Effects of Epidermal Growth Factor, Platelet Derived Growth Factor and Growth Hormone
on Cultured Rat Keratinocytes Cells in vitro
M.D. Babak Hossein Zadeh Zoroufchi
Some growth factors, such as Epidermal Growth Factor (EGF), Growth Hormone (GH) and Platelet Derived Growth Factor (PDGF) have beneficial effects on keratinocyte proliferation and wound healing. Although the mechanism of these factors is unclear. In response to injury, growth factors are secreted by kinds of cutaneous cells. The goal of this project is to investigate the factors that could cause proliferate of the keratinocyte cells in vitro. The keratinocytes were removed from rat pups (10 days). Cultured in media with different concentration of GH, PDGF and EGF separately. The proliferation of cells was evaluated by the method of MTT and 3H-thymidine incorporation. Proliferation of keratinocytes was significantly higher in experimental groups than in control group. EGF maximally stimulated at 10 and 25 ng mL-1. PDGF-BB maximally stimulated at 50 ng mL-1, respectively. And maximal stimulation of GH was 2.5 IU L-1. GH, PDGF-BB and EGF stimulate keratinocyte cells proliferation in different concentration. These growth factors could play in healing of the skin.
Received: November 10, 2013;
Accepted: January 23, 2014;
Published: March 29, 2014
The outermost layer of skin is epidermis and contacts the outside environment
directly. Rapid and efficient wound healing is essential for normal life. Wound
repair is a multiple step process, included cell proliferation, differentiation,
formation of granulation tissue and synthesis of various collagen. In wound
healing keratinocyte migration causes re-epithelization of the skin (Lal
et al., 2000). Several growth factors are essential for skin repair.
Therefore, much research's have been done on these factors.
Growth Hormone (GH) widely used in children. There are serious effects, that
this hormone has great effects on keratinocyte repair (Rasmussen
et al., 1994; Jorgensen and Oxlund, 1996).
Several studies reported that some factors such as insulin-like growth factor
(IGF1) intercede the action of GH in wound repair (Pell
and Bates, 1992).
These studies showed that the Epidermal Growth Factor (EGF), Platelet-Derived
Growth Factor-BB (PDGF-BB) naturally produced peptides that important for preserving
of skin integrity. EGF stimulates proliferation of human epithelium (Imanishi
et al., 2000). Other researchers have shown than EGF have several
therapeutic effects, including promoting vascularization, increasing of cell
turnover and healing of epithelial and endothelial lesions (Knorr
et al., 1997). EGF was found to increase epidermal thickness and
stimulate proliferation of epidermal keratinocytes (Jost
et al., 2000).
Locally release of PDGF promotes proliferation, vascularization and migration
of connective tissue cells and then synthesis of extracellular matrix components.
Cells of keratinocyte, endothelial, fibroblast and smooth muscle, contribute
to PDGF generation in lesion site (Rollman et al.,
2003). Mesenchymal cells in the skin express PDGF protein (Heldin
and Westermark, 1999). Epidermal cells under physiological condition dont
express transmembrane receptors that recognize any type of classical PDGF isoform
(Yan et al., 2007). Animal data have shown that
delayed in skin repair is associated with reduce expression of PDGF protein
and their receptors (Vavken et al., 2010). Human
studies in diabetic ulcer have shown that topical performance of PDGF-BB promotes
granulation tissue and increase vascularization (Wieman
et al., 1998). PDGF is a principle mitogen for cell culture with
Mesenchymal origin such as fibroblast, smooth muscle (Shin
et al., 2012). The biological activity of PDGF is mediated via binding
to cell-surface receptors. Two receptors for PDGF have been identified alpha
and beta (Lederle et al., 2010).
Due to several activities which stimulated by the PDGF on cells, it has been
implicated as a main components involved in dermal wound repair (Denk
and Knorr, 2002). PDGF is a chemotactant factor for monocytes, neutrophils
and fibroblast cells (Xian et al., 1997). It
stimulates production of extra cellular matrix by fibroblast (Marikovsky
et al., 1996). Studies have shown that PDGF-BB treatment accelerate
the rate of ulcer healing in human and rats (Wong et al.,
2012). All of these growth factors can stimulate proliferation of keratinocytes
in vitro but the exact dose of these factors has not been identified. In this
study it was assumed that adding of these factors with determine doses cause
rapid proliferation of keratinocytes cells in vitro.
MATERIALS AND METHODS
Pups keratinocyte isolation and culture: All experiments in this study,
especially separation and isolation of keratinocytes were accomplished according
to the manifest of Helsinki. Ethical approval was taken from Semnan University
of Medical Science. The cells of keratinocyte were isolated and cultured according
to the protocol that described by Bevan et al. (1997).
Briefly, Normal keratinocytes cells were obtained from dorsum of rat pups (10
days). They washed with warm Hanks balanced salt solution (HBSS) [sigma] containing
1.25 μg mL-1 of amphotericin. Samples were sliced into small
pieces and washed again, then incubated at 37°C in 0.25% trypsin/EDTA Co2
5% for 1 h. The epidermis was removed from dermis. Resultant epidermis re-suspended
in HBSS for 10 min. The remaining cells were centrifuged for 10 min at 1200
rpm and resuspended in culture medium (Dulbecco's modified Eagles medium
[DMEM]/F12,3:1) [sigma] containing penicillin (100 IU L-1), streptomycin
(100 μg mL-1), fetal bovine serum10%, hydrocortisone(0.525 μg
mL-1), cholera toxin (10-10 M). The cell suspension (5x105
cells) was in 75 cm2 culture flasks. Culture flasks were sustained
at 37°C containing 5% CO2. Culture medium was replaced every
Cell proliferation (3H-thymidine incorporation) assay: Keratinocyte cells were treated with trypsin /EDTA 0/1% at 37°C for 10 min. The reaction was then stopped by adding Fetal Bovine Serum (FBS) [sigma]. The cells were counted by trypan blue exclusion and then re-plated into 24 well plates at a concentration (1x105) with respected medium. The plates were placed in incubator at 37°C overnight. The next day, culture medium was replaced with serum free medium. On the third day, 0.5 mL of serum free medium, 1 μCi 3H-thymidin, and respective growth factor with different concentration (PDGF 0, 1, 5, 10, 25, 50, 75 ng mL-1) (EGF: 0, 1, 2, 5, 10, 25, 50 ng mL-1) (GH: 0, 0.5, 2.5, 5, 10, 50 IU L-1) were added to each well of plate. The plates were placed in incubator for 24 h. On the fourth day, cells of each well were rinsed with HBSS, and then washed by 5% trichloroacetic acid. The cells of each well were dissolved with 5 mL well-1 of 1 N NaOH. Added 0.1 mL acid glacial acetic to each well. A sample aliquot of 0.1 mL was moved into scintillation vials in duplicate and counted in the scintillation counter.
Keratinocyte proliferation and effects of growth factors: Proliferation of keratinocyte cells was measured by tetrazolium bromide based colorimetric assay (MTT cell assay). All subcultures were done into the 96 well plate with 3x103 cell well-1. Above factors were added according to the protocolPDGF 0, 1, 5, 10, 25, 50, 75 (ng mL-1), EGF: 0, 1, 2, 5, 10, 25, 50 (ng mL-1), GH: 0, 0.5, 2.5, 5, 10, 50 (IU L-1). The final volume of media in each well was 100 μL. The cells were incubated for3 days in 37°C, 5% CO2 incubator. On day 3, 20 μL from MTT solution with a concentration of 5 mg mL-1 was added to the media of each well, and then was incubated for 4 h in a 37°C, 5% CO2 incubator. Culture medium was removed and then added 200 μL of dimethylsulphoxide (DMSO) to wells and stirred for 10 min in a shaker. Cell increasing was evaluated from the absorbance of 540-560 nm, using an Enzyme-Linked Immunosorbent Assay (ELISA) readerstat fox.
Statistical analysis: The results of this study are expressed as the
Mean±standard error. Difference between proliferation of the cell responses
following treatment with PDGF-BB, EGF and GH were compared together. Data were
analysed using the one way ANOVA, and post hoc comparisons were done using Tukeys
test at 0.05 level of significance (SPSS ver.11).
Keratinocytes cells: Various concentrations of PDGF-BB, VEGF and GH on proliferation
of keratinocytes were studied. PDGF-BB has a stimulatory effect on keratinocyte
||Keratinocytes cell response to PDGF. Concentration 25-50 and
75 ng mL-1 significantly increased cell number. No significant
effect were observed in the concentration 1-10 ng mL-1. maximum
proliferation was at 50 ng mL-1 concentration
||Keratinocytes cell response to EGF. Concentration 10-25 and
50 ng mL-1 significantly increased cell number. No significant
effect were observed in the concentration 1-2-5 ng mL-1 maximum
proliferation was at 25 ng mL-1 concentration
Concentrations of 25-50 and 75 ng mL-1 had the most stimulatory
effect for cell proliferation (p<0.05). Cell proliferation was dose dependent.
The maximum proliferation was at 50 ng mL-1. There were no any significant
differences between concentrations of 1-5 and 10 ng mL-1 (Fig.
1) Compared to control group, concentrations of 10-25 and 50 ng mL-1
of EGF caused a significant increase in cells proliferation. Cell proliferation
was in a dose dependent manner. The maximal proliferation was at 25 ng mL-1.
There was no any significant difference between concentrations 1-2 and 5 ng
mL-1 (Fig. 2). The concentrations of 2.5-5 and
10 IU L-1 of GH significantly increase cell proliferation (p<0.05)
(Fig. 3). The maximum stimulation was at 2.5 IU L-1
and there was no significant difference between concentrations 0/5 and 50 IU
L-1. All groups were compared in Fig. 4.
||Keratinocytes cell response to GH. Concentration 2.5-5 and
10 IU L-1 significantly increased cell number. No significant
effect were observed in the concentration 0/5-50 and 75 IU L-1
maximum proliferation was at 2.5 IU L-1 concentration
||Keratinocytes cell response to GH, PDGF and EGF were compared
to each other. Concentration 50 ng mL-1 PDGF have maximum effect
on keratinocytes cell proliferation
DISCUSSION AND CONCLUSION
The present study has confirmed that the proliferation of the keratinocyte cells can be increased or decreased, depending on the concentration and kind of growth factors. In this study some concentrations of EGF, PDGF-BB and GH significantly increased the proliferation of keratinocytes.
The proliferative effects of growth factors are well known, for example fibroblast
proliferation increased by PDGF and FGF (Lee et al.,
2010; Sun et al., 2004). Study of Wu
et al. (2010) demonstrated that growth factors enhance chemo taxis
at proliferative concentrations. Some growth factors such as, PDGF-BB, EGF,
GH to be very important peptides in the repair and maintenance of the in many
species (Haber et al., 2003). EGF concentrations
10-25 and 50 ng mL-1 have a positive effect while other concentrations
such as 1-2 and 5 ng mL-1 have no stimulatory effect. The results
were similar to results from other researchers and other species. In some species
such as rabbit model, EGF at 1 ng mL-1 increase keratinocyte cells
proliferation (Hongo et al., 1992). After stimulation
with 50 ng mL-1, human keratinocytes increased by 366% (Pancholi
et al., 1998). In this study, rat keratinocytes number increased
after stimulation with 25 ng mL-1 of EGF. These results imply that
the factor EGF in cultured human keratinocytes is useful and can be used in
clinical and may have beneficial effects in clinic. Other study has shown an
increase in healing when using 50 μg mL-1 of EGF (Burling
et al., 2000). Administration 5 μg mL-1 has neither
inflammation nor improvement of wound healing. results have shown keratinocytes
cells are stimulated at the dose between 10-25 and 50 ng mL-1 but
maximum stimulation was 25 ng mL-1. So using these doses within drugs
can have beneficial effects on repair of lesion site on skin. There are two
types of receptors on the epithelial and endothelial cells (High and low affinity).
However, other researchers have shown to have low affinity receptors that are
found in the keratinocyte cells. This result accord with the growth promotion
effects in keratinocyte cells (Hongo et al., 1993).
PDGF-BB similar to EGF significantly increased keratinocytes proliferation
in some concentration. But maximum stimulation of keratinocytes was at a concentration
of 50 ng mL-1. This concentration was more effective than those reported
results of other studies (Hoppenreijs et al., 1993).
They reported that PDGF-BB at concentration of 50 to 100 ng mL-1
maximally promote human keratinocytes proliferation between (150-246%).
The effects of PDGF-BB on keratinocyte cells depending on the type, distribution,
number of receptor and receptor sites. In some species such as human and bovine,
expression of receptors are limited but in rabbits, β subunit are more
numerous, then wound repaired is accelerated by PDGF (Denk
et al., 2003). Results of the study indicated that stimulation of
PDGF-BB at higher concentration would be important.
The addition of PDGF to various wound healing models accelerate wound healing
including re-epithelialization and granulation issue formation (Ansel
et al., 1993; Penha-Goncalves et al.,
1997). Platelets and macrophages that enter into the wound have been proposed
as the largest sources of PDGF in wound healing. These studies have shown that
some concentrations of PDGF accelerate proliferation of keratinocyte hence addition
of these doses of PDGF can help in wound healing. Also cultured keratinocytes
produce high level of PDGF suggesting that this cell is sensitive to exogenous
PDGF (Pastore et al., 2008).
Treatment of ulcers in diabetic mellitus of rats with growth factor PDGF-BB
increase wound repair (Li et al., 2008). PDGF-BB
is produced by many different kind of cells. This production is in responses
to some stimuli, such as damages, another cytokines and some growth factors.
This exerts were done by binding of factors to transmembrane receptors of tyrosine
kinase. After binding, tyrosine was phosphorylated in PDGF receptor. Then some
proteins are produced in intracellular matrix Subsequently, PDGF receptor-mediated
induction of extracellular matrix metalloproteinase production and these factors
are essential for wound healing (Li et al., 2008).
These actions of PDGF demonstrated proliferation of keratinocyte cell in culture
GH stimulates the development of multiple organs, not by cellular hypertrophy
but by cell proliferation. GH acts both directly and indirectly on the specific
cells. It induces the differentiation of progenitor cells and also increases
the level of IGF-I which coincides with the results of this study, where there
is a GH increase cell mitogenic effect. This implies that GH causes promotion
of wound healing. Other research studies demonstrated that fibroblast proliferation
decreases above the concentration of 5 IU L-1 and Li
et al. (2008) have shown that GH has a dose dependency on fibroblasts
(Reuterdahl et al., 1993; Cook
et al., 1999). In the present study, it was found that keratinocyte
proliferation increased between 2.5 to 10 IU L-1. However, the effective
dose of GH on cell proliferation requires further study.
In conclusion, growth factors such as PDGF-BB, GH and EGF facilitate keratinocyte proliferation in vitro. Much remains to be studied in vivo. However, this study provides evidence that PDGF-BB, EGF and GH could be applied topically in clinic to accelerate wound repair and severe lesion.
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