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1.
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
2.
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
3.
Terramani TT Eton D Bui PA Wang Y Weaver FA Yu H 《In vitro cellular & developmental biology. Animal》2000,36(2):125-132
Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial
cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells
(HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated
in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were
also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly
used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell
proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth
supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement
in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were
grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types
I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented
with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage. 相似文献
4.
Brigitte A. van der Haegen Richard G. Ham Tamiko Kano-Sueoka 《In vitro cellular & developmental biology. Plant》1989,25(2):151-157
Summary An improved serum-free medium has been developed that supports growth of rat mammary tumor line 64–24 with far less protein
supplementation and with a much smaller inoculum than previously possible. An initial survey showed that MCDB 202 supported
clonal growth with 1% dialyzed serum. The remaining serum was then replaced with 5 μg/ml insulin, 10 ng/ml epidermal growth
factor (EGF), 1 μg/ml hydrocortisone, 50 ng/ml ovine prolactin, and 5 μg/ml liposome B (a mixture of soy lecithin, sphingomyelin,
cholesterol, vitamin E, and vitamin E acetate in liposome form). Insulin and EGF are required and growth is improved by hydrocortisone
and prolactin. Estradiol is stimulatory in the absence of liposome B. With adequate iron supplementation, transferrin has
no effect. Liposome B increases growth rate substantially. Most of the growth stimulation can be replaced with phosphatidylethanolamine
or sphingomyelin.
This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by B.A.VdH.
in partial fulfillment of the requirements for the Ph.D. degree. This research was supported by grant CA-15305 to R.G.H. and
grant CA-30545 to T.K.-S., both from the National Institutes of Health, Bethesda, MD. 相似文献
5.
Simultaneous occurrence of pregnancylike lobuloalveolar morphogenesis and casein-gene expression in a culture of the whole mammary gland 总被引:1,自引:0,他引:1
Nivedita Ganguly Ranjan Ganguly Nozer M. Mehta Linda R. Crump M. R. Banerjee 《In vitro cellular & developmental biology. Plant》1981,17(1):55-60
Summary Entire second thoracic mammary glands of estrogen- and progesterone-treated immature virgin BALB/c mice were stimulated to
pregnancylike lobuloalveolar morphogenesis after 6 days of incubation with insulin (5 μg/ml), aldosterone (1 μg/ml), growth
hormone (5 μg/ml), cortisol (5 μg/ml), and prolactin (80 ng/ml, present as a contaminant in 5 μg/ml growth hormone). The alveolar
growth in the glands, as judged by morphological studies, was accompanied by an increase in cell number as a function of incubation
time in the hormonal medium. Hybridization of the total RNA from these glands to the casein mRNA specific complementary DNA
probe (cDNAcsn) revealed that the level of casein mRNA rises from 0.00012 to 0.005% between 1 and 6 days of incubation. Estimates showed
that the concentration of casein mRNA per cell rises 17-fold from 70 molecules on Day 1 to 1200 molecules on Day 6, whereas
the number of epithelial cells increases only twofold during the same incubation time. When the growth hormone preparation
was totally replaced by 80 ng of prolactin during the 6-day incubation, casein-mRNA levels were found to be 0.0083%. These
results demonstrate that a pregnancy-like morphogenesis and concurrent expression of the casein gene in vitro can be achieved
in a controlled hormone environment containing high cortisol and low prolactin concentrations. This one-step mammogenesis-lactogenesis
culture model should be useful for studying the mechanisms of hormonal regulation of casein-gene expression observed in prepartum
mammary gland in vivo.
This work was supported by Department of Health, Education and Welfare Grants CA11058 and CA25304 from the National Cancer
Institute. 相似文献
6.
Ming-Sound Tsao Judith D. Smith Joe W. Grisham 《In vitro cellular & developmental biology. Plant》1985,21(5):249-253
Summary The ability of a normal rat liver epithelial cell line with phenotypic characteristics of “oval” cells to grow in calcium-poor
medium has been investigated. The growth of these cells could be arrested in medium containing 0.03 mM Ca2+, a concentration below which cell necrosis began to occur 24 h postexposure. With increasing calcium concentration, progressive
cell proliferation was observed. Epithelial growth factor (EGF) (10 ng/ml) increased the survival and proliferation of cells
in calcium-poor medium and the response was inversely correlated with the extracellular calcium concentration. In contrast,
phenobarbital (0.2 to 2 mM), 12-0-tetradecanoylphorbol-13-acetate (0.01 to 1 μg/ml), or retinoic acid (0.001 to 0.1 μg/ml) depressed growth of cells
in calcium-poor medium. The results confirm the ability of EGF to lower the calcium requirement for proliferation of normal
cells, but such an effect does not seem to be a universal property of tumor promoters.
This research was supported by National Institutes of Health Grant CA 29323. 相似文献
7.
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
8.
C Péchoux L Frappart 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1992,315(10):365-372
A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal human fetal mammary epithelial cells within an extracellular matrix preparation. Human fetal mammary epithelial cells were isolated from the mammary glands of human female fetuses, 17 to 39 weeks-old. The "organoids" were embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm (EHS) sarcoma according to the method of Hahm and Ip. "Organoids" were grown in either serum-free medium or in medium with fetal calf serum (FCS). The "organoid" proliferated over a 2 to 3 weeks culture period and remained viable for 1 or 2 months within the basement membrane matrix in serum free medium. Several types of colonies were observed; including alveolar-like budding clusters obtained from cultures of mammary gland from fetuses of over 20 weeks age, units with ductule-like projections and stellate-type colonies. Cell proliferation was dependent on the culture medium (with FCS no proliferation was obtained) and on the substratum (without matrix, significantly less growth and development occurred). These types of colonies are obtained when a glandular differentiation of cells budding from the malpighian epithelium is observed. Light microscopic and transmission electron microscopic studies were undertaken at the time of culture. This unique system using normal fetal mammary epithelial cells thus provides a model in which the regulation of human mammary development can be investigated. 相似文献
9.
Summary A new culture method was used to study increases in wet and dry weight and soluble protein during normal development of the
transparent lens. Seven different media with more than ten different additives were tested for their effects on cultured lens
transparency.In vivo, rat lenses increased 53% in soluble protein content between 3 and 5.5 days of age. Only HL-1 serum-free medium containing
15 μg/ml insulin plus 1–2 ng/ml BB platelet-derived growth factor (PDGF), or 5–7 ng/ml epidermal growth factor (EGF) allowed
similar growthin vitro, during the same time period. Normal lens grwoth occurred in culture when fresh medium was delivered to lenses as a pulse
every 4–6 hours. Lenses decreased in dry weight and soluble protein content, and became opaque when the same medium was delivered
continuously. Lenses increased only 26% and 32% in soluble protein content when delivered pulses of HL-1 medium containing
BB PDGF or EGF in the absence of insulin. We suggest that pulsatile delivery of medium containing insulin and PDGF or EGF
stimulates lens growth during developmentin vitro. This pulsatile organ culture system is presented as a new approach for studying the effects of growth factors on cell proliferation,
differentiation, and receptor regulation in a developing tissue.
This work was supported by grants from EY-07031 and EY-04542 from the National Eye Institute and a grant from the Oculon Corporation.
Editor's statement This report documents an in vitro system that may mimic lens development and response to growth regulators
and hormones. The system may be useful for application to other organs and provide a foundation for cell and molecular level
analysis. 相似文献
10.
Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium 总被引:1,自引:0,他引:1
Summary An improved procedure has been developed for clonal growth of normal human epidermal keratinocytes (HK) without feeder cells
or conditioned medium. The use of medium 199, supplemented with 0.4 μg/ml hydrocortisone (HC) and 20% (v/v) whole fetal bovine
serum (wFBS) and conditioned overnight by 3T3 cells, eliminated the need for a feeder layer of lethally irradiated 3T3 cells
for HK growth. Several other media with equivalent conditioning and supplementation failed to support satisfactory multiplication
of HK, including Dulbecco's modified Eagle's medium, which is normally used for growth of HK with a feeder layer. Increasing
the concentration of HC to 10 μg/ml (2.8×10−5
M) made possible clonal growth of HK without any conditioning of the medium. The addition of 10−5
M putrescine, 10−5
M vitamin B12, or 3.7×10−6
M β-estradiol further enhanced growth in unconditioned medium. Substantially greater improvement was obtained by the addition
of pituitary extract or fractions prepared from pituitary extract. In medium 199 supplemented with 10 μg/ml HC, 20% (v/v)
wFBS, and 0.15 mg/ml each of two pituitary fractions, single HK attach with a colony-forming efficiency equal to that in conditioned
medium and form stratified, keratinized colonies that grow to confluency and can be subcultured. These results make it clear
that HK do not require special “conditioning factors” from fibroblasts for clonal growth and differentiation in culture. Thus,
factors directly involved in growth and the expression of differentiation can be analyzed without the interfering effects
of any other type of cell. Preliminary studies with epidermal growth factor (EGF), which stimulates growth and extends life
span of HK grown in the presence of fibroblasts, have shown that, in the absence of fibroblasts, EGF has no effect either
on clonal growth or on cumulative multiplication potential of HK.
This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna
M. Peehl in partial fulfillment of the requirements for the Ph.D. degree.
This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute
on Aging. 相似文献
11.
Lawrence E. Shapiro Neil Wagner 《In vitro cellular & developmental biology. Plant》1988,24(4):299-303
Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium
is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum
containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these
two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium
was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the
absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium
from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free
culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells.
This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes
of Health grants CA 24604-09 and CA 16463-14. 相似文献
12.
Mark L. Johnson Joseph Levy Jeffrey M. Rosen 《In vitro cellular & developmental biology. Plant》1985,21(8):439-444
Summary Milk protein gene expression was studied in cell subpopulations of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinoma cells enriched or depleted for casein production grown on attached collagen gels.
Culture of these cells in the presence of 10% fetal bovine serum, insulin (5 μg/ml), hydrocortisone (10 μg/ml), and prolactin
(5 μg/ml) maintained α-, β-, and γ-casein and whey acidic protein mRNAs at levels identical to cells isolated from perphenazine-treated
rats. Whey acidic protein mRNA levels in the tumor cells relative to the 14-d lactating gland were greater than those of the
casein mRNAs. Withdrawal of prolactin from the casein-producing cells resulted in the loss of all four milk protein mRNAs.
Subsequent addition of prolactin to the withdrawn cells caused a rapid accumulation of these mRNAs to prewithdrawal levels.
Milk protein gene expression in this tumor cell subpopulation is modulated by prolactin (in the presence of insulin and hydrocortisone)
in a similar manner to that observed in the normal mammary gland when these tumor cells are cultured on attached collagen
gels.
This work was supported by National Institutes of Health grant CA 16303. M. L. Johnson was the recipient of NIH Fellowship,
HD 06157. 相似文献
13.
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
14.
David G. Chilton Betty H. Johnson Laurence Danel-Moore Simon Kawa E. Brad Thompson 《In vitro cellular & developmental biology. Plant》1990,26(6):561-570
Summary CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously
cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium
of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine
serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar
to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for
3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell
morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived
lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited
increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine
synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be
completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially
sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone
in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results
suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid
hormone action may be pursued more precisely in a clearly defined culture medium.
This work was conducted in conjunction with the Walls Medical Research Foundation. 相似文献
15.
Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media 总被引:3,自引:0,他引:3
David Danielpour Terry L. Riss Masami Ogasawara David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(1):42-52
Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated
DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When
ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate
was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect
on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors
in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal
growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium
equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth
rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion
may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone.
This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255. 相似文献
16.
In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI)
to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes
were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI
transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method).
The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression
levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the
ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several
different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained
with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA
concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared
to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared
to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1
(CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression
in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production
of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments,
however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
17.
Susumu Yoshida Shigeo Kasuga Yuzo Hirao Tohru Fuwa Shizutoshi Nakagawa 《In vitro cellular & developmental biology. Plant》1987,23(7):460-464
Summary Biosynthetic human epidermal growth factor (Bh-EGF) induced dose-dependent synthesis and secretion of neutral mucin glycoprotein
when the fundal cells isolated from rabbit stomach were cultured in serum-free medium containing Bh-EGF at concentrations
as high as 10 to 100 ng/ml. At these high concentrations, Bh-EGF had no effect on the cell growth. In marked contrast, much
lower concentrations from 0.1 to 1.0 ng/ml of Bh-EGF failed to stimulate mucin synthesis, but enhanced proliferation of the
cells. Electrophoretic pattern of the mucin secreted from the cultured mucosal cells was very similar to that of the authentic
mucin obtained from rabbit stomach. Maximal secretion of the mucin from the cells was observed at Hour 96 of the culture.
Although fetal bovine serum (5%) and insulin (0.5 μg/ml) also stimulated the mucosal cells, both in growth and in mucin synthesis
and release, the enhancing activity of the mucin synthesized and released by Bh-EGF at a concentration of 100 ng/ml per microgram
DNA of cultured cells was far superior to that of 5% fetal bovine serum and 0.5 μg/ml insulin. 相似文献
18.
The serum-free growth of primary cultures of normal human epithelial-like cells from amniotic membranes was accomplished. The synthetic medium consists of a 1 : 1 basal nutrient mixture of Dulbecco's modified Eagle medium (DMEM) and Ham's F-12 supplemented with 2.5 μg/ml insulin, 50 ng/ml epidermal growth factor (EGF), 5 μg/ml transferrin, and 0.1 ng/ml triiodothyronine (T3). EGF is the primary mitogen and is essential for cell proliferation in this system. 相似文献
19.
Masayoshi Ono John W. Perry Takami Oka 《In vitro cellular & developmental biology. Plant》1981,17(2):121-128
Summary Cortisol was previously shown to elicit a concentration-dependent inhibition of α-lactalbumin accumulation in midpregnant
mouse mammary gland cultured in medium containing optimal concentrations of 5 μg/ml prolactin and insulin. In contrast, casein
accumulation under these conditions was progressively stimulated by addition of increasing amounts of cortisol (Ono, M.; Oka,
T. Cell 19: 473–480; 1980). In the present study we found that in the presence of a suboptimal concentration of 0.5 μg/ml
prolactin, 2.8×10−9
M to 2.8×10−7
M cortisol stimulated α-lactalbumin accumulation. Furthermore, higher concentrations of cortisol produced a smaller inhibition
of α-lactalbumin accumulation as compared to that obtained in cultures containing 5 μg/ml prolactin. The maximal increase
in α-lactalbumin accumulation attained in the presence of 1.4×10−8
M cortisol, 0.5 μg/ml prolactin, and insulin was comparable to that observed in culture containing 5 μg/ml prolactin and insulin.
Similar results were obtained in a cortisol concentration-response study of α-lactalbumin accumulation in cultures containing
a suboptimal concentration of 0.5 μg/ml human placental lactogen. Measurement of the rate of α-lactalbumin synthesis in cultured
tissue indicated that the opposing effects of low and high concentrations of cortisol on α-lactalbumin accumulation involved
an alteration in the rate of synthesis of the milk protein. In contrast to α-lactalbumin, the synthesis of casein was stimulated
in a concentration-dependent manner by addition of cortisol that acted synergistically with either 0.5 μg/ml or 5 μg/ml prolactin.
The maximal increases were obtained in the presence of 2.8×10−6
M cortisol. These results indicated that the action of cortisol on α-lactalbumin accumulation can be modulated by the concentration,
of prolactin and suggest that the interplay between cortisol and prolactin in regulation of α-lactalbumin synthesis may be
different from that involved in casein synthesis. 相似文献
20.
Moll SJ Jones CJ Crocker IP Baker PN Heazell AE 《Apoptosis : an international journal on programmed cell death》2007,12(9):1611-1622
Pre-eclampsia and intrauterine growth restriction are associated with increased apoptosis of placental villous trophoblast.
This may result from placental hypoperfusion, leading to the generation of reactive oxygen species (ROS). Apoptosis can be
induced in villous trophoblast following exposure to oxidative stress. Epidermal growth factor (EGF) reduces trophoblast apoptosis
resulting from exposure to hypoxia. We hypothesised that exposure to hydrogen peroxide, a potent generator of ROS, would induce
apoptosis in term placental villous explants and that this could be reduced by treatment with EGF. Placental explants were
taken from normal term pregnancies and exposed to increasing doses of hydrogen peroxide (0–1,000 μM) or to a combination of
increasing doses of hydrogen peroxide and EGF (0–100 ng/ml) for either 6 or 48 h. Apoptosis was assessed by TUNEL, proliferation
by Ki-67 immunostaining, necrosis by lactate dehydrogenase activity and trophoblast differentiation by human chorionic gonadotrophin
(hCG) secretion in conditioned culture media. Immunoperoxidase staining was performed to identify phosphorylated-AKT (p-AKT)
and phosphorylated-PI3 kinase (p-PI3k). Exposure to 1,000 μM hydrogen peroxide for 48 h induced apoptosis in placental explants.
The increase in TUNEL positive nuclei predominantly localised to syncytiotrophoblast. The amount of apoptosis was reduced
to control levels by treatment with 10 and 100 ng/ml EGF. Proliferation of cytotrophoblasts within villous explants was significantly
reduced following exposure to 1,000 μM hydrogen peroxide, this was restored to control levels by simultaneous treatment with
10 or 100 ng/ml EGF. Neither exposure to hydrogen peroxide or EGF altered the amount of necrosis. There was increased immunostaining
for pPI3K following treatment with EGF. This study shows that apoptosis may be induced in villous trophoblast following exposure
to ROS, and demonstrates the anti-apoptotic effect of EGF in trophoblast, the maintenance of which is essential for normal
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