫苗设计或单克隆抗体抗制备中具有重要价值。 相似文献
50.
纳豆菌、甘露寡糖对仔猪肠道pH、微生物区系及肠黏膜形态的影响 总被引:24,自引:1,他引:24
选取144头18日龄断奶,体重5.6kg的杜大长三元杂仔猪,按体重和性别分成6个处理,每组3个重复,每重复8头仔猪,研究纳豆菌(Natto)和甘露寡糖(MOS)对早期断奶仔猪肠道pH、微生物区系和小肠黏膜形态的影响。结果为:(1)Natto、MOS均有降低仔猪肠道pH的趋势(P>0.05),Natto与MOS联用时显著降低仔猪空肠、回肠、盲肠和结肠内容物的pH(P<0.05);(2)Natto提高了仔猪结肠内容物中乳酸杆菌和双歧杆菌数量(P<0.05),提高结肠黏膜中乳酸杆菌数量(P<0.05),MOS提高了仔猪结肠内容物和黏膜中乳酸杆菌数量(P<0.05),2g/kg的MOS组使结肠内容物和黏膜中大肠杆菌数下降(P<0.05)。Natto与MOS联用时内容物中大肠杆菌数量下降(P<0.05),乳酸杆菌数量显著高于金霉素组(P<0.05),黏膜中乳酸杆菌数量上升,大肠杆菌数下降,与空白组、金霉素组有显著差异(P<0.05);(3)Natto及联用组都显著提高了小肠黏膜的绒毛高度(P<0.05),但处理组间隐窝深度没有显著差异(P>0.05),Natto组、1g/kg的MOS组及联用组的绒毛高度/隐窝深度比值显著上升(P<0.05)。试验说明,纳豆菌和甘露寡糖通过调节肠道内容物及黏膜中微生物区系,降低肠道pH,以维持仔猪肠黏膜正常的形态结构。 相似文献