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991.
符云峰 《中国生物化学与分子生物学报》1988,4(1):84-87
本研究确定了在0℃条件下,(Na~++K~+)-ATP酶纯化制备物与5mmol/L Na~+或Mg~(2+)在5mmol/L咪唑(pH7.4)环境中预保温30分钟,然后进行磷酸化,可以获得最高磷酸化水平,Na~+或Mg~(2+)的K_(0.5)值分别为0.29mmol/L或0.35mmol/L;以ADP代替Na~+和Mg~(2+)与酶预保温,对E_2向E_1转变无任何影响,而与Na~+、Mg~(2+)一起存在时则能加强Na~+及Mg~2的预保温效果。 相似文献
992.
A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro 总被引:4,自引:0,他引:4
Timothy A. McCaffrey Lily A. Agarwal Babette B. Weksler 《In vitro cellular & developmental biology. Plant》1988,24(3):247-252
Summary Many research efforts require the accurate determination of cell density in vitro. However, physical cell counting is inaccurate,
time-intensive and requires removal of the cells from their growth environment, thereby introducing a host of potential artifacts.
The current studies document a very simple method of determining cell density in microtiter wells via DNA-enhanced fluorescence.
Fixed cells are stained with the A-T intercalating DNA stains DAPI or Hoechst 33342 and then fluorescence is quantified in
a plate fluorometer. Fluorescence is shown to be linearly related to cell density as determined by two physical counting methods.
The validity of the method is established in determining serum-stimulated growth of smooth muscle cells and in mitogen-induced
growth of endothelial cells. The fixed cells can be stored for prolonged periods, thus allowing time-course proliferation
assays without interassay variations. The fixed cells are also suitable for determinations of antigens of interest by ELISA.
This method is potentially valuable in many in vitro systems where the quantification of cell density and proliferation is
necessary.
This work supported in part by NIH Cardiovascular Training Grant HL07423 and a grant from the American Federation for Aging
Research to T. M. and HL35724 to B. W.
EDITOR’S STATEMENT The technique described in this paper represents an approach to quantifying cell density in adherent monolayers
of cultured cells in microtiter wells that is rapid and simple and does not require radioisotopes or removal of cells. 相似文献
993.
C. L. Bell H. S. Tenenhouse C. R. Scriver 《In vitro cellular & developmental biology. Plant》1988,24(7):683-695
Summary Primary cultures of murine renal epithelial cells were established from a preparation of proximal tubule fragments. Confluent
cultures exhibited multiple dome formation, indicating the presence of tight junctions and an intact transcellular transport
process. Ultrastructural analysis revealed a monolayer of polarized cells, with a sparse but clearly defined microvillar surface
facing the growth medium and a basolateral surface attached to the substratum. Cultures grown on collagen gels did not show
domes. The epithelial monolayer exhibited several differentiated functions of the proximal tubule: a) parathyroid hormone
(PTH)-stimulated cAMP synthesis; b) production of 24,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3; c) high alkaline phosphatase activity; and d) Na+-dependent transport of phosphate (Pi) and α-methylglucoside (α-MG). The sugar uptake was selectively inhibited by phlorizin,
a competitive inhibitor of glucose uptake at the luminal membrane. Kinetic analysis revealed independent transport systems
for Pi and α-MG, with Km values corresponding to the high affinity systems identified in brush border membrane vesicles derived
from the proximal tubule. Pi uptake by the epithelial monolayers was regulated by the concentration of Pi in the growth medium.
Phorbol esters and PTH did not exert an effect on Pi and α-MG transport in mouse primary cultures. The present study demonstrates
that primary cultures provide a useful in vitro preparation to investigate renal proximal tubular function.
Cindy Bell was the recipient of an MRC Studentship Award. This work was supported by the MRC (Group in Medical Genetics).
This is publication number 88011 of the McGill University-Montreal Children's Hospital Research Institute. 相似文献
994.
Terry L. Riss Kenneth P. Karey B. Daniel Burleigh David Farker David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(11):1099-1106
Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant
insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on
poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's
modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin,
100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free
growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold
more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The
concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED50), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like
growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin,
ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED50, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth
factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures.
This work was supported by a contract from IMCERA Bioproducts, Inc. 相似文献
995.
Allison A. Welder Tina Machu Steven W. Leslie Richard E. Wilcox June Bradlaw Daniel Acosta 《In vitro cellular & developmental biology. Plant》1988,24(8):771-777
Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models
for investigating cellular transsarcolemmal45Ca++ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding,
beta-adrenergic receptor mediated cellular calcium (45Ca++) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained
from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude
membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [125I]iodopindolol ([125I]IPIN). The suspensions had a significantly lower B
max
(42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K
D
of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after
3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10−7
M isoproterenol resulted in a significant increase in45Ca++ exchange as early as 15 s. In contrast,45Ca++ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess
saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated45Ca++ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable
than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart.
This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology
Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration. 相似文献
996.
Cyprian Weaver Robert L. Sorenson Brian Kobienia 《In vitro cellular & developmental biology. Plant》1988,24(2):108-116
Summary The purpose of this study was to develop a nonenzymatic method of isolating adult islets using atrophied pancreata from copper-deficient
rats and to analyze their morphologic characteristics and behavior in culture. This unusual model of isolation was studied
because islets remain intact in the course of dietary copper deficiency while the acinar glandular component of the pancreas
undergoes selective atrophy and lipomatosis. Small fragments containing islets were readily microdissected from atrophied
glands and placed in culture. Within 24 h the fragments congealed into small irregular- to spherical-shaped masses within
which the darker profile of islets could be distinguished. Within a period of 3 to 5 d, islet tissue began to bud from the
lipocytic mass until by Day 7 spherical aggregates of intact islet tissue separated from the residual fragments. Subsequent
to further in vitro treatment, these islets could be maintained as free viable spherical masses if periodically agitated,
as attached stationary islets which developed monolayer growth if left undisturbed and as aggregated masses of islet tissue
forming megaislets if combined in small groups. Grouped islets treated with actinomycin D and cycloheximide did not exhibit
aggregation when incubated with these inhibitors. This suggests that megaislet formation was an active process requiring protein-RNA
synthesis rather than passive clumping or aggregation that can accompany metabolically altered or dying islets undergoing
cellular shedding and adhesion. Immunohistochemical localization demonstrated that insulin, glucagon, somatostatin, and pancreatic
polypeptide-immunoreactive cell types were present within the islets derived from this technique. The cellular topography
of these islets was not unlike that described by others for islets cultured from enzymatic isolation. This culture model may
serve as a resource for mature, viable islets isolated without mechanical or enzymatic disaggregation which can have attenuating
effects on islet function.
This work was supported by a research grant from the Diabetes Research and Education Foundation. 相似文献
997.
Kenneth P. Karey Terry L. Riss B. Daniel Burleigh David Parker David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(11):1107-1113
Summary The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized,
resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (125I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation
at 15°C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding
sites with K
d
=59.6 pM and an estimated 1.57×105 receptors/cell. Half-maximal displacement of bound125I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective
competitors, providing halfmaximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 μg/ml, respectively. Epidermal
growth factor, transforming growth factor type α, and acidic and basic fibroblast growth factors did not compete for125I-IGF-I binding at 1 μg/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more
tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that125I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol.
This work was supported by a contract from IMCERA Bioproducts, Inc. 相似文献
998.
John F. Foley Byron Th. Aftonomos 《In vitro cellular & developmental biology. Plant》1988,24(9):900-904
Summary Colonies of HeLa cells cultured in media supplemented with human or bovine serum or both can be morphologically described
as three types: diffuse, intermediate, and compact, with their modal distribution depending on the serum or sera added to
the growth medium. We have observed that for a particular medium or serum system, the percentage of compact colonies remains
fairly constant under normal culture conditions, 0.2%, whereas the diffuse and intermediate colonies vary over a much wider
range. The presence of certain substances as trypsin, heparin and Darvan in the medium favor the increase of compact colonies
at the expense of other types. Furthermore, we have discovered that colonial morphology is influenced by cocultivation of
the HeLa cells with human fibroblastlike cells, the compact colonies increasing as the density of the fibroblast element introduced
into the mixed cultures is increased. Subsequent investigation revealed that conditioned medium from confluent fibroblast
and HeLa cell cultures contained a factor(s), that significantly increased the percentage of compact colonies. The factor
is nondialyzable, heat-stable and can be neutralized by serum. Recorded in this presentation are preliminary observations
on the kinetics of colony formation and the interaction among the three HeLa cell colony types, the diffuse, the intermediate,
and the compact. The factor's effect on HeLa cell colonial morphology is time dependent and rapidly reversed if the factor(s)
is removed and fresh medium added. 相似文献
999.
Marsha L. Green Ronald G. Green William Santoro 《Applied psychophysiology and biofeedback》1988,13(3):187-199
This study investigated the effect of daily relaxation on concentrations of serum immunoglobulins A, G, and M and secretion rates of salivary immunoglobulin A (S-IgA). Twenty-four volunteers were randomly assigned to practice a relaxation technique daily for 3 weeks and 16 to a waiting list control condition. Blood and saliva samples were collected before and after a supervised 20-min relaxation session at the beginning and end of the 3-week practice period. S-IgA secretion rate increased significantly (p<.001) after 20 min of relaxation. A longer-term practice effect also occurred in that the increase in secretion rate in before to after relaxation samples was higher (p=.014) in subjects who had practiced relaxation once a day for 3 weeks than in waiting list control subjects practicing for the first time. Serum IgA (p<.001), IgG (p<.001), and igM (p<.05) increased significantly over the 3-week practice period. Relaxation may be a self-regulating strategy affecting both humoral and cellular divisions of the immune system.Parts of this paper were presented at the annual meeting of the Biofeedback Society of America, March 1987. Materials for the IgA assays were provided by Cooper Biomedical, Malvern, Pennsylvania. 相似文献
1000.
Usually the toxicity of superoxide is attributed lo its ability to reduce metal ions and subsequently reoxidation of the metal by hydrogen peroxide yields deleterious oxidizing species. As many other nontoxic biological reductants reduce metal compounds, we suggest that part of the mechanism of superoxide toxicity results from its ability to oxidize metal ions bound to biological targets, which subsequently degrade the target via an intramolecular electron Transfer reaction. 相似文献