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31.
Marshall H. Montrose Geraldine Bebernitz George A. Kimmich 《The Journal of membrane biology》1985,88(1):55-66
Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H+ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K+ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H+ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na+/H+ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na+ gradient. The kinetics of the Na+/H+ exchange reaction at pH 6.0 [K
t
for Na+=57mm,V
max=42 mmol H+/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K
t
for Na+=15mm,V
max=1.7 mmol H+/liter 3OMG space/min). Experiments involving imposed K+ gradients suggest that these cells have negligible K+/H+ exchange capability. They exhibit limited but measurable H+ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate. 相似文献
32.
无论是自发的、病毒引起的或致癌物诱发的恶性转化的哺乳类细胞的体外培养,其形态多发生改变,总是变得近似圆形,边缘突起短而少,细胞致密和折光性强,同时失去生长接触抑制,降低细胞与细胞之间和细胞与生长底物之间的粘着性等特性。近年报道了关于短链脂肪酸如丁酸(或丁酸钠)对细胞能产生明显的影响,能抑制培养细胞的分裂,可诱发一些上皮性细胞产生形态的改变,可使转化的细胞 相似文献
33.
抗胃癌细胞系单克隆抗体PD4的初步研究 总被引:1,自引:0,他引:1
以胃癌细胞系MGC803免疫Balb/c小鼠,取其脾细胞与小鼠骨髓瘤细胞NS-1融合。经选择培养、筛选及克隆化,获得恒定地分泌抗胃癌细胞系单克隆抗体(MoAb)的杂交瘤细胞系PD4。MoAb PD4与3/4胃癌细胞系有强结合反应,与4/4肺癌细胞系有弱结合反应,但与淋巴细胞、ABO红细胞、骨髓细胞、二倍体成纤维细胞及经检测的其他肿瘤细胞均无反应。PD4抗原主要表达于靶细胞的膜上,不耐热,为分子量40kD的蛋白性抗原。该抗原与HLA抗原系统,血型抗原系统无关,亦不同于其他作者所报告的其他胃癌相关抗原。 相似文献
34.
Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains 总被引:8,自引:0,他引:8
Kazunori Furukawa Tomiko Shimada Patricia England Yohichi Mochizuki Gary M. Williams 《In vitro cellular & developmental biology. Plant》1987,23(5):339-348
Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible
establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats
by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells
different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded
numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority
of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation
at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in
the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity,
whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics
of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial
cells propagable in culture were derived from a cell type other than the hepatocyte. 相似文献
35.
Philip S. Rudland Christine M. Hughes Sharon A. Ferns Michael J. Warburton 《In vitro cellular & developmental biology. Plant》1989,25(1):23-36
Summary Parenchymal organoidal structures that were obtained from collagenase digestion of reduction mammoplasty specimens of apparently
normal human breasts have been grown in short-term primary cultures, either on plastic or on floating gels of polymerized
rat-tail collagen. Three morphologically distinct major cell types are readily observed in both systems: cuboidal cells, which
occupy apical positions on collagen gels; larger, epithelioid, or basal cells on gels; and elongated cells which penetrate
into the gel. In addition, a fourth cell type, that of a large, flat cell, is observed less readily by phase contrast microscopy
on the surface of cultures grown on plastic. Immunofluorescent and immunocytochemical staining of cultures on plastic or histologic
sections of cultures on gels have been undertaken with antisera and other histochemical reagents that stain the different
parenchymal cell types in vivo. Thus antisera to epithelial membrane antigen(s), monoclonal antibodies (MABs) to the defatted
mammary milk fat globule membrane, peanut lectin, and keratin MAB LE61, which preferentially stain the epithelial cells of
ducts in vivo, also stain the cuboidal/apical cells in vitro. The large, flat cells are stained intensely by the first three
reagents but not by the last one. Antisera to collagen IV, laminin, fibronectin, actin, keratin MAB LP34, MABs to the common
acute lymphoblastic leukemia antigen, and MAB LICR-LON-23.10, which showed enhanced staining for the ductal myoepithelial
cells in vivo, also stain the epithelioid/elongated cells in vitro. However, the effect of the last four reagents is reduced
considerably in most elongated cells, and MAB LP34 stains the large, flat cells intensely. Heterogeneous cells of intermediate
morphologies and staining patterns between the cuboidal/flat cells and large epithelioid cells have also been identified.
The results suggest that the cuboidal cells and large, flat cells are related to mammary epithelial cells, whereas the large
epithelioid/elongated cells have some characteristics of myoepithelial cells, and that intermediate forms may exist in culture
between the two parenchymal cell types.
This work was supported in part by the Ludwig Institute for Cancer Research and the Cancer and Polio Research Fund. Dr. M.
J. Warburton is supported by the Cancer Research Campaign. 相似文献
36.
Jeffery R. Cook Barbara E. Crute Laura M. Patrone Joseph Gabriels Maureen E. Lane Robert G. van Buskirk 《In vitro cellular & developmental biology. Plant》1989,25(10):914-922
Summary We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics
of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous
membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater
than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial
basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate
gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs.
microporous membranes. When proteins were pulse-labeled with [35S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic
substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous
membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous
membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell
HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment.
A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where
the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of
the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that
both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of
the basement membrane. 相似文献
37.
Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel
Timothy Turner Howard A. Bern Peter Young Gerald R. Cunha 《In vitro cellular & developmental biology. Plant》1990,26(7):722-730
Summary Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved
in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1∶1 (vol/vol), supplemented with
bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5α-dihydrotestosterone, cortisol,
putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells
occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture.
Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics.
Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated
and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male
mice. These tissue recombinants showed distinctive prostatic histologic characteristic (alveoli and ducts lined with cuboidal
or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258,
epithelial cells of mouse origin were distinguishable from stromal cells of rat origin.
Aided by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD, and by M. A. R. C. fellowship
GM08730 to T. T. 相似文献
38.
Hillary A. Hahm Margot M. Ip Kathleen Darcy Jennifer D. Black Wendy K. Shea Suzanne Forczek Masami Yoshimura Takami Oka 《In vitro cellular & developmental biology. Plant》1990,26(8):803-814
Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within
a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and
secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within
an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting
of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor,
bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic
level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins.
Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was
observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of
casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot
analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein
mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels.
Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike
colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the
RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs
from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid
when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve
as an excellent model in which the regulation of mammary development and gene expression can be investigated.
This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health,
Bethesda, MD. 相似文献
39.
Teresa L. Johnson Mary Pat Moyer 《In vitro cellular & developmental biology. Plant》1990,26(11):1095-1100
Summary Normal human colon mucosa cells and cells obtained from histologically normal tissues near that cancer were fused with human
colon cancer cells. Resultant hybrid populations of normal and malignant cell fusions behaved as nonmalignant cells in culture,
were unable to grow in soft agar, did not express tumor-associated antigens, and were nontumorigenic in nude mice. Autofusion
of the cancer cell population led to a phenotype intermediate between normal and malignant cells. That is, the cultures had
a much lower plating efficiency in soft agar, and the tumors had a longer latency and slower growth rate in nude mice. This
is the first cell culture system to demonstrate that normal epithelial cells can suppress malignancy of their autologous cancer
cells, and is a prelude to more extensive studies of genetic events involved in malignant conversion of human colonic epithelium.
This study was supported by The University of Texas Health Science Center at San Antonio Center for Human Cell Biotechnology
and a graduate student stipend (T. J.) from the Department of Cellular and Structural Biology. 相似文献
40.
小鼠胚胎与子宫单层上皮细胞共培养的研究 总被引:11,自引:0,他引:11
本文报道建立了小鼠胚胎与小鼠子宫单层上皮细胞体外共培养系统。结果揭示;小鼠胚胎与 子宫单层上皮细胞共培养可以促进胚胎的发育、粘附和扩展;如果培养液中加入 3、67 × 10-6mol/L 17β-雌二醇,可以显著提高胚胎在共培养系统中的发育率、粘附率和扩展率。以上结果表明:小鼠 胚胎与小鼠子宫单层上皮细胞共培养系统是研究胚泡着床机理较理想的研究手段。 相似文献