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Growth and differentiation of periodontal ligament-derived cells in serum-free defined culture 总被引:4,自引:0,他引:4
Tetsuji Okamoto Nobuhiro Yatsuzuka Yoshiharu Tanaka Mikio Kan Takenori Yamanaka Akihiko Sakamoto Takashi Takata Yasumasa Akagawa Gordon H. Sato J. Denry Sato Kazuaki Takada 《In vitro cellular & developmental biology. Animal》1997,33(4):302-309
4.
Proliferation of epithelial cells derived from rat dorsolateral prostate in serum-free primary cell culture and their response to androgen 总被引:4,自引:0,他引:4
Nozomu Nishi Yuhsi Matuo Takahisa Nakamoto Fumio Wada 《In vitro cellular & developmental biology. Plant》1988,24(8):778-786
Summary Primary cultured epithelial cells derived from the rat dorsolateral prostate proliferated in serum-free nutrient medium WAJC
404 supplemented with mitogens: insulin (650 nM), cholera toxin (120 pM), epidermal growth factor (EGF) (2.5 nM), dexamethasone (300 nM), and bovine pituitary extract (25 μg/ml). The culture consisted of two types of epithelial cell colonies: one originated
from single cells or small cell aggregates and the other was epithelial cell outgrowth from small tissue fragments attached
to a substratum. There were differences in requirements for the mitogens between the two types of colonies. Requirements for
cholera toxin, bovine pituitary extract, and dexamethasone were higher in the former type of colonies, and those for EGF were
higher in the latter type of colonies. Proliferation of the epithelial cells in either type, of colony was suppressed more
than 50% by 1 nM dihydrotestosterone. This suppressive effect was not mediated by stromal component in the tissue fragments, and was counteracted
by cyproterone acetate, indicating specific and direct action of the androgen on prostate epithelial cells. The results suggest
that there is discrete participation of polypeptide growth factors and androgen in proliferation and differentiation, respectively,
of prostate epithelial cells in vivo. 相似文献
5.
Kristina Sundqvist Yun Liu Kristina Arvidson Kari Ormstad Lennart Nilsson Rune Toftgård Roland C. Grafström 《In vitro cellular & developmental biology. Animal》1991,27(7):562-568
Summary Human buccal epithelial cells have been reared from explants maintained in supplemented MCDB 153 medium. Primary epithelial
outgrowths show typical structural features and uniformly express keratins; subunit analyses demonstrate expression of keratins
5, 6, 14, 16/17, and 19. The cells exhibit up to 6% colony forming efficiency and divide at about 0.8 population doublings
per day on fibronectin/collagen-coated dishes at clonal density. Studies of markers of proliferation and differentiation in
buccal epithelial cells indicate that epidermal growth factor, cholera toxin, retinoic acid, and pituitary extract each exhibit
a distinctive ability to enhance growth and variably affect cell migration and cell surface area. Transforming growth factorβ-1 inhibits growth and increases surface area without affecting migration, involucrin expression, and cross-linked envelope
formation. Moreover, exposure of cells to fetal bovine serum, the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate or an elevated Ca2+ concentration (from 0.1 to 1 mM) inhibits growth and induces squamous differentiation as indicated by inhibition of migration, increases in surface area,
involucrin expression, or formation of cross-linked envelopes. The results show that epithelial cells can be reproducibly
derived from explant cultures of human buccal mucosa specimens and the cells transferred under serum-free conditions. Buccal
epithelial cells in culture undergo a pattern of growth and differentiation that mimics parakeratinization in vivo and variably
respond to several agents shown to modulate growth of cells that originate from other types of epithelia.
This work was supported in part by grants from the Swedish National Board of Laboratory Animals, the Swedish Medical Research
Council, the Swedish Natural Science Research Council, the Swedish Fund for Scientific Research Without Animal Experiments,
the Swedish Cancer Society, the Swedish Tobacco Company, and Lions Club International, Djurg?rden, Stockholm, Sweden. 相似文献
6.
Carsten Ropke Bo van Deurs Ole W. Petersen 《In vitro cellular & developmental biology. Plant》1990,26(7):671-681
Summary Thymic epithelial cells were grown in defined medium without unknown serum factors and without concurrent growth of other
cell types. Thymic tissue was obtained from 1- to 4-wk-old mice, disaggregated, and incubated in a mixture of collagenase-dispase-DNAse.
The resulting organoids were seeded on collagen-coated flasks. The culture medium consisted of DME-F12 with low or high concentration
of Ca2+ supplemented with insulin, epidermal growth factor, cholera toxin, hydrocortisone, and transferrin. Under these conditions,
explants attached to the substrate within 2 d, and expanding epithelioid monolayer islets emerged from the organoids during
the following days. [3H]Thymidine incorporation revealed a growth fraction of the cells close to 5%. By omitting either epidermal growth factor,
insulin, or cholera toxin from the medium, pronounced reduction in sizes of islets and in [3H]thymidine incorporation was found. Throughout the culture period, the islets appeared as continuous sheets of polygonal
cells. The epithelial nature of the expanding cell islets was confirmed by demonstration of cytokeratins and of desmosomes.
Ultrastructural evaluation of early cultures revealed clusters of epithelial cells intermixed with lymphocytes, and late cultures
showed a typical pattern of stratified keratinizing epithelium. However, squamous metaplasia was avoided by the use of low
Ca2+ medium, which also proved essential for cell transfer. MHC class II antigen was detected on the majority of the cultured
cells, and culture supernatants contained co-mitogenic activity for thymocytes and GM-colony stimulating activity.
This work supported by The Danish Research Council, grant 12-8148. 相似文献
7.
Masami Ogasawara David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(9):911-920
Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed
of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate.
Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained
MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA,
insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED50 values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors
in Tf-BSA but required 10-fold higher concentrations for ED50. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml
concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic
for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA.
This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells
without interfering activities known to be present in serum.
This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American
Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc. 相似文献
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Tomokiyo A Maeda H Fujii S Wada N Shima K Akamine A 《Differentiation; research in biological diversity》2008,76(4):337-347
Abstract The periodontal ligament (PDL) that anchors the tooth root to the alveolar bone influences the lifespan of the tooth, and PDL lost through periodontitis is difficult to regenerate. The development of new PDL-regenerative therapies requires the isolation of PDL stem cells. However, their characteristics are unclear due to the absence of somatic PDL stem cell lines and because PDL is composed of heterogeneous cell populations. Recently, we succeeded in immortalizing human PDL fibroblasts that retained the properties of the primary cells. Therefore, we aimed to establish a human PDL-committed stem cell line and investigate the effects of basic fibroblast growth factor (bFGF) on the osteoblastic differentiation of the cells. Here, we report the development of cell line 1–17, a multipotent clonal human PDL cell line that expresses the embryonic stem cell-related pluripotency genes Oct3/4 and Nanog , as well as the PDL-related molecules periostin and scleraxis. Continuous treatment of cell line 1–17 with bFGF in osteoblastic induction medium inhibited its calcification, with down-regulated expression of FGF-Receptor 1 ( FGF-R1 ), whereas later addition of bFGF potentiated its calcification. Furthermore, bFGF induced calcification of cell line 1–17 when it was co-cultured with osteoblastic cells. These results suggest that cell line 1–17 is a PDL-committed stem cell line and that bFGF exerts dualistic (i.e., promoting and inhibitory) effects on the osteoblastic differentiation of cell line 1–17 based on its differentiation stage. 相似文献
10.
Growth and characterization of epithelial cells from normal human uterine ectocervix and endocervix 总被引:3,自引:0,他引:3
M. E. Turyk T. R. Golub N. B. Wood J. L. Hawkins G. D. Wilbanks 《In vitro cellular & developmental biology. Plant》1989,25(6):544-556
Summary The human uterine cervix consists of an endocervical canal lined with a single layer of columnar mucus-secreting cells and
an outer ectocervix covered by a stratified squamous epithelium. We report here the culture of human endocervical epithelial
cells (HEnE) and human ectocervical epithelial cells (HEcE) in serum-free medium (KGM). Both HEnE and HEcE cultures were composed
of keratinocytelike cells which formed desmosomal contacts and stratified in the presence of high concentrations of calcium
ions. Cells with a pleomorphic epithelial morphology were observed in HEnE cultures, but not in HEcE cultures. Keratin 18,
which is characteristic of endocervix in vivo, was detected by indirect immunofluorescent staining in all HEnE cells but was
never detected in cultured HEcE. HEcE expressed keratin 13 which is characteristic of ectocervix in vivo. Although keratin
13 was never detected in primary HEnE cultures, it was expressed in passaged HEnE cultures grown in medium with high concentrations
of calcium and in late passage HEnE cultures. HEnE underwent an average of 15.1 population doublings during serial culture.
Mean colony-forming efficiency during Passages 2 to 3 was 14.7% and mean population doubling time was 17.8 h. HEcE cultures
underwent significantly more population doublings (29.0) than HEnE cultures, whereas colony-forming efficiencies and doubling
times were similar to those determined for HEnE. HEnE and HEcE cells may be useful in developing in vitro models of cervical
squamous metaplasia and for exploring the interactions between target cell differentiation, carcinogens, and papillomaviruses
in the development of cervical neoplasia.
This study was supported in part by the Rush University Committee on Research and by the Lester B. and Francis Knight and
the S. Charles and Marsha Papageorge research funds. 相似文献
11.
A cloned rat thymic epithelial cell line established from serum-free selective culture 总被引:6,自引:0,他引:6
Arthur Piltch Paul Naylor Jun Hayashi 《In vitro cellular & developmental biology. Plant》1988,24(4):289-293
Summary A serum-free system has been developed for selective growth and long-term culture of rat thymic epithelial cells. The growth
media is a modification of McKeehan's WAJC 404, plus insulin, cholera toxin, dexamethasone, and epidermal growth factor. Cultures
have been continuously passaged and maintained for over 6 mo., and a cloned cell line, TEA3A1, has been established. These
cells are epithelial, judging by morphology and ultrastructure, and are positive for A2B5 and thymosin α markers for thymic
endocrine cells.
This work was partly supported by grant PCM-834 0582 from the National Science Foundation, Washington, DC, and grant P01 CA
37589-2 from the National Cancer Institute, Bethesda, MD. 相似文献
12.
This study examined the kinds of desmosomal proteins in the human periodontal ligament fibroblasts (PDLFs). The PDLFs obtained from young and older patients were cultured and the amounts of desmosomal proteins were measured by ELISA with antibodies to desmoplakins, desmogleins, and desmocollins. Cultured cells and tissue sections of the human periodontal ligament were immunostained with the same antibodies. Expression of desmosomal proteins in the PDLFs was clearly demonstrated both by ELISA and the immunohistochemical studies, suggesting the existence of desmosome-like junctions in the PDLFs. The junctions are considered to protect gap junctions in the PDLFs against cell transformation caused by cell contraction, which may relate to tooth eruption and repair of periodontal tissue, and/or strong occlusal forces. Statistically significant differences (P < 0.0001) in the expression of desmoplakins and desmogleins between younger and older patients were observed in this study. 相似文献
13.
Cultures of fibroblast-like cells (PLF) and epithelial rest cells (PLE) prepared from explants of porcine periodontal ligament synthesized and secreted four glycosaminoglycans (GAG) in differing proportions. The PLF produced predominantly chondroitin sulfate (greater than 60%) with smaller amounts of hyaluronic acid (HA) (17%), dermatan sulfate (13%), and heparan sulfate (7%), whereas PLE produced predominantly HA (greater than 80%). In coculture and under conditions of reciprocal transfer of conditioned media neither cell type affected the other's GAG synthesis. Endothelial cells (EC), however, or their conditioned growth media, were able to stimulate increased GAG synthesis, especially HA, in PLF. A similar result was obtained with smooth muscles cells (SMC) cultured in EC growth media but here again PLE were unable to stimulate GAG synthesis by SMC. These findings suggest that the spectrum of GAG found in whole ligament results both from independent production by, and from interaction between, the different cell types within the ligament. The results also provide support for a general hypothesis that loose connective tissues, which are rich in HA, are formed and maintained under the influence of epithelial, including endothelial, cells. 相似文献
14.
Growth of mouse vaginal epithelial cells in culture: Effect of sera and supplemented serum-free media 总被引:3,自引:0,他引:3
Taisen Iguchi Francis-Dean A. Uchima Howard A. Bern 《In vitro cellular & developmental biology. Plant》1987,23(8):535-540
Summary Normal mouse vaginal epithelial cells isolated from ovariectomized ca. 35-d-old BALB/cCrgl mice were grown in primary culture
using collagen gel metrix and a serum-free medium composed of a 1∶1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s
F12 (D:H) medium supplemented with insulin (IN), epidermal growth factor (EGF), cholera toxin, transferrin, and bovine serum
albumin V (BSA). Three-dimensional cellular outgrowths occurred inside the collagen gel matrix. The contribution of each factor
to cell growth was examined by individual addition to the basic D:H medium and by individual deletion from the complete serum-free
medium. When added individually, only IN promoted growth. Deletion of IN from the complete serum-free medium markedly, diminished
growth; deletion of EGF or BSA slightly diminished growth. When horse, fetal bovine, or chicken serum was added to the basal
D:H medium, only with increasing doses of horse serum was there enhanced cell growth. The effect of 17?-estradiol and diethylstilbestrol
on the growth of cells was also tested, using a suboptimal medium of D:H supplemented with BSA and IN, or a minimal medium
supplemented with IN alone. During the 8-d time period, addition of estrogen did not enhance cell growth in either medium.
To date, we have been unable to demonstrate a mitogenic effect of estrogen; rather a dose-dependent inhibition of proliferation
is seen.
This investigation was supported by grants CA-05388 and CA-09041, awarded by the National Institutes of Health, DHHS, Bethesda,
MD. 相似文献
15.
David G. Thomassen 《In vitro cellular & developmental biology. Plant》1989,25(11):1046-1050
Summary The colony-forming efficiency of rat tracheal epithelial (RTE) cells was determined in serum-free media containing different
types of commercially available bovine serum albumin (BSA): crude fraction V, essentially globulin-free, essentially fatty-acid-free,
and essentially globulin- and fatty-acid-free BSA. RTE cells exhibited a concentration-dependent increase in colony-forming
efficiency in response to crude fraction V BSA. Similar results were obtained using essentially globulin-free BSA. However,
deletion of cholera toxin from the medium resulted in a decrease in the colony-forming efficiency for cells plated in high
concentrations (>2 mg/ml) of globulin-free, but not one type of fraction V, BSA. Essentially fatty-acid-free or essentially
fatty-acid- and globulin-free BSA stimulated RTE cell colony formation at low concentrations (less than 2.5 to 5 mg BSA/ml)
but resulted in concentration-dependent decreases in colony-forming efficiency at higher concentrations. The response of cells
to these BSAs was not dependent on cholera toxin. Finally, commerically available fraction V BSA prepared by heat shock, dialysis,
charcoal treatment, and deionization was stimulatory at low concentrations but inhibitory at high concentrations. These data
suggest that impure preparations of BSA can, under different conditions, stimulate or inhibit cell proliferation and that
the expression, of these activities is affected by the method of BSA preparation, the concentration of BSA used, and, in some
cases, by the presence or absence of cholera toxin.
Research conducted with support from the Office of Health and Environmental Research, U.S. Department of Energy, Washington,
DC, under contract no. DE-AC04-76EV01013 in facilities fully acredited by the American Association for Laboratory Animal Care. 相似文献
16.
Yasuhiro Tomooka Stephen E. Harris John A. McLachlan 《In vitro cellular & developmental biology. Plant》1985,21(4):237-244
Summary Epithelial cells from mouse seminal vesicles were enzymatically dissociated enriched by gradient centrifugation, and maintained
in collagen gel cultures with defined (serum-free) media. The epithelial origin of the cells was determined morhologically,
immunocytochemically, and biochemically. Cells formed three-dimensional colonies with a lumen in collagen gels. Cell number
was increased eight-fold within a 8 to 12-d culture period in a medium supplemented with epidermal growth factor (EGF) (10
ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml), and hydrocortisone (0.1 μg/ml). The cells required
eGF and insulin; the growth-promoting effects of these two peptide hormones were optimized by transferrin, cholera toxin,
and hydrocortisone. Fetal bovine serum did not support growth; rather, it suppressed the stimulated growth observed in serum-free
media. A time-course study revealed that a lag period preceded rapi growth. The collagen gel, serum-free culture provides
a powerful tool to study the effects of hormones on proliferation and differentiation of androgen sensitive cells. 相似文献
17.
Kazuhiro Takeuchi Ikuro Maruyama Sinichi Yamamoto Toshimichi Oki Yukihiro Nagata 《In vitro cellular & developmental biology. Animal》1991,27(9):720-724
Summary In the present study we have established a pure monolayer culture system of human fallopian tube epithelial cells. The cells
were isolated using collagenase digestion, and were cultured in Medium 199 supplemented with 15% fetal bovine serum. The epithelial
cells derived from primary and secondary culture were characterized using immunocytochemical staining and electron microscopy.
The cells continued to grow for 2 to 3 wk once the monolayer culture of the cells was established. It is currently possible
to maintain the cultures until the third generation. Proliferation of these cells was enhanced by epidermal growth factor
but not by basic-fibroblast growth factor, insulin, transferrin, estradiol-17β, or progesterone. This culture system offers a good model for determining characteristics of the tubal epithelium and would
permit effective study of co-culture with embryos. 相似文献
18.
Human oral epithelial cell culture I. Improved conditions for reproducible culture in serum-free medium 总被引:6,自引:0,他引:6
Summary Gingival tissue from healthy adult human donors was used as a source of epithelial cells for culture. An overnight incubation
of this tissue with dispase facilitated the mechanical separation of the surface epithelium from the underlying fibrous connective
tissue. This step minimized culture contamination with fibroblasts. The epithelium was then trypsinized to prepare a single
cell suspension. The cell pellets were collected by centrifugation and resuspended in keratinocyte growth medium, incubated
at 37° C and 5% CO2 in a humid atmosphere. Primary cultures grew in small islands that coalesced at confluency. Immunohistochemistry demonstrated
uniform staining of the cells with antibodies to keratins of stratified squamous epithelium. Ultrastructurally, the cells
contained distinct intermediate filaments. When cells were grown in media with low calcium (0.15 mM), cell-to-cell contacts were via interlacing papillary projections with no desmosomes. However, when cells were grown under
physiologic calcium (1.2 mM), desmosomes were prominent and well developed. Cells were maintained in culture for over 100 d (7 passages).
This work was supported by Biomedical Research grant RR 05346 from the National Institute of Health, Bethesda, MD, and the
University of Washington Graduate School Research Fund. 相似文献
19.
Sayuri Hamano Atsushi Tomokiyo Daigaku Hasegawa Asuka Yuda Hideki Sugii Shinichiro Yoshida Hiromi Mitarai Naohisa Wada Hidefumi Maeda 《Journal of cellular biochemistry》2020,121(12):4798-4808
Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue. 相似文献
20.
Sharma R Srivastava S Bajpai VK Balapure AK 《In vitro cellular & developmental biology. Animal》2002,38(5):293-297
A repertoire of hormonal signals including estrogen regulate the growth, differentiation, and functioning of diverse target tissues, including the ovary, the mammary gland, and skeletal tissue. A serum-free culture system derived from rabbit endometrium explants has been devised and is reported here to explore estrogen action in vitro. The system involves aseptically harvesting the uterus from a virgin rabbit, dissecting the endometrium, explanting it into 1- to 2-mm(3) pieces weighing approximately 1-2 mg each, and incubating these pieces in serum-free Medium-199. The culture is carried out for a period of 4 d in a humidified CO(2) incubator at 37 degrees C with 5% CO(2). The effect of extraneously added estrogen (1 microg/ml) was investigated by histological and ultrastructural procedures. It was observed that estrogen could induce specific changes, such as abundant mitochondria, rough endoplasmic reticulum, golgi complex, and intracellular collagen deposition, in both the epithelial and the fibroblast cell components of the explanted tissue. The study, therefore, indicates that the proposed system is an ideal tool for exploring and demonstrating estrogen responsiveness under in vitro conditions. 相似文献