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1.
Spontaneous establishment and characterization of mouse keratinocyte cell lines in serum-free medium 总被引:6,自引:0,他引:6
M. E. Kaighn R. F. Camalier F. Bertolero U. Saffiotti 《In vitro cellular & developmental biology. Plant》1988,24(8):845-854
Summary Mouse keratinocytes cultures readily develop into established cell lines without undergoing a “crisis” in a newly-developed
serum-free medium, LEP/MK2. LEP/MK2 consists of calcium-free MEM with non-essential amino acids supplemented with 8 factors.
Two lines, MK1 and MKDC4, have been isolated and have now doubled more than 400 and 200 times respectively. In MK1 cells,
Giemsa banding has revealed significant karyotypic changes as early as the 4th passage, leading to a near-tetraploid karyotype
with random loss and gain of individual chromosomes. Minute chromosomes, but no stable markers have been observed. After these
initial changes, examination of cultures at several passage levels has shown that the karyotype has remained essentially stable.
The MKDC4 line, also sub-tetraploid at the 7th passage, had 4 marker chromosomes by the 47th passage. The rapid increase in
chromosome number may have contributed to the “immortalization” of these lines.
The response of these established keratinocyte lines to growth factors and serum-derived inhibitors changed with increasing
passage level. Most notable of these changes were a reduction in the requirement for bovine pituitary extract (an absolute
requirement for growth of secondary MK1 cells) and a decreased sensitivity to serum and serum-derived inhibitors, e.g., transforming
growth factor-β. The established lines, like primary and secondary keratinocytes, remain responsive to calcium-induced terminal
differentiation and are non-tumorigenic in athymic, nude mice. This serum-free system is suitable for transformation studies
with oncogenes and chemical carcinogens.
Editor's Statement Keratinocytes are useful and important models for studies of carcinogenesis and tumor promotion and differentiation.
This paper provides a solid in vitro basis for examination of the cellular endocrinology of these phenomena in vitro, and
implicates TGF beta as a regulator of these cells. 相似文献
2.
Donald J. Sarubbi Ramaswamy Narayanan Nitin T. Telang Michael J. Newman 《In vitro cellular & developmental biology. Plant》1990,26(12):1195-1201
Summary Novel or modified serum-free media were developed for the anchorage-dependent growth of nontransformed murine mammary epithelial
cells (MMEC) and Balb/MK murine keratinocytes respectively. Growth rates for both cell lines were similar in serum-containing
and serum-free media. The serum-free media were used to evaluate potential mechanisms of epithelial cell growth regulation
by type 1 transforming growth factor β(TGF-β1). The growth of MMEC and Balb/ MK cells was reversibly inhibited 40–65% in a
time- and dose-dependent fashion by TGF-β1 under both serum-containing and serum-free conditions. Constitutive over-expression
of a stranfected c-myc oncogene inMMEC did not result in loss of sensitivity to growth inhibition by TGF-β1. In addition, Balb/MK and MMEC growth
inhibition by TGF-β1 was not potentiated by polynsaturated fatty acids or reversed by vitamin E. Expgenous type V collagen
was able to mimic the inhibitory effects of TGF-β1 on the serum-free growth of Balb/MK and MMEC. In contrast, collagen type
I and IV, fibronectin and laminin did not inhibit the growth of these cells. The type V collagen used was not contaminated
with TGF-β, and subsaturating, but not saturating concentrations of type V collagen and TGF-β1 were additive with respect
to Balb/MK and MMEC growth inhibition. These results demonstrate that nontransformed epithelial cell growth inhibition by
TGF-β1 is mediated by mechanisms distinct from those observed with certain carcinoma and melanoma cells. Our results also
suggest the possible involvement of type V collagen in Balb/MK and MMEC growth inhibition by TGF-β1.
This work was supported, in part, by grant #R29 CA 44741 from the National Institutes of Health, Bethesda, MD to NTT. 相似文献
3.
To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive
compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 × 106 cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent
fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite
cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 × 103 cells per well. Cells were grown for 1 day in MEMα plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by
2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well
plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus
10% FBS and 1 μM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation
medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing
this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds. 相似文献
4.
Clonal BALB/c mouse epidermal keratinocyte (BALB/MK) cell lines were established in tissue culture. Despite their aneuploid nature, the lines were nontumorigenic, and retained in vitro properties similar to those of primary diploid keratinocytes. These included the constitutive expression of keratin and terminal differentiation in response to a calcium concentration greater than 1.0 mM in the medium. The cells also demonstrated an absolute requirement for nanomolar concentrations of epidermal growth factor (EGF) for their proliferation. BALB or Kirsten murine sarcoma viruses are acute transforming retroviruses, which have been shown to transform fibroblastic and hematopoietic cells. Infection of BALB/MK or its clonal sublines with either virus leads to the rapid acquisition of EGF-independent growth. The cells concomitantly lose their sensitivity to calcium-induced terminal differentiation. Thus these retroviruses can rapidly confer upon epithelial keratinocytes in culture growth properties that resemble those of malignant epidermoid carcinoma cells. 相似文献
5.
Hidenobu Okumura Kunio Matsumto Koji Hashimoto Kunihiko Yoshikawa 《Experimental cell research》1991,192(2):647-650
Involucrin is a precursor protein of detergent-insoluble cornified envelope and a marker of terminal differentiation of epidermal keratinocytes. To quantify differentiation of cultured human keratinocytes, the population of involucrin-positive cells was estimated by immunofluorescent staining using anti-involucrin antibody and flow cytometry. Normal human keratinocytes were cultured under three conditions for induction of differentiation: low Ca2+ concentration (0.1 mM Ca2+), high Ca2+ concentration (1.8 mM Ca2+), and high Ca2+ concentration with 10% fetal bovine serum (FBS). The relationship between fluorescence intensity and involucrin synthesis was confirmed by visual examination of sorted cells. The population of involucrin-positive cells increased from 7.2 to 18.1% by elevating Ca2+ concentration and to 37.0% by adding FBS. The extent of cornified envelope formation under the same culture conditions was consistent with the estimation of involucrin-positive cells. The cytofluorographic analysis of involucrin synthesis made it possible to determine the number of differentiated cells in a large number of samples precisely and reliably. Thus, it is a useful method for quantifying keratinocyte differentiation. 相似文献
6.
Background aimsHuman mesenchymal stromal cells (hMSC) are of enormous interest for various clinical applications. For the expansion of isolated hMSC to relevant numbers for clinical applications, 10% fetal bovine serum (FBS)-supplemented medium is commonly used. The main critical disadvantage of FBS is the possibility of transmission of infectious agents as well as the possibility of immune rejection of the transplanted cells in response to the bovine serum. Therefore, we tested a commercially available medium, Panserin 401, that was specifically developed for serum-free cell cultivation.MethodshMSC were isolated from bone marrow (BM) and expanded in either Dulbecco's modified Eagle medium (DMEM) or Panserin 401 alone, or combined with FBS (2% or 10%), with or without supplementary growth factors. Cell proliferation and cytotoxicity were monitored twice a week for 3 weeks.Results and ConclusionsNo proliferation was observed in any of the serum-free media. However, DMEM/10% FBS (the conventional culture medium for hMSC) and DMEM/2% FBS with growth factors revealed moderate proliferation. Interestingly, the best proliferation was obtained using Panserin 401 supplemented with 2% FBS and growth factors (as well as with 10% FBS). Analysis of cell growth in Panserin 401 supplemented with 2% FBS only or with growth factors only revealed no proliferation, demonstrating the necessity of the combination of 2% FBS and growth factors. Efficient isolation and expansion of hMSC from cancellous bone could also be performed using Panserin 401 with 2% FBS and growth factors. Furthermore, these isolated cultures maintained multipotency, as demonstrated by adipogenic and osteogenic differentiation. 相似文献
7.
Jeniffer Farias dos Santos Nathália Ruder Borçari Mariana da Silva Araújo Viviane Abreu Nunes 《Journal of cellular biochemistry》2019,120(8):13141-13155
Epidermal differentiation is a complex process in which keratinocytes go through morphological and biochemical changes in approximately 15 to 30 days. Abnormal keratinocyte differentiation is involved in the pathophysiology of several skin diseases. In this scenario, mesenchymal stem cells (MSCs) emerge as a promising approach to study skin biology in both normal and pathological conditions. Herein, we have studied the differentiation of MSC from umbilical cord into keratinocytes. MSC were cultured in Dulbecco's modified Eagle's medium (DMEM) (proliferation medium) and, after characterization, differentiation was induced by culturing cells in a defined keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor (EGF) and calcium chloride ions. Cells cultivated in DMEM were used as control. Cultures were evaluated from day 1 to 23, based on the cell morphology, the expression of p63, involucrin and cytokeratins (KRTs) KRT5, KRT10 and KRT14, by quantitative polymerase chain reaction, Western blot analysis or immunofluorescence, and by the detection of epidermal kallikreins activity. In cells grown in keratinocyte serum-free medium with EGF and 1.8 mM calcium, KRT5 and KRT14 expression was shown at the first day, followed by the expression of p63 at the seventh day. KRT10 expression was detected from day seventh while involucrin was observed after this period. Data showed higher kallikrein (KLK) activity in KSFM-cultured cells from day 11th in comparison to control. These data indicate that MSC differentiated into keratinocytes similarly to that occurs in the human epidermis. KLK activity detection appears to be a good methodology for the monitoring the differentiation of MSC into the keratinocyte lineage, providing useful tools for the better understanding of the skin biology. 相似文献
8.
T Hirobe 《The Journal of experimental zoology》1991,257(2):184-194
Suspensions of disaggregated epidermal cells from skins of newborn C57BL/10JHir mice were plated in a growth medium that consisted of Ham's F-10 plus bovine pituitary extract (BPE), insulin, and transferrin. Fetal bovine serum (FBS) was added to the culture medium at a concentration of 4% at the time of plating. On the second day of culture, a small number of melanocytes was randomly distributed among large sheets of keratinocytes. From the third day onward, FBS was excluded from the culture medium to prevent the proliferation of keratinocytes and fibroblasts. The melanocytes began to grow preferentially, and after 12 days pure and enriched populations of melanocytes could be harvested. In the absence of the proliferation of keratinocytes and fibroblasts, melanocytes could be serially passaged in the growth medium supplemented with a conditioned medium (CM) prepared from keratinocyte-enriched cultures, namely, those at the early stages of the primary culture. FBS was added at a concentration of 1% for the first day. These results suggest that both BPE and keratinocyte CM contain growth factors required for proliferation of melanocytes. 相似文献
9.
10.
K Matsumoto K Hashimoto M Hashiro H Yoshimasa K Yoshikawa 《Journal of cellular physiology》1990,145(1):95-101
The effect of transforming growth factor-type beta 1(TGF-beta) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TGF-beta markedly inhibited the growth of keratinocytes at the concentrations greater than 2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [3H]thymidine incorporation. The decrease of [3H]thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main factors in the process of TGF-beta-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-beta under high Ca2+ conditions (1.8 mM Ca2+, differentiation-promoting culture environment) was examined. TGF-beta inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions. Thus, the effect of TGF-beta on keratinocyte differentiation is Ca2+ dependent. It enhances differentiation of human keratinocytes under high Ca2+ conditions, but inhibits differentiation under low Ca2+ conditions. Taken together, there is a clear discrepancy between TGF-beta effects on growth inhibition and induction of differentiation in human keratinocytes. These data indicate that growth inhibition of human keratinocytes by TGF-beta is direct and not induced by differentiation. 相似文献
11.
Ming Chow Monica Kong Harry Rubin 《In vitro cellular & developmental biology. Animal》1997,33(10):809-818
Summary We have reported that nontransformed sublines of NIH 3T3 cells that are incubated under the growth constraint of confluence
for 10 d or longer exhibit heritable reductions of growth rate upon serial subculture at low density, which simulate the effects
of aging in vivo on cell growth. There is also a marked increase in the likelihood of neoplastic transformation. After switching to a new
batch of calf serum (CS), we found the reduced growth rate was no longer produced within the previously established timeframe.
However, substitution of fetal bovine serum (FBS) for CS during the period of recovery from confluence or the following tests
of growth rate resulted in profound inhibition of growth in cells serially subcultured from confluent cultures. In some cases,
fewer than one in a thousand cells from subcultures of confluent cultures formed colonies in FBS although they cloned at relatively
high efficiency in CS. The reduced growth in FBS was retained in the postconfluent subcultures after many generations of multiplication
at low density in CS. Generally, similar results with individual variations were obtained with three other batches of FBS.
The numbers of cells per 3-d colony initiated from subcultures of confluent cultures were lower than those of control cultures
that had never been confluent. Supplementation of FBS-containing medium with CS fully restored the growth of the postconfluent
subcultures to the rate in CS medium, indicating that there is a deficiency of growth factor(s) in FBS rather than the presence
of an inhibitor. The results show that prolonged incubation at confluence induces a populationwide heritable increase in requirement
for growth factor(s) in short supply in FBS. Because clonal studies have shown that the reduction in growth rate is irreversible
and varies in degree from clone to clone, we propose it arises from damage to DNA at any of many different genetic loci or
from chromosome aberrations. Such genetic damage is also consistent with the increased tendency for neoplastic transformation
in subcultures from the long-term confluent cultures. 相似文献
12.
钙离子对鼠角质细胞生长和分化的影响 总被引:3,自引:0,他引:3
用无血清培养基培养角质细胞,研究了Ca2+对鼠角质细胞生长和分化的影响。实验结果表明,培养基中钙离子最佳浓度为0.2mmol/L。在此浓度下,细胞克隆形成率达到10.8%,细胞的贴壁率达到28.7%,细胞的分化比例和老化比例分别为5.4%和26.3%;当Ca2+浓度达到0.6mmol/L以上时,则会引起角质细胞显著的分化和老化。 相似文献
13.
H C Lin J T Piing Y M Tseng C F Chao 《Proceedings of the National Science Council, Republic of China. Part B, Life sciences》1988,12(4):215-221
Epidermal growth factor is an important element in maintaining keratinocyte proliferation and maturation. To evaluate its effect on keratinocyte growth in vitro, human foreskins were cultured. The epidermal keratinocyte growth in culture was separated into two stages by a conditional medium: the proliferation stage, in which the cells were maintained in a monolayer; and the differentiation stage, in which the cells grew to stratification and keratinization. The keratinocytes were cultured in various concentrations of epidermal growth factor, and their morphology and growth behavior were closely observed. Our results demonstrated that cultured keratinocytes grew in a confluent layer under the influence of epidermal growth factor. In contrast, in a culture without epidermal growth factor, the proliferation rate of cultured keratinocytes slowed down and eventually the cells stopped growing. During serum stimulation, with or without additional exogenous epidermal growth factor, the cultured keratinocytes grew continuously to the normal terminal differentiation. Under this two-stage culture model, the cultured keratinocytes could grow into an intact sheet of graftable epidermis. 相似文献
14.
A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin. 总被引:20,自引:2,他引:18 下载免费PDF全文
P W Cook P A Mattox W W Keeble M R Pittelkow G D Plowman M Shoyab J P Adelman G D Shipley 《Molecular and cellular biology》1991,11(5):2547-2557
A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate. 相似文献
15.
Growth factor-independent proliferation of normal human neonatal keratinocytes: production of autocrine- and paracrine-acting mitogenic factors 总被引:10,自引:0,他引:10
When normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 x 10(3)/cells cm2, the cells proliferated continuously and the addition of IGF-I, EGF, TGF alpha, bFGF, or aFGF did not significantly alter growth rate. Heparin sulfate, TGF beta, or suramin inhibited keratinocyte growth factor-independent proliferation. The addition of EGF, TGF alpha, or aFGF reversed heparin-induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte-derived conditioned medium (CM) indicated the presence of TGF alpha peptide at a concentration of approximately 235 pg/ml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte-derived CM replaced EGF in stimulating keratinocyte clonal growth, and an anti-EGF receptor mAb inhibited CM-induced keratinocyte clonal growth. In addition to its effect on keratinocytes, keratinocyte-derived CM stimulated the incorporation of [3H]thymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR-2B cells, and EGF-receptorless mouse NR6 cells. CM-stimulated [3H]thymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti-EGF receptor mAb. Heparin sulfate partially inhibited CM-induced keratinocyte clonal growth and [3H]thymidine incorporation into quiescent AKR-2B cells. We hypothesize from these data that autocrine and paracrine-acting factors produced by keratinocytes mediated their effect through the activation of both EGF receptor-dependent and EGF receptor-independent mitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin. 相似文献
16.
Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free medium 总被引:9,自引:0,他引:9
Growth and differentiation of keratinocytes in a serum-free medium (keratinocyte growth medium or KGM) was studied and compared to that under conditions in which serum and feeder cell layers were used. Cells were grown in KGM containing 0.1 mM calcium (KGM/low calcium), KGM containing 1.2 mM calcium (KGM/normal calcium), or Dulbecco's modified Eagles medium containing 5% fetal calf serum and 1.8 mM calcium in presence of mitomycin treated 3T3 M cells (DMEM/5% FCS). Plating efficiency and rate of growth were similar in the three media till confluence. In postconfluent cultures, protein and DNA content of cells attached to the plate in KGM/low-calcium dishes decreased as an increased number of cells were shed into the medium. Cell shedding was much less evident in the presence of normal calcium. Cells grown in KGM/low calcium had a higher rate of cell proliferation (3H-thymidine incorporation into cellular DNA) than cells grown in normal calcium. Transglutaminase activity, involucrin content, and cornified envelope formation were greatest in cells grown in KGM/normal calcium, intermediate in cells grown in DMEM/5% FCS, and least in cells grown in KGM/low calcium. Keratin profiles from cells grown in KGM/low calcium showed a lower percentage of high molecular weight bands compared to the keratin profiles from cells grown in the presence of normal calcium. Keratinocytes in KGM/low calcium grew as a monolayer of cuboidal cells with few features of differentiation, whereas cells grown in KGM/normal calcium stratified into multilayered islands (3-5 layers) surmounted by 2-4 layers of enucleated cells with thickened cornified envelopes. Cells grown in KGM/normal calcium also contained tonofilaments and lamellar bodies unlike cells grown in KGM/low calcium. Cells grown in DMEM/5% FCS also formed stratified layers comparable to cells grown in KGM/normal calcium but lacked cornified cells, keratohyalin granules, tonofilament bundles, and lamellar bodies. These studies indicate the usefulness of serum-free conditions for the culture of human keratinocytes and confirm the importance of extracellular calcium in keratinocyte differentiation. 相似文献
17.
Growth of human keratinocytes on fibronectin -coated plates 总被引:9,自引:0,他引:9
Keratinocytes derived from the skin of newborns and of adults aged 19 to 57 years were grown on plates coated with human fibronectin (HFN) in the absence of a 3T3 monolayer. The cells grew well, attained confluence and could be sub-cultivated at densities approximately 10% of those necessary for successful cultivation of human keratinocytes on collagen coated dishes. Growth was excellent at concentrations of fetal bovine serum (FBS) as low as 5%, and appreciable growth occurred over a six day period even in the complete absence of serum. Growth was enhanced by addition of cholera toxin to the medium. Fibroblast overgrowth of the keratinocyte colonies was not observed. The observation that keratinocytes grow well on fibronectin in the absence of a fibroblast feeder-layer should simplify further study of this fastidious cell type and increases our understanding of keratinocyte growth requirements in vitro. 相似文献
18.
19.
John A. McLane Marion Katz Nana Abdelkader 《In vitro cellular & developmental biology. Plant》1990,26(4):379-387
Summary 1,25-Dihydroxyvitamin D3 (1,25-(OH)2-D3) is known to decrease the proliferation and increase the differentiation of different cell types including human keratinocytes.
The growth and differentiation of keratinocytes in the presence of 1,25-(OH)2-D3 using serum-free media formulations has been described previously. This investigation extends these studies to describe various
culture conditions with human foreskin keratinocytes to determine the optimal antiproliferative activity of 1,25-(OH)2-D3. Keratinocytes were plated onto tissue culture dishes using one of three basic serum-free media protocols; a) with no feeder
layer in keratinocyte growth medium (KGM); b) onto mitomycin C-treated 3T3 mouse embryo fibroblasts; or c) onto mitomycin
C-treated dermal human fibroblasts. The last two protocols utilized Dulbecco's modified Eagle's Medium (DMEM) supplemented
with growth factors. Keratinocyte cell growth was greatest in the KGM medium. Although the growth of keratinocytes on either
feeder layer was similar, there were differences in the ability of the cells to form envelopes in the presence of 1,25-(OH)2-D3. The addition of hydrocortisone and cholera toxin to the medium also affected the response of the keratinocytes to 1,25-(OH)2-D3. The antiproliferative effect of 1,25-(OH)2-D3 was not altered by varying the extracellular calcium levels from 0.25 to 3 mM. The antiproliferative activity of 1,25-(OH)2-D3 is attenuated in cells at low density. Our results suggest that an optimal condition to investigate the ability of 1,25-(OH)2-D3 to inhibit keratinocyte proliferation is at preconfluent cell density in the presence of KGM supplemented with 1.5 mM calcium without a feeder layer. These conditions are not appropriate for investigating the enhancement of differentiation
by 1,25-(OH)2-D3, but can be used to assay other agents that modulate keratinocyte proliferation.
Portions of this work were presented and abstracted at the April 1988 meeting of the Society of Investigative Dermatology
(J. Inv. Derm. 90(4): 586; 1988) and the February 1988 meeting of New York Academy of Sciences (Ann NY Acad. Sci. 548: 341–342;
1988). 相似文献
20.
Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor 总被引:12,自引:0,他引:12
G D Shipley W W Keeble J E Hendrickson R J Coffey M R Pittelkow 《Journal of cellular physiology》1989,138(3):511-518
Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin. 相似文献