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1.
In vitro cultivation of human renal cell cancer 总被引:3,自引:0,他引:3
R. D. Williams A. Y. Elliott N. Stein E. E. Fraley 《In vitro cellular & developmental biology. Plant》1978,14(9):779-786
Summary Two cell lines derived from primary human renal-cell cancers (RCC) have been established and characterized. Cell line 786-O has been in culture for longer than 1 year and has been subcultured more than 50 times. It has a doubling time of 45 hr and a hypertriploid karyotype and possesses a Y chromosome. Cell line 769-P also has been in culture for longer than 1 year. It has been subcultured 50 times and has a doubling time of 35 hr and a hypodiploid karyotype. Cells from both lines are epithelial, and they produce tumors in the cheek pouches of immunosuppressed hamsters. Neither cell line is contaminated withMycoplasma. Cells of the two lines can be distinguished from HeLa cells both by their karyotypes and by the mobility patterns of their isoenzymes of glucose-6-phosphate dehydrogenase. American Cancer Society Clinical Faculty Fellow, 1977–1980. Supported in part by Grants CA 13095-03 and CA 15551-03 from the National Cancer Institute. 相似文献
2.
The oral absorption of a compound is a critical factor for the future of the compound as a drug. This absorption is mainly controlled by the passage across, the intestinal epithelium. Thus, the prediction of the intestinal absorption by means of anin vitro model may represent a powerful tool for the early selection of molecules during the process of drug development. In the present study, the differentiated human intestinal epithelial cell line HT29-18-C1, was grown on permeable filters in dual chambers. These cells formed tight monolayers that were used to measurein vitro the transepithelial permeability coefficient (Pc) of various molecules. The results were compared within vivo data of oral absorption. A threshold value ofin vitro permeability of 2×10–6 cm/s was found. Molecules having a permeability coefficient higher than this value were absorbed orally more than 80%, while drugs withPc values lower than 2×10–6 cm/s were poorly absorbed. By mathematical simulation, it was found that thisPc value, when extrapolated to the surface area and volume of the small intestine, corresponds to an absorption of 80% for a compound with a transit time through the small intestine of 5 h. This demonstrates the predictive utility of the threshold value of the permeability coefficient derived from thein vitro model of intestinal epithelium.Abbreviations Pc transepithelial permeability coefficient - MTX methotrexate 相似文献
3.
Eileen A. Friedman Paul J. Higgins Martin Lipkin Hiromi Shinya Alvin M. Gelb 《In vitro cellular & developmental biology. Plant》1981,17(7):632-644
Summary Human colonic epithelial cells from three classes of benign tumors have been reproducibly cultured free of fibroblasts for
8 wk using a supplemented Medium 199 (M 199S). The cultured colonic cells were identified as epithelial by the presence of
junctional complexes (tight junctions, gap junctions, and desmosomes), a brush border on the apical surface, keratin fibrils,
and by both a close-packed columnar or cuboidal morphology and the capability to transport water and ions to form hemicysts.
Colony formation was initiated by groups of epithelial cells, not by single cells, and was inhibited by cocultivation with
either lethally irradiated 3T3 cells or human diploid fibroblasts. Enhancement of epithelial colony formation was observed
following culture on nonadherent, “floating” substrates compared with substrates attached directly to the bottom of the culture
dish.
Replication of epithelial cells in M 199S from the class of benign colonic tumors least prone to malignancy, the tubular,
was significantly enhanced by epidermal growth factor (EGF). In contrast, EGF did not stimulate the growth of cells in M 199S
from the other classes of benign tumors, the villotubular and the villous, which exhibit more malignant potential. These data
imply that premalignant colonic epithelial cells lose responsiveness to growth modulation by EGF as they progress toward frank
carcinoma.
This study was supported by NCI Contract N01-CP43366 to M. L. and NCI Grant 1-R26-CA 28822 to E. F. 相似文献
4.
In vitro cultivation of human renal cell cancer 总被引:3,自引:0,他引:3
R. D. Williams A. Y. Elliott N. Stein E. E. Fraley 《In vitro cellular & developmental biology. Plant》1976,12(9):623-627
Summary A technique for initiating and propagating epithelial cell cultures of human renal cell cancer and adjacent nontumor kidney
is described. Seventy-five percent of the tumors and 79% of the adjacent kidney specimens cultured with this method have shown
initial outgrowth and have been subcultured at least once. Two renal cell cancer cultures initiated by this method have now
been in continuous culture more than 6 months, have been subcultured 27 and 19 times, and now appear to be stable lines.
The ability to establish long-term in vitro cultures of human renal cell cancers will facilitate studies concerning the immunoreactivity,
cholesterol metabolism, the isolation of renal-cell-cancer-specific antigens, and in vitro chemotherapy testing and will further
our understanding of the basic biology of human renal cell cancer.
American Cancer Society Clinical Fellow 1975–1976.
Supported in part by Grants CA 13095-03 and CA 15551-03 from the National Cancer Institute. 相似文献
5.
New human breast cell lines were developed from metastatic breast cancer tissues and normal breast tissues. Primary cultures
were initiated from cellular outgrowths of explanted tissues or from mechanically isolated cells in two serum-free media.
Cell cultures derived from both cancer and normal tissues were immortalized with pRSV-T plasmid to generate permanent breast
cell lines that exhibited an epithelial morphology. Cell lines generated in this study were characterized with respect to
morphology, growth rate, karyotype, presence of specific genes, and the expression of epithelial and breast markers. The cell
lines expressed the epithelial cell markers, cytokeratins 8 and 18, and retained the capacity to produce human milk fat globulin.
They also express the BRCA-1, erbB2, and EGF receptor genes and possess the H-ras, K-ras, and p53 genes. Preliminary data
showed that one of the new cancer cell lines was highly sensitive to the cytotoxic action of taxol. It is envisioned that
the new breast cell lines will be useful as targets for identification of therapeutic agents against breast cancer and as
models for carcinogenesis studies.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
6.
Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated
cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in
mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid
bodies’, are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and
mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into
differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation,
tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled
pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro,
the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics.
Furthermore, the differentiation system has been used for genetic analyses by ‘gain of function’ and ‘loss of function’ approaches
in vitro.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
7.
8.
We have screened primary cultures of human prostate for the expression of markers reported to be characteristic of specific cell lineages in vivo, in order to ascertain whether human prostate cells in vitro maintain and reflect their in vivo differentiated phenotypes and to evaluate the homogeneity of the populations of cells that can be derived from this tissue. Using single and dual stain immunofluorescent microscopy to analyse very early organoid and subsequently derived monolayer stage cultures, we have observed that expression of markers characteristic of human prostate epithelial cells in vivo is deregulated within 48h, indicating that dissociation of human prostate tissue and cultivation of prostate epithelial cells in culture can result in promiscuous expression of cell type specific markers of prostate epithelial cells. These observations have important implications for studies of cell lineage and differentiation of prostate cells in vitro. 相似文献
9.
S. J. D. Vitkus S. A. Hanifin D. W. McGee 《In vitro cellular & developmental biology. Animal》1998,34(8):660-664
Summary Intestinal epithelial cells (IEC) have previously been shown to produce several cytokines including interleukin-6 (IL-6).
However, many factors which may regulate IL-6 secretion by human IEC still remain a mystery due in part to the lack of appropriate
model cell lines and the difficulty of culturing human IEC over long periods of time. We have determined that the human colonic
carcinoma cell line Caco-2 is capable of secreting IL-6 when stimulated by the inflammatory cytokines IL-1β or tumor necrosis
factor-α (TNF-α), and stimulation of these cells with IL-1β plus TNF-α induced a synergistic enhancement of IL-6 secretion.
The inflammatory cytokine-induced enhancement in IL-6 secretion was greatest when the cells were cultured in a 10% CO2 atmosphere as compared to cells grown in 5% CO2, suggesting that environmental CO2 levels may affect IEC cytokine secretion. Finally, long-term culture of the Caco-2 cells to induce cellular differentiation
had no effect on the capacity of these cells to produce IL-6, indicating that the regulation of IL-6 secretion was not affected
by differentiation. Taken together, these studies provide important information on the factors which regulate IL-6 secretion
by human IEC as they may contribute to the cytokine network during a mucosal inflammation. The results also suggest that the
Caco-2 cell line is an appropriate model for further studies on the regulation of cytokine secretion by human IEC. 相似文献
10.
The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications 总被引:13,自引:0,他引:13
The gastrointestinal tract remains the most popular and acceptable route of administration for drugs. It offers the great advantage of convenience and many compounds are well absorbed and thereby provide acceptable plasma concentration-time profiles. Currently there is considerable interest from the pharmaceutical industry in development of cell culture systems that would mimic the intestinal mucosa in order to evaluate strategies for investigating and/or enhancing drug absorption. The intestinal epithelial cells of primary interest, from the standpoint of drug absorption and metabolism, are the villus cells, which are fully differentiated cells. Anin vitro cell culture system consisting of a monolayer of viable, polarized and fully differentiated villus cells, similar to that found in the small intestine, would be a valuable tool in the study of drug and nutrient transport and metabolism.The Caco-2 cell line, which exhibits a well-differentiated brush border on the apical surface and tight junctions, and expresses typical small-intestinal microvillus hydrolases and nutrient transporters, has proven to be the most popularin vitro model (a) to rapidly assess the cellular permeability of potential drug candidates, (b) to elucidate pathways of drug transport (e.g., passive versus carrier mediated), (c) to assess formulation strategies designed to enhance membrane permeability, (d) to determine the optimal physicochemical characteristics for passive diffusion of drugs, and (e) to assess potential toxic effects of drug candidates or formulation components on this biological barrier. Since differentiated Caco-2 cells express various cytochrome P450 isoforms and phase II enzymes such as UDP-glucuronosyltransferases, sulfotransferases and glutathione-S-transferases, this model could also allow the study of presystemic drug metabolism. 相似文献
11.
Michael S. Risley 《Molecular reproduction and development》1983,7(4):331-346
Culture conditions that support the in vitro development of many spermatogenic stages from the frog Xenopus laevis are described. Spermatogenic cells were dissociated with collagenase and preelongation stages aseptically isolated by density gradient centrifugation in Metrizamide. The cells were then cultured in modified forms of defined nutrient oocyte medium (DNOM). The development of spermatogenic cells was affected significantly by changes in fetal calf serum concentration, cell density, energy sources, and NaCl concentration. Optimum in vitro spermatid development was obtained when spermatogenic cells were cultured at relatively high densities (3–7 × l07 cells/25 cm2) in DNOM modified to contain 10% heat-inactivated, dialyzed fetal calf serum, 2 mM 1-glutamine, 0.1 % glucose, 15 mM HEPES buffer (pH 7.4), and 38.3–48.3 mM NaCl. These culture conditions also supported the differentiation of preelongation spermatids and spermatocytes isolated by density-gradient centrifugation in Metrizamide and subsequent unit gravity sedimentation in gradients of bovine serum albumin. Approximately 95 % of such isolated spermatids and spermatocytes continued differentiating in vitro for 14 days at in vivo rates. Phase-contrast and electron microscopy of the cultured cells demonstrated that in vitro differentiation was morphologically normal between the leptotene and elongate spermatid stages. Autoradiographic studies of preleptotene development demonstrated that spermatogonia proliferated and preleptotene spermatocytes developed to zygotene in 12-day cultures. The results suggest that many spermatogenic stages in Xenopus can develop independent of Sertoli cells, and demonstrate that spermatogenic cell cultures can now be used for in vitro studies of spermatogenesis. 相似文献
12.
K. E. Driscoll J. M. Carter P. T. Iype H. L. Kumari L. L. Crosby M. J. Aardema R. J. Isfort D. Cody M. H. Chestnut J. L. Burns R. A. LeBoeuf 《In vitro cellular & developmental biology. Animal》1995,31(7):516-527
Summary We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37° C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham’s F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number. However, at later passages the 6T cells became polyploid, while the 6TN genotype remained stable. The RLE-6T and 6TN cells were not tumorigenic in nude mice. The cell isolation methods reported and the novel cell lines produced represent potentially useful tools to study the role of pulmonary epithelial cells in neoplastic and nonneoplastic lung disease. 相似文献
13.
Vaughan MB Ramirez RD Wright WE Minna JD Shay JW 《Differentiation; research in biological diversity》2006,74(4):141-148
A therapeutic approach being investigated for a variety of pathologies is tissue regeneration using a patient's own cells. Such studies have been hampered due to the difficulty in growing epithelial cells for prolonged periods in culture. Replicative senescence due to short telomeres and p16 induced by culture stress work together to inhibit cell growth. Forced expression of telomerase (hTERT) can prevent replicative senescence, and expression of the cell cycle protein cdk4 can sequester p16, thereby immortalizing epithelial cells in culture. In the present study, we used this method to immortalize human bronchial epithelial cells (HBECs) to determine whether immortalized HBECs retain the ability to differentiate normally. HBECs were plated atop contracted collagen gels containing lung fibroblasts. This three-dimensional (3D) tissue model was cultured initially submerged, then raised to the air/liquid interface for up to 28 days. Normal differentiation was assessed by the presence of ciliated cells, goblet (mucin-producing) cells, and basal epithelial cells. Scanning electron microscopic observations revealed both ciliated and non-ciliated cells in these 3D tissues. Histological examination revealed the presence of mucin-producing cells, and immunohistochemistry using antibodies against p63 and keratin 14 showed the presence of basal cells. These results demonstrate that immortalized HBECs retain the capacity to differentiate into each of three cell types: basal, mucin-producing, and columnar ciliated epithelial cells. Such cells will be useful cellular reagents for research in aging, cancer progression, as well as normal bronchial epithelial differentiation and will help progress the use of engineered cells to enhance tissue regeneration. 相似文献
14.
Melatonin inhibits the proliferation of retinal pigment epithelial (RPE) cells in vitro 总被引:4,自引:0,他引:4
Hing-Sing Yu Veronica Hernandez Mark Haywood Corinne G. Wong 《In vitro cellular & developmental biology. Animal》1993,29(5):415-418
Summary The possible antiproliferative effect of melatonin on retinal pigment epithelial (RPE) cells in vitro was investigated. Bovine RPE cells cultured in Ham’s F12 medium supplemented with 10% fetal bovine serum had a nuclear density of 73.6 ± 6.1 nuclei/mm2 at 72 h after seeding. The nuclear density at this time-point was doubled if either 50 or 100 ng/ml human epidermal growth factors (hEGF) was added to the culture medium. When these hEGF-stimulated cells were treated with melatonin from 10 to 500 pg/ml, the proliferation was suppressed with a dose-response relationship. At 250 and 500 pg/ml melatonin, the nuclear densities of the melatonin-treated cells were similar to those of the control cells. Using mitotically active SV-40 transformed human fetal RPE cells cultured in a serum-free medium, melatonin was also shown to be antiproliferative. In the presence of 500 pg/ml melatonin, the proliferation of these cells was inhibited to 77% as compared to the control. These results were further supported by the reduced [H3]thymidine uptake in the melatonin-treated cells. We propose that melatonin, at physiologic concentrations, has an antiproliferative effect, and that cultured RPE cells stimulated to proliferate by either hEGF treatment or SV-40 transfection are responsive to melatonin. Melatonin may either inhibit mitosis in actively dividing cells or modulate hEGF action. 相似文献
15.
In vitro differentiation of human embryonic neural stem cells 总被引:1,自引:1,他引:1
16.
Schwerdt G Freudinger R Mildenberger S Silbernagl S Gekle M 《Cell biology and toxicology》1999,15(6):405-415
To test the apoptotic potential of the nephrotoxic mycotoxin ochratoxin A (OTA), we exposed human proximal tubule-derived
cells (IHKE cells) for various times to OTA concentrations close to those occurring during dietary exposure (from 2 to 100
nmol/L) and investigated caspase 3 activation, chromatin condensation, and DNA fragmentation. OTA induced a time- and concentration-dependent
activation of caspase 3: concentrations as low as 5 nmol/L OTA caused a slight but significant increase in caspase 3 activity
after 7 days of OTA exposure. Exposure to 10 nmol/L OTA for 72 or 24 h led to a significantly increased activity of caspase
3 in human proximal tubule-derived cells. Radical scavengers such as N-acetylcysteine had no effect on OTA-induced caspase 3 activation. Chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethylester) (BAPTA-AM) also showed no effect. Exposure to 30 nmol/L or more OTA led to
DNA fragmentation and chromatin condensation in IHKE cells. Cultured renal epithelial MDCK-C7 and MDCK-C11 or OK cells also
showed increased caspase 3 activity after OTA exposure. We conclude that exposure to low OTA concentrations can lead to direct
or indirect caspase 3 activation and subsequently to apoptosis in cultured human proximal tubule cells and in other renal
epithelial cell lines of different origins.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
17.
人胚胎干细胞(human embryonic stem cells,hESCs)由囊胚期胚胎内细胞团分离培养获得,具有保持未分化状态的无限增殖能力。hESCs具有多向分化潜能,在体内和体外均可分化形成所有三个胚层(外胚层、中胚层、内胚层)的衍生物。hESCs一般在鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)饲养层上培养和扩增。为了优化培养条件,目前人们已发展了多种人类细胞饲养层和无饲养层、非条件培养基体系。hESCs可以在体外定向诱导分化为多种细胞类型,为揭示人胚早期发育机制和发展多种疾病的细胞移植治疗奠定了基础。hESCs可以在体外进行遗传修饰,将有助于揭示特定基因在发育过程中的调控和功能。对hESCs的深入研究将极大地推动医学和生命科学的进展,并将最终应用于临床,造福人类。 相似文献
18.
Akiramasuda Kenzo Ohtsuka Mutsushi Matsuyama 《In vitro cellular & developmental biology. Plant》1990,26(7):713-721
Summary Seven clonal epithelial cell lines from a thymoma of an (ACI/NMs×BUF/Mna)F1 rat and seven clonal epithelial cell lines from
an ACI/NMs rat thymus were established in a medium containing 1 μM dexamethasome (DM) and were characterized cytologically. Long-term treatment of DM stabilized the epithelial nature of these
epithelial cells irreversibly. The established cell lines showed a polygonal shape, were positively stained with antikeratin
antiserum and had tonofilaments and desmosomes. Species of their keratin paptides were the same as those of normal thymic
epithelial cells in primary cultures. The cell lines were positively stained with Th-4 monoclonal antibody which preferentially
stains the medullary epithelial cells of the thymus, but not with Th-3 which preferentially stains the subcapsular and cortical
epithelial cells of the thymus. The cells from the rat thymoma were much large than those from the normal thymus, as reflected
in their primary cultures. No transformed phenotypes, such as high growth rate, high saturation density anchorage independency,
low serum dependency and so on, were found on the cell lines from the thymoma as in the cell lines from the normal thymus
by in vitro assays. DNA synthesis of the thymic lymphocytes was stimulated by culturing with a line of rat thymoma with no
lectins. Thymic lymphocytes strongly bound on the cell lines from the thymoma and changed the shape of the cells. These cell
lines may be useful to investigate the mechanism of thymomegenesis and the interactions between epithelial cells and thymocytes
in the rat thymoma. 相似文献
19.
20.
Shashi Shrivastav Yousuf Sharief John Day Charles F. Reich Robert A. Bonar 《In vitro cellular & developmental biology. Plant》1981,17(12):1117-1124
Summary A new cell line, SS78, was established from a primary renal cell carcinoma of a Caucasian male. The tissue was dispersed with
collagenase, and viable cells were separated by flotation on a Ficoll-Hypaque gradient. In culture, the SS78 cells retained
a distinct epithelial morphology, and no fibroblastlike cells were seen. The cultured cells were aneuploid with a modal chromosome
number of 80 and had several marker chromosomes. Inoculation of the cultured cells into athymic nude mice caused tumors at
the sites of inoculation.
This research was supported in part by Grants CA 15972 and CA 14930 from the National Cancer Institute through the National
Bladder Cancer Project and by the Medical Research Service of the Veterans Administration. 相似文献