全文获取类型
收费全文 | 704篇 |
免费 | 85篇 |
国内免费 | 17篇 |
出版年
2023年 | 15篇 |
2022年 | 13篇 |
2021年 | 16篇 |
2020年 | 38篇 |
2019年 | 37篇 |
2018年 | 17篇 |
2017年 | 28篇 |
2016年 | 17篇 |
2015年 | 15篇 |
2014年 | 36篇 |
2013年 | 45篇 |
2012年 | 32篇 |
2011年 | 34篇 |
2010年 | 17篇 |
2009年 | 26篇 |
2008年 | 21篇 |
2007年 | 28篇 |
2006年 | 23篇 |
2005年 | 20篇 |
2004年 | 20篇 |
2003年 | 22篇 |
2002年 | 14篇 |
2001年 | 9篇 |
2000年 | 13篇 |
1999年 | 17篇 |
1998年 | 9篇 |
1997年 | 11篇 |
1996年 | 14篇 |
1995年 | 15篇 |
1994年 | 23篇 |
1993年 | 22篇 |
1992年 | 18篇 |
1991年 | 19篇 |
1990年 | 11篇 |
1989年 | 9篇 |
1988年 | 8篇 |
1987年 | 5篇 |
1986年 | 7篇 |
1985年 | 12篇 |
1984年 | 10篇 |
1983年 | 5篇 |
1982年 | 6篇 |
1981年 | 9篇 |
1980年 | 8篇 |
1979年 | 2篇 |
1978年 | 3篇 |
1977年 | 1篇 |
1976年 | 3篇 |
1974年 | 1篇 |
1973年 | 2篇 |
排序方式: 共有806条查询结果,搜索用时 171 毫秒
91.
Effects of oxygen on engineered cardiac muscle 总被引:6,自引:0,他引:6
Carrier RL Rupnick M Langer R Schoen FJ Freed LE Vunjak-Novakovic G 《Biotechnology and bioengineering》2002,78(6):617-625
Concentration gradients associated with the in vitro cultivation of engineered tissues that are vascularized in vivo result in the formation of only a thin peripheral tissue-like region (e.g., approximately 100 microm for engineered cardiac muscle) around a relatively cell-free interior. We previously demonstrated that diffusional gradients within engineered cardiac constructs can be minimized by direct perfusion of culture medium through the construct. In the present study, we measured the effects of medium perfusion rate and local oxygen concentration (p(O2)) on the in vitro reconstruction of engineered cardiac muscle. Neonatal rat cardiomyocytes were seeded onto biodegradable polymer scaffolds (fibrous discs, 1.1 cm diameter x 2 mm thick, made of polyglycolic acid, 24 x 10(6) cells per scaffold). The resulting cell-polymer constructs were cultured for a total of 12 days in serially connected cartridges (n = 1-8), each containing one construct directly perfused with culture medium at a flow rate of 0.2-3.0 mL/min. In all groups, oxygen concentration decreased due to cell respiration, and depended on construct position in the series and medium flow rate. Higher perfusion rates and higher p(O2) correlated with more aerobic cell metabolism, and higher DNA and protein contents. Constructs cultured at p(O2) of 160 mm Hg had 50% higher DNA and protein contents, markedly higher expression of sarcomeric alpha-actin, better organized sarcomeres and cell junctions, and 4.5-fold higher rate of cell respiration as compared to constructs cultured at p(O2) of 60 mm Hg. Contraction rates of the corresponding cardiac cell monolayers were 40% higher at p(O2) of 160 than 60 mm Hg. The control of oxygen concentration in cell microenvironment can thus improve the structure and function of engineered cardiac muscle. Experiments of this kind can form a basis for controlled studies of the effects of oxygen on the in vitro development of engineered tissues. 相似文献
92.
Transport characteristics of certain inorganic elements such as copper, magnesium, selenium and iron have been studied in maternal-fetal direction in normal pregnancies, using in vitro perfusion of isolated placental lobules. Copper, selenium, magnesium and iron salts corresponding to twice physiological concentrations were injected as a 100 l bolus, into the maternal arterial perfusate. Serial perfusate samples were collected from venous outflows for a study period of 5 min. Concentrations of various inorganic elements and their transport kinetics were determined. Transport fractions of copper, selenium, magnesium and iron averaged 0.14, 0.19, 0.06 and 0.23% of maternal load respectively. The pharmacokinetic parameters such as area under the curve, clearance, elimination constant, and time for maximum response showed some significant differences between the various elements. We speculate that copper and selenium share the same transport pathway along a concentration gradient in maternal-fetal direction, while for iron and magnesium, active transport plays a predominant role for element transfer across the human placental membrane. 相似文献
93.
Kai Iding Dirk Lütkemeyer Elisabeth Fraune Kurt Gerlach Jürgen Lehmann 《Cytotechnology》2000,34(1-2):141-150
Since 1969 much attention has been devoted to the useof spinfilter systems for retention of mammalian cellsin continuous perfusion cultivations. Previousinvestigations dealt with hydrodynamic conditions,fouling processes and upscaling. But hydrodynamicconditions and fouling processes seem to have asecondary importance in spinfilter performance duringauthentic perfusion cultivations. Obviously,alterations in culture condition are more relevantespecially during long-term processes. Therefore, ourpratical approach focussed on the performance qualityof a commercially available 20 m spinfilterduring a perfusion cultivation of a recombinant CHOcell line in pilot scale regarding the followingissues: 1) retention of viable cells in thebioreactor; 2) removal of dead cells and cell debrisfrom the bioreactor; 3) alterations in culturecondition; and 4) changes in perfusion mode.Furthermore, we tested the performance of 20 mspinfilters in 2 and 100 l pilot scale using solidmodel particles instead of cells. Our investigationsshowed that retention of viable cells in pilot scalewas independent of spinfilter rotation velocity andperfusion rate; the retention increased from 75 to 95%corresponding to operation time, enlarging celldiameter and enhanced formation of aggregates in theculture during the perfusion cultivation. By means ofthe Cell Counter and Analyzer System (CASY) anoperation cut off of 13 m was determined forthis spinfilter. Using solid model particles in 2 lscale, optimal retention was achieved at a tip speedof 0.43 m s-1 (141 rpm) – furtherenhancement of spinfilter rotation velocity up to0.56 m s-1 (185 rpm) decreased the retentionrapidly. In pilot scale best retention performance wasobtained with tip speeds of 0.37 m s-1(35 rpm) and 1.26 m s-1 (120 rpm). Hence,significant retention in pilot scale could already beachieved with low agitation. Therefore, the additionof shear force protectives could be avoided so thatthe purification of the target protein from thesupernatant would be facilitated. 相似文献
94.
Monocyte-colony inhibition factor (M-CIF) was produced in microcarrier perfusion cultures from engineered Chinese hamster
ovary (CHO) cells. Three and fifteen liter microcarrier perfusion bioreactors equipped with internal spin filters were operated
for over two months. Approximately 60 L and 300 L of culture filtrate were harvested from the 3L and 15L microcarrier perfusion
bioreactors respectively. During the perfusion operation, cell density reached 2–6 × 106 cells/ml. Importantly, stable expression of M-CIF from the CHO cells under non-selection condition was maintained at a level
of 4–10 mg/L. Specific productivity was maintained at 1.8–3.4 mg/billion cells/day. The ability of the recombinant CHO cells
to migrate from microcarrier to microcarrier under our proprietary HGS-CHO-3 medium greatly facilitated microcarrier culture
scale-up and microcarrier replenishment. Future directions for microcarrier perfusion system scale-up and process development
are highlighted.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
95.
An alternative culture system has been developed based on a conventional tissue culture plate (3.5 cm diameter) which is changed into a closed perfusion chamber. The system can easily be scaled up from one to several chambers. The shape and the size of the area of cell growth may be designed to individual experimental demands. The whole culture chamber is optically accessible, so cell growth and morphology can be evaluated by light microscopy. Furthermore the cellular physiology can be characterised by any fluorimetric assay using a bottom type fluorescence reader. A peristaltic pump sustains a constant medium flow through the chamber thus creating true homeostasis. The use of HPLC-valves and connectors allows the switching between different media or assay solutions. Thus it is possible to perform in situ assays also measuring transient effects. A protocol for vitality tests using calcein-AM is worked out for an adherent cell line and for a suspension cell line. The lower detection limits are 7 × 102 cells cm-2 for the adherent cells and 5 × 104 cells mL-1 for the suspension cells. The upper limits are 1–2 × 105 cells cm-2 respectively 8 × 106 cells mL-1. 相似文献
96.
Bote G. Bruinsma James H. Avruch Pepijn D. Weeder Gautham V. Sridharan Basak E. Uygun Negin G. Karimian Robert J. Porte James F. Markmann Heidi Yeh Korkut Uygun 《Journal of visualized experiments : JoVE》2015,(98)
There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 °C. 相似文献
97.
Izuno H Kobayashi Y Sanada Y Nihei D Suzuki M Kohyama N Ohbayashi M Yamamoto T 《Life sciences》2007,81(15):1183-1192
Rat organic solute carrier protein 1 (rOscp1) was isolated from a rat testis cDNA library. Isolated rOscp1 cDNA consisted of 1089 base pairs that encoded a 363-amino acid protein, and the amino acid sequence was 88% and 93% identical to that of human OSCP1 (hOSCP1) and mouse Oscp1 (mOscp1), respectively. The message for rOscp1 is highly detected in rat testis. When expressed in X. oocytes, rOscp1 mediated the high affinity transport of p-aminohippurate (PAH) with a Km value of 15.7+/-1.9 microM, and rOscp1-mediated organic solutes were exhibited in time- and Na+-independent manners. rOscp1 also transported various structurally heterogenous compounds such as testosterone, dehydroepiandrosterone sulfate (DHEA-S), and taurocholate with some differences in substrate specificity compared with hOSCP1. Immunohistochemical analysis revealed that the rOscp1 protein is localized in the basal membrane side of Sertoli cells as observed in mouse testis [Kobayashi et al., 2007; Kobayashi, Y., Tsuchiya, A., Hayashi, T., Kohyama, N., Ohbayashi, M., Yamamoto, T., 2007. Isolation and characterization of polyspecific mouse organic solute carrier protein 1 (mOscp1). Drug Metabolism and Disposition 35 (7), 1239-1245]. Thus, the present results indicate that a newly isolated cDNA clone, rOscp1, is a polyspecific organic solute carrier protein with some differences in substrate specificity compared with human and mouse OSCP1. 相似文献
98.
99.
Kaczmarczyk A Blom AM Alston-Smith J Sjöquist M Fries E 《Molecular and cellular biochemistry》2005,271(1-2):61-67
Bikunin is a chondroitin sulfate-containing plasma protein synthesized in the liver. In vitro, it has been shown to inhibit proteases and to have additional activities, but its biological function is still unclear. Here we have studied the dynamics of plasma bikunin in rats and mice. A half-life of 7 ± 2 min was obtained from the time course of the decrease of the plasma level of bikunin following hepatectomy. Clearance experiments with intravenously injected radiolabeled bikunin with or without the chondroitin sulfate chain showed that the polysaccharide had little influence on the elimination rate of the protein. The uptake of bikunin by different tissues was studied using bikunin labeled with the residualizing agent 125I-tyramine cellobiose; 60 min after intravenous injection, 49% of the radioactivity was recovered in the kidneys and 6–11% in the liver, bones, skin, intestine and skeletal muscle. The uptake in the liver was analyzed by intravenous injection of radiolabeled bikunin followed by collagenase perfusion and dispersion of the liver cells. These experiments indicated that bikunin is first trapped extracellularly within the liver before being internalized by the cells. (Mol Cell Biochem 271: 61–67, 2005) 相似文献
100.
The mechanism of sexualization of the tubular gonad in seawater bivalves is unknown, and no information regarding the genes involved in this process is yet available, except for the identification of esterase (Est)-like "male-associated polypeptide" in the male gonad of Mytilus galloprovincialis. Our present work reveals distinct protein profiles specific for the testicular or ovarian portion of the ovotestis of Pecten maximus. Two proteins exhibiting testis- or ovary-dependent enrichment in the ovotestis have been identified and partially characterized as Est-like and fibronectin (Fn)-like polypeptides, respectively. Immunofluorescence has demonstrated a close association between the localization of these polypeptides and the gonad tubule network and interstitial stroma of the ovotestis of P. maximus. We also present evidence of Est-like and Fn-like protein enrichment, respectively, in testicular and ovarian tissue in hermaphroditic, sex-reversal, and gonochoric species of seawater bivalves. Together, the results (1) strongly suggest that sex-cell-biased expression of Est-like and Fn-like polypeptides in gonad tissue is a widespread phenomenon among bivalve mollusks, despite the high diversification of their sexual patterns, (2) confirm and expand our previous demonstration of sex-biased protein expression in M. galloprovincialis, and (3) indicate a direct link between germ cell differentiation and sexual specialization of the bivalve somatic gonad. 相似文献