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The Effects of Interleukin-1 on Skin Cells in
Monolayer, Polymer, and Raft Cultures
Thesis Proposal
April 2004
Christopher Folts
Department of Biology
Clarkson University, Potsdam, NY 13699-3980
Mentor: Craig D. Woodworth, Ph.D.
Associate Professsor, Department of Biology
Clarkson University, Potsdam, NY 13699
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The Effects of Interleukin-1 on Skin Cells in Monolayer, Polymer,
and Raft Cultures
Christopher Folts
Department of Biology
Clarkson University, Potsdam, NY 13699-3980
Mentor: Craig D. Woodworth, Ph.D.
Associate Professsor, Department of Biology
Clarkson University, Potsdam, NY 13699
PURPOSE
Skin has several functions, but when its integrity is breached – e.g., by an incurred wound
– and it ceases to be functional, it must be repaired as quickly as possible. The process of wound
repair is a complex orchestration of cellular activity – including an immune response mediated
by pro-inflammatory molecules to wound contraction and fibrosis. The aim of this thesis work is
to assay the effects of an important protein, interleukin-1, which has been shown to initiate a primary inflammatory response and modulate the expression of certain genes in skin cells that will
promote the restoration of normal functionality to the tissue. Interleukin-1 should increase the
expression of those genes involved with an immune response – e.g., NF-B – and those genes
associated with wound contraction – e.g., keratins.
The pupose of this thesis will be to quantify the extent to which the important pro-inflammatory cytokine, interleukin (IL)-1, affects gene expression in skin cells – human keratinocytes
and fibroblasts – grown in monolayer, polymer, and organotypic (raft) cultures. Genetic expression in treated and control populations of skin cells will be assayed by reverse transcription polymerase chain reaction (RT-PCR) and gel electrophoresis, and then quantified with a computer
program and a statistical analysis program that will determine numerically the reletive levels of
gene expression. This research is a component of a larger project whose aim is to provide a
biosynthetic skin replacement tissue for burn victims that will minimize hypertrophic scarring
and restore lost ectodermal appendages. The goal is to produce a degradable polymer-based
scaffolding with incorporated peptides, which will modulate the differentiation and proliferation
of the skin cells seeded on to it, and restore the wound bed to a prototypic, functional state.
BACKGROUND
Skin – the largest organ of the human body – serves multiple purposes: (1) protection
against the assault of injurious UV radiation; (2) retention of necessary fluids, electrolytes, and
proteins and the prevention against dessication; (3) an immunological barrier between the body
and surrounding environment; (4) thermoregulation; and (5) sensory reception. A breach of this
organ’s integrity translates into a breach of its functions; that is to say, an incurred wound
threatens its efficacy and, as a result, jeopardizes the survival of the affected individual. For this
reason, the immediate repair of damaged tissue is required. Frequently in wound repair, function
is placed above form in terms of importance; the physical appearance and function of a repaired
wound is often compromised by the celerity with which it is mended. This compromise has
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generated an immense amount of interest in wound repair and tissue regeneration – specifically
in a rapid mechanism of repair that minimizes the fibrotic or hypertrophic scar, which naturally
results from such processes (Balasubramani et al., 2001; Bello et al., 2001; Gröne, 2002; Janes et
al., 2002; Lee, 2000; Martin, 1997; Young & Heath, 2000).
The process of wound repair and tissue regeneration involves both the epidermis and
dermis – the two layers into which the skin is divided. The former is exposed to the environment
in which the organism lives and the latter attached to subcutaneous tissues by a basement
membrane. Each is stratified into various layers and composed of different cells, each with
diverse functions. The epidermis is traditionally divided into five sublayers – stratum basale,
stratum spinosa, stratum granulosa, stratum lucidum, and stratum cornea – and is primarily
composed of keratinocytes; other cells – i.e., melanocytes, Merkel, and Langerhans cells – are
present, but only constitute two or three percent of epidermal resident cells. The dermis, by
contrast, is divided into three sublayers – papillary dermis, reticular dermis, and hypodermis –
and its resident cell population is primarily composed of fibroblasts; adipocytes are also present
in small numbers (Young & Heath, 2000). The structure and function of skin are maintained by
macromolecular secretions of keratinocytes and fibroblasts, which produce keratin and collagen,
respectively. Keratin forms the cornified upper epidermal layer that protects the lower layers of
skin – and, as a result, the body – from desiccation and the absorption of deleterious UV
radiation. Collagen – acting in conjunction with other ultrastructural molecules, e.g., laminin,
fibronectin, and glycosaminoglycans – forms the extracellular matrix to which cells adhere and
on which the morphology of the tissue as a whole is dependent (Evans et al., 2003; Gröne, 2002;
Holland et al., 2002; Janes et al., 2002; Lee, 2000; Niemann & Watt, 2002; Martin, 1997;
Rombouts et al., 2002; Watt, 2001).
The layers of this tissue are studded with three ectodermal appendages: hair follicles,
sweat glands, and sebaceous glands. Hair follicles or pilosebaceous units are found on all
epidermal surfaces except on the soles of the feet or on the palms of the hand; their functions
include insulation and moderate protection from UVB irradiation. Sweat glands are responsible
for secreting a hypotonic saline solution that controls systemic thermoregulation by evaporative
cooling. Sebaceous glands are found at the follicular bulge region of the hair and secrete sebum,
an oil that covers the cornified epidermis and contributes to its antidesiccatory properties.
(Jankovic & Jankovic, 1998; Rusting, 2001; van Steensen et al., 2001; Young & Heath, 2000).
While epithelial cells of the sebaceous gland (sebocytes) and follicular cells are of keratinocytic
lineage, the origins of sweat glands are less clear (Young & Heath, 2000).
Normal wound repair is a carefully orchestrated process that involves most dermal and
epidermal sublayers, and frequently these ectodermal appendages. It is traditionally divided into
three overlapping phases: (1) inflammation; (2) proliferation; and (3) regeneration (Balasubramani et al., 2001; Harding et al., 2002; Shakespeare, 2001). In the first phase, neutrophils and
eventually macrophages invade the wound bed; these consume any invading bacteria and other
exogenous material. The release of specific cytokines – namely, PDGF, IL-1, and TNF-α – by
macrophages has been shown to facilitate the transistion from the first phase to the second
(Cooper et al., 1994). The second phase is characterized by the rapid proliferation of fibroblasts
and the polymerization of ultrastructural macromolecules, e.g., collagen, fibronectin, and α-actin,
which compose the granulation tissue. This is responsible for the contraction of the wound and
the formation of a fibrotic sheath for temporary protection. Simultaneously, a new extracellular
matrix is produced, a highway by which keratinocytes on the wound’s marginal regions are able
to migrate and ultimately differentiate into necessary cell lineages. The final phases may occur
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over a period of months or years; in it, the wound site returns to a prototypic, functional tissue by
the processeses of neovascularization, degranulation, and by the reduction of cell number – specifically, those cells produced in phase two. This phase may – and frequently does – result in
fibrotic scar tissue (Balasubramani et al., 2001; Eickelberg, 2001; Harding et al., 2002; Harty et
al., 2003; Martin, 1997; Shakespeare, 2001).
Figure 1.
Fig. 1. Interleukin-1 signal transduction pathways (adapted form Auron, 1998).
IL-1 is a cytokine whose transduction pathways has been shown to mediate fibroblast
proliferation, thymocyte and T cell activation, induction of acute-phase proteins, and both local
and systemic inflammatory reactions. There are two IL-1 receptors (IL-1R); the type I receptor
(IL-1RI; CD121a) transduces a signal, whereas the type II receptor (IL-1RII) binds IL-1, but due
to its shorter cytoplasmic domain (approximately 20 residues), it is unable to transduce a signal. ;
It has been termed a “dummy” or “decoy” receptor. Since IL-1 has a high affinity for IL-1RII,
the receptor competes with IL-1RI. The type I IL-1 receptor (IL-1RI) is expressed by T cells,
thymocytes, endothelial cells, fibroblasts, keratinocytes, and other cells. The IL-1R signal transduction system is extremely efficient, requiring fewer than ten ligated receptors per cell to induce
a notable response. Ligation leads to formation of a complex with the accessory protein
(IL1RacP) and the binding of a serine/threonine kinase (IRAK). This leads to activation of different pathways (MAPk cascades, PI(3)K pathways, and NF-B, which is shown in Fig. 1) and
other transcription factors. Each of these pathways is important for the generation of an inflammatory response (Sims, 2002; Uchi et al., 2000; Dinarello, 2003; Dunne & O’Neill, 2003).
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The process of tissue repair becomes complicated when a wound is thermally induced,
i.e., a burn, especially if the affected area is large. One complication with skin replacements in
burn and lesion patients is the absence of the three epidermal appendages. Cells of epidermal
and dermal layers, i.e., keratinocytes and fibroblasts, can be replaced, but the re-installation of
these appendages has been rarely accomplished in vivo. Methods of replacement are varied. For
small and superficial burns (first-degree or epidermal burns), autologous keratinocyte samples
can be excised from a donor site, cultured, and placed in the denuded wound. This is not
effective or practical for larger or deeper burns (second- or third-degree) for several reasons.
First and foremost, a larger burn requires a larger grafted sample from the donor site; this
increases the risk of infection and exposes the patient to excessive pain and discomfort. Second,
deeper burns that involve the denudement of both epidermal and dermal layers cannot be as
easily repaired or replaced as with superficial burns, i.e., a combined dermis-epidermis cannot be
cultured; in most cases, the dermis does not attach to subcutaneous membranes and as a result
the engrafted tissue ceases to be a viable replacement.
For these reasons, artificial tissues have been developed. They are varied – using
autogenic or allogenic cell samples, or both – cultured on various substrates. Essentially, these
engineered tissues are a polymeric scaffolding that tries to mimic the complex extracellular
matrix of the dermis, which is seeded with fibroblasts, keratinocytes, and factors that aid (or
attempt to aid) in the processes of re-epithelialization, revascularization, and fibrosis. (Bello et
al., 2001; Bello & Fallabella, 2001; Bianco & Robey, 2001; Bottaro et al., 2002; Boyce, 2001;
Langer, 2000; Lee, 2000; Machens et al., 2000; Schultz et al., 2000; Shakespeare, 2001). The
science of drug delivery by degradable polymeric systems has been modified to form the
necessary base analogs or substrates for these new tissue models. Some polymers being assayed
include poly(lactide-co-glycolide), tyrosine-derived polycarbonates, and poly(ethylene glycol)
(Agrawal & Ray, 2001; Bourke & Kohn, 2003; Holmes, 2002; Kost & Langer, 2001; Langer,
1999; Langer, 1995; Marler et al., 1998; Murphy et al., 2000; Pins et al., 2000; Shastri et al.,
2003; Tangpasuthadol et al., 2000; Yu & Kohn, 1999; Yu et al., 1999).
The lab of Dr. Anja Meuller intends to provide a degradable, enzymatically synthesized
polymer scaffolding and the lab of Dr. Craig Woodworth will provide biological/genetic data.
The union of the two will hopefully be a replacement tissue that avoids the complications of
current models. The long-term aims of this project include (i) to provide a degradable, polymeric scaffolding seeded with chemokines or cytokines needed for modulated keratinocyte differentiation into the desired ectodermal appendages (e.g., hair follicles); and (ii) to provide a
replacement or subsitute tissue that reduces hypertrophic scarring and the associated morbidity.
The aim of this thesis is to assay the activity of a key pro-inflammatory cytokine – interleukin
(IL)-1 – and its effects on gene expression in keratinocytes and fibroblasts in monolayer, polymer, and raft cultures.
PROPOSED WORK
The research will be broken into four stages. First, skin cell lines must be established
from human neonatal foreskin grafts and preserved for future use. Second, experimental populations of keratinocytes and fibroblasts in the three cultures will be treated with a determined
concentration of lyophylized IL-1 protein; control (untreated) populations of the same cell lines
in identical culture conditions will be simultaneously maintained. Third, the RNA of each experimental and control populations will be isolated, reverse transcribed into cDNA, amplified by
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polymerase chain reaction (PCR), and tested with a number of chosen genes using gel electrophoresis. Third, the data will be semi-quantitatively analyzed using statistical programs to determine the extent to which IL-1 affects the expression of pro-inflammatory genes. The final stage
will be an extenion of the third, in which PCR will be repeated for selected inflammatory genes
and with other genes associated with wound repair, e.g., those responsible for wound contraction
and fibrosis.
Stage 1: Establishment of Cell Lines and Cultures
Human fibroblasts and keratinocytes will be cultured from neonatal foreskin grafts and
cryopreserved in liquid nitrogen for future use. Fibroblasts are to be cultured in Dulbrecco’s
modified Eagle’s medium (DMEM)/HAM’s F-12 and supplemented with 50 mL of fetal bovine
serum, 5 mL penicillin/streptomycin (Gibco), and 2 mL fungizone (Gibco) per 500 mL of media.
Keratinocytes are to be cultured in keratinocyte serum-free media supplemented (KSFM) with 5
mL penicillin/streptomycin (Gibco) and 2 mL fungizone (Gibco) per 500 mL of media. The first
population of low-passge skin cells will be cultured to confluent monolayers in 100mm culture
plates (Falcon) at 37°C with a 5% CO2 injection. The polymer populations will be seeded onto
100mm culture plates, which will have been spincoated with a suitable polymer that has been
shown to not be cytotoxic (e.g., methylmethacrylate), and grown to confluency in a manner identical to the monolayer cultures. The third population of skin cells will be grown into stratified,
three-dimensional organotypic (raft) cultures as previously described by Visalli et al. (1997).
Stage 2: Treatment and RNA Purification
One-half of each cell culture (monolayer, polymer, and raft) will be treated with a nanomolar solution of lyophylized interleukin-1 in respective medium – i.e., KSFM for keratinocytes
and DMEM for fibroblasts – for a determined time period (24–36 hours). The other half of
culture plates, the controls, will receive equal amounts of respective medium without IL-1 for the
same time period. After this period of time, mRNA will be extracted from all experimental and
control plates. For each sample set, the plates will be rinsed with phosphate-buffered saline
(PBS) and then thoroughly drained. To one plate, 8 mL of Trizol is to be added; cells are then
distrupted and removed from the plate’s bottom with a cell scraper. The trizol and cell lysates
will be scraped into the next plate of the same sample set. This will be repeated until each plate
had been scraped; the resultant trizol-lysate mixture is to be stored in a 15-mL conical tube and
preserved at –70°C until RNA purification.
To purify the RNA, the high molecular weight DNA in each sample tube must be sheared
with a 21.5 gauge needle; the sample is then to be poured in to a 15-mL round bottom
polypropylene centrifuge tube to which 1.6 mL of chloroform will be added. Each sample will
be shaken and then placed in a centrifuge at 12 000 xg and 8ºC for ten minutes. After
centrifugation, the aqueous phase containing the RNA is to be removed with a sterile pipette and
transferred into a new centrifuge tube; to this, 4 mL of isopropanol will be added. The samples
will be allowed to incubate at room temperature for ten minutes before being placed in the
centrifuge under the same conditions for ten minutes. After a second centrifugation, the
supernatant will be poured off and the resulting pellet will be allowed to dry. This is to be later
resuspended in 200 μL of sterile distilled water. The concentration of RNA will be measured
with a BioRad SmartSpec 3000 at a wavelength of 260 nm; from these readings, the amount of
RNA in each sample will be calculated; 10 μg aliquots of each sample are to be frozen at –70°C.
The samples of RNA will be fractionated on a formaldehyde-containing agarose gel and
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photographed with a transiluminator (Spectroline, Model TR-302) to ensure successful and
equivalent aliquotting.
Stage 3: Reverse Transcription, PCR, and Gel Electrophoresis
Complimentary DNA (cDNA) will be synthesized from the sample mRNA by reverse
transciption, as described in the Invitrogen protocol included in the RT-PCR kit, using 5 μg RNA
aliquots. The samples are to be then amplified using polymerase chain reaction (PCR); 2 μL of
each sample will be added to 2 μL of both sense and antisense primers (to be determined) in a
0.2-mL thin-walled PCR tube. To this, 34 μL of PCR Platinum Supermix are to be added. Each
40 μL reaction will be then placed in a thermocycler and exposed to 25-30 cycles of amplification, which will ensure that the amplification will be in the linear range of the PCR curve.
After PCR, 10 μL of 6X loading buffer – 0.25% Bromoøblue, 0.25% xylene cyanol, 15% Ficoll
– will be added to better visualize the band locations in the gel. About 20 μL of each reaction are
to be loaded into a well in a 1.4% agarose gel with 5 μL ethidium bromide per 100 mL of gel
added, poured to a depth of 4-5 mm. The gels will run in 1X TAE buffer for 45 to 60 minutes at
60V; thereafter, each will be photographed with a transiluminator. The intensities of the bands
are to be analyzed using the Kodak 1D program, and compared with those of β-Actin, a housekeeping gene whose expression should not be affected by treatment with interleukin-1.
Stage 4: Extended Analysis with Additional Genes
The effect of expression of genes not involved with an immune response in skin cells will
also be evaluated, using the methods employed in Stage 3. These genes include those that are
responsible for cell morphology and mediate wound contraction, e.g., actin, fibronectin, and keratins. The aim of this stage is to determine whether or not the given treatment of interleukin-1
will modulate the expression of these genes, which are not directly involved with cytokine-mediated immune responses.
PRELIMINARY DATA
The research work is currently in Stage 1; cell lines have been established from foreskin
grafts and cryopreserved for future use. This stage is the most time-consuming because these
skin cells are fastidious; an optimized set of isolation and culture conditions were needed to maximize their growth rates and survival. As a result of this, the research has remained in this stage
for an extended length of time that had not been anticipated. As one may suspect, no data has
been generated by the later Stages.
However, the tools needed to complete these Stages have been mastered over the course
of the past 12 months. During the past summer, the effects of another cytokine, transforming
growth factor (TGF)-, on gene expression in skin cells in monolayer cultures were studied. The
research report, which resulted from last summer’s laboratory work, demonstrates proficiency in
various cell cultures, RNA extraction and purifcation, reverse transcription, gene amplification
using polymerase chain reaction, and gel electrophoretic analysis. The results are reproduced
here:
Genotypic Expression
The analyses of genotypic expression fall into two categories: (1) those that demonstrate which
genes are expressed in which cell type (i.e., fibroblasts, keratinocytes, both, or neither); and (2) those that
demonstrate the effects of TGF-β1 on treated samples in comparison with control populations.
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Figure 2.
HKC
–
–
+
HFB
–
+
HKC
–
+
–
+
+
HFB
–
+
–
+
–
+
200
A.
β-Actin
B.
γ-Actin
C.
Fibronectin
0
1
2
3
4
5
6
7
8
200
0
1
2
3
4
5
6
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8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
200
0
200
Vinculin
D.
0
Fig. 2. Control (–) and treated (+) human keratinocytes (HKC) and fibroblasts (HFB):
treated populations received a 3-ng·ml–1 solution of TGF-β1 for 24 hours. Relative intensities per sample on the left, gel electrophoresis results on the right. (A) β-Actin at 25
cycles; (B) γ-Actin at 30 cycles;(C) fibronectin at 30 cycles; (D) vinculin at 30 cycles.
The low intensity of the treated HFB (lane 6) in (B), (C), and (D) is attributed to a low
concentration of cDNA in this sample at the time of analysis.
The standard housekeeping gene used for reference was β-Actin, as it is expressed in both
normal keratinocytes and fibroblasts; in addition to constuitive expression, this gene appears to
be unaffected by TGF-β1 – at least at the low concentrations that were used. Ideally, the variance
in expression between control/experimental samples and keratinocyte/fibroblast samples should
be negligible. Statistically, the housekeeping gene should have the smallest standard deviation.
Figure 4 compares the relative intensity means – as measured by Kodak 1D – and standard deviation for each assayed gene; as expected, β-Actin has the smallest variance (± 14.3).
Figure 3.
HKC
–
+
HFB
–
+
–
+
HKC
–
–
+
+
HFB
–
+
–
+
–
+
200
A.
IL-1α
B.
IL-1β
C.
Keratin-1
D.
Keratin-10
E.
IL-6
0
1
2
3
4
5
6
7
8
200
0
1
2
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8
1
2
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6
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3
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7
8
200
0
200
0
1
2
200
0
1
2
Fig. 3. Control (–) and treated (+) human keratinocytes (HKC) and fibroblasts (HFB):
treated populations received a 3-ng·ml–1 solution of TGF-β1 for 24 hours. Relative intensities per sample on the left, gel electrophoresis results on the right. (A) IL-1α at 30
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cycles; (B) IL-1β at 30 cycles;(C) K-1 at 30 cycles; (D) K-10 at 30 cycles; (E) IL-6 at 30
cycles). The low intensity of the treated HFB (lane 6) in (E) is attributed to a low concentration of cDNA in this sample at the time of analysis.
Genes associated with ultrastructural proteins did not appear to be affected by the concentration of TGF-β1, as seen in Fig. 2 (B–D) and Fig. 3 (C–D), after 30 cycles of PCR. Gene
expression in each sample is of comparable intensity. (The low intensity of the first treated
fibroblast sample (lane 6) in Fig. 2 (B–D) is be attributed to a low concentration of cDNA at the
time of analysis, which could be the result of poor storage and subsequent evaporation. PCR
analysis using β-Actin made prior those in Fig. 2 demonstrated a cDNA concentration that was
comparable to its untreated counterpart.) The six genes of Fig. 2 and Fig. 3 yielded mean relative intensities with relatively small standard deviations, as seen in Fig. 4, ranging from 96.42 ±
14.3 (β-Actin) to 141.2 ± 30.4 (fibronectin).
Genes involved with inflammation – those belonging to the interleukin family – showed
greater variation between keratinocytes and fibroblasts. Interleukin (IL)-1α and -1β were more
expressed in keratinocytes than fibroblasts after both 25 (not shown) and 30 cycles of PCR (Fig.
3, A–B); statistical analyses support this qualitative data (Fig. 4), which yielded mean relative
intensities with high standard deviations ranging from 114.8 ± 57.0 (IL-1β) to 93.51 ± 59.5 (IL1α). Keratin (K)-1 and -10 – although associated with epidermal differentiation and not with
immunological reponses – showed a similar result after 30 cycles of PCR (Fig. 3, C–D). DNA
bands were only detected in keratinocyte lanes and not in fibroblasts, thus indicating an absence
of expression. In comparison, interleukin (IL)-6 showed upregulation in fibroblasts and not in
keratinocyte samples (Fig. 3E).
Figure 4.
Mean Relative Intensisty With Standard Deviation Per Assayed Gene
200
Relative Intensity
150
100
50
0
β-Actin
γ-Actin
Fibronectin
Vinculin
IL-1α
IL-1β
Keratin-1
Keratin-10
IL-6
Fig. 4. Graph of mean relative intensities ± standard deviations for studied genes (β-Actin, γActin, fibronectin, etc.); analyses completed with the Kodak 1D program.
The two keratin genes (1 and 10) also demonstrate what appears to be a response to TGFβ1 with the given concentration, treatment time, and number of PCR cycles in the keratinocytes
(Fig. 3, C–D). Keratin-1, specifically, illustrates this: the relative intensities of untreated sam-
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ples were 99.58 and 116.34, and the treated samples yielded relative intensities of 0.00 and
76.71, respectively – showing at least a 34 percent decrease in expression in the second sample.
The results from this research demonstrate a proficieny in the methods and tools needed
to complete this thesis.
PROPOSED TIME TABLE
Summer 2004: collect more primary research sources while away from lab
September 2004: choose an appropriate polymer for the polymer cultures, one that is
sufficiently thin such that cells may be seen with phase-contrast microscopy,
degradable, and not cytotoxic. Genes will be chosen for PCR analysis, and new
gene primers will be ordered if necessary.
October 2004: Stage 2 – Treatment with IL-1 in the three culture types; while cells are
growing, followed by Stage 3 – RNA extraction/purification and reverse transcription into cDNA, which will be preserved.
October/November 2004: Stage 3, continued – PCR and gel electrophoresis.
November 2004: Stage 4 – PCR and gel electrophoresis with genes not directly associated with an immunoresponse.
December 2004: compilation and analysis of data; search for additional literature.
Preliminary draft of thesis should be written by mid-month
January/February 2005: revision of thesis; a final draft should be completed by mid-February.
March 2005: thesis oral presentation.
April 2005: final thesis is due.
PROPOSAL SUMMARY
Skin has several functions, but when its integrity is breached – e.g., by an incurred wound
– and it ceases to be functional, it must be repaired as quickly as possible. The process of wound
repair is a complex orchestration of cellular activity – including an immune response mediated
by pro-inflammatory molecules to wound contraction and fibrosis. The aim of this thesis work is
to assay the effects of an important protein, interleukin-1, which has been shown to initiate a primary inflammatory response and modulate the expression of certain genes in skin cells that will
promote the restoration of normal functionality to the tissue. Three types of cell cultures commonly used in a laboratory setting will be treated with interleukin-1 and the genetic effects will
be quantified by a process known as reverse transcription polymerase chain reaction – in which
the mRNA corresponding to activated genes can be amplified. These amplified gene products
can be later visualized with gel electrophoresis. Interleukin-1 should increase the expression of
those genes involved with an immune response – e.g., NF-B – and those genes associated with
wound contraction – e.g., keratins.
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