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1.
由成年转基因山羊体细胞而来的克隆山羊   总被引:22,自引:0,他引:22       下载免费PDF全文
在已经获得的乳腺特异性表达人促红细胞生成素 (rhEPO)成年转基因山羊 (Caprahircus)的基础上 ,取其耳尖成纤维细胞和卵巢颗粒细胞 ,进行体外传代培养 ,然后将这种培养的转基因山羊的体细胞移入去核的处于第Ⅱ次减数分裂中期的卵母细胞中 ,并进行电融合 ,构建重构胚胎 ,重构胚胎在体内培养 6d ,再将发育至囊胚或桑椹胚的重构胚胎移入同步情期的寄母羊子宫内。结果 ,有 2只寄母羊妊娠并最终产下 2只成活的克隆山羊。她们分别来自同一成年母羊的耳尖成纤维细胞和卵巢颗粒细胞。克隆羊经PCR RFLP图谱分析显示 :以克隆羊组织DNA为模板的PCR产物与相应的提供体细胞的基因羊的PCR产物的酶切图谱完全一致 ;并且经PCR对外源hEPO基因检测表明 2只克隆山羊均携带hEPO外源基因。由此证明获得了转基因成年体细胞的克隆山羊  相似文献
2.
D—半乳糖对神经元和成纤维细胞拟老化作用的实验研究   总被引:21,自引:0,他引:21  
实验用体内、外相结合的方法,观察了D-半乳糖(D-galactose,D-gal.)对细胞的促老化作用。结果表明,D-gal使培养大鼠胎脑神经元出现生长发育缓慢、突起脱落、死亡率增高等退行性变;大鼠肺成纤维细胞的体外分裂代数减少;人胎儿肺二倍体成纤维细胞(HBS)分裂周期中G2-M期细胞减少、G-G1期细胞增加、DNA含量下降,细胞增殖速度减慢;大鼠胎脑神经细胞和HBS中SOD含量降低,丙二醛(MDA)水平升高,这些结果与次黄瞟呤(HPX)-黄瞟呤氧化酶(XOD)系统产生的超氧阴离子自由基(O-2·)作用一致。提示,D-gal抑制细胞生长发育和分裂繁殖,加速细胞老化,其机理可能与O-2·等活性氧(ROS)过荷引起的脂质过氧化有关。  相似文献
3.
To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G0/G1 phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G1/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G1 phase or G2 phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G2 phase. Little lethality was observed in cultures in G1 phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.  相似文献
4.
Cloned goats(Capra hircus)from adult ear cells   总被引:11,自引:0,他引:11       下载免费PDF全文
The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MII chromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female dining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5μ mol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no signifi  相似文献
5.
云南矮马耳缘组织成纤维细胞系的建立及其生物学特性   总被引:10,自引:0,他引:10  
随着全球生物多样性及其遗传资源丧失程度的加剧 ,使得各国纷纷投入大量人力和财力 ,并以多种方式收集、保存和整理大自然赋予人类的宝贵财富 ,如美国典型培养物保藏中心 (AmericanTypeCultureCollection ,ATCC) ,欧洲动物细胞培养物保藏中心 (EuropeanCollectionofAnimalCellC  相似文献
6.
检测人成纤维细胞增殖的XTT比色法   总被引:10,自引:0,他引:10       下载免费PDF全文
利用3种不同来源的成纤维细胞(大鼠肾上皮细胞,人胎肺成纤维细胞,人成纤维细胞)的活细胞线粒体脱氢酶在电子偶联剂硫酸酚嗪甲酯(phenazine methosulfate, PMS)的协同作用下,还原四氮唑复合物(XTT)形成可溶性的棕黄色甲簪(formazan)产物,测定细胞甲簪的生成量来反映细胞的生长与活性状态,并与传统的四甲基偶氮唑盐(MTT)方法作比较.结果表明,XTT方法直接测定水溶性的甲簪产物,敏感度高于MTT法,具有操作简单、快速、灵敏度高、结果准确的优点,为成纤维细胞的研究建立了新的检测方法.  相似文献
7.
5 氨基γ 酮戊 (ALA)及其己酯 (He ALA)具有内源生成光敏剂的特点 ,在肿瘤光动力探测及治疗中显示出了优势。ALA及He ALA对神经母细胞瘤、肝癌细胞及成纤维细胞的光动力作用被研究比较。由特征荧光光谱证实 ,经ALA或He ALA培养后 ,三种细胞内均可生成原卟啉 (PpIX)产物。激光扫描荧光显微镜显示 ,在经ALA或He ALA培养后的神经母细胞瘤中 ,PpIX均以弥散方式分布在细胞质中。PpIX在三种细胞中的积聚动力学过程不同 ,随着ALA或He ALA培育时间的增长 ,PpIX在肝癌细胞及成纤维原细胞中的积累增加 ,而在神经母细胞瘤中PpIX在 8h后已达到饱和。此外 ,在同样的培育条件下 ,神经母细胞瘤中PpIX的生成浓度明显高于肝癌细胞及成纤维细胞。经ALA培养及光照射后 ,可使近 90 %的神经母细胞瘤失活 ;而在同样条件下却只能杀伤 5 0 %左右的肝癌细胞及成纤维细胞。揭示了神经母细胞瘤对ALA光动力作用有极高的敏感性 ,并适于光动力治疗。与ALA相比 ,He ALA可在三种细胞内造成与ALA相近的杀伤率 ,但所用的药物浓度却比ALA低 10倍 ,显示He ALA具有极高的光动力灭活效率。因此在内源光动力治疗中 ,He ALA是一种极具开发前景的新药物。  相似文献
8.
In an effort to regulate the behavior of mammalian cell entrapped in a gel, the gels were functionalized with the putative cell-binding (-Arg-Gly-Asp-) (RGD) domain. The adhesion molecules composed of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides and the cell recognition ligands were inculcated into the thermo-reversible hydrogel composed of N-isopropylacrylamide, with a small amount of succinyl poly(ethylene glycol) (PEG) acrylate (MW 2000) used as the biomimetic extracellular matrix (ECM). The GRGDS-containing p(NiPAAm-co-PEG) copolymer gel was examined in vitro for its ability to promote cell spreading and to increase the viability of the cells by introducing PEG spacers. ECM poorly adhered to hydrogel lacking adhesion molecules permitting only a 20% spread of the seeded cells after 10 days. When the PEG spacer arms, which were immobilized by a peptide linkage, had been integrated into the hydrogel, the conjugation of RGD improved cell spreading by 600% in a 10-day trial.  相似文献
9.
兔体细胞核移植的初步研究   总被引:4,自引:0,他引:4       下载免费PDF全文
实验以兔胎儿成纤维细胞为核供体,对兔体细胞核移植技术的融合,激活和发育等环节进行了初步研究。实验通过比较不同电场强度对兔2细胞胚胎卵裂球融合以及卵母细胞激活的影响,证实200和260V/mm的电场强度可有效地诱导2细胞胚胎的融合和兔卵母细胞的孤雌激活。然后将200和260V/mm电场强度用于体细胞核移植,融合率分别为44.4%和48.4%,卵裂率分别为58.8%和53.8%,桑椹胚/囊胚发育率分别为5.9%和5.5%。但112枚核移植胚胎移植到5只受体后没有幼子出生。结果表明,实验中所建立的程序至少可以支持兔体细胞克隆胚胎的早期发育。  相似文献
10.
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