Expression Of Satb1 And Satb2 At Different Stages Biology Essay

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The skin is the largest organ in the body, forming part of the integument system. The skin is about 2mm thick Martini, 2006. The skin diversifier's in function these including immunologic, endocrine, metabolic, psycho- social, and neuro immunologic(Chuong. CM et al., 2002). However, the most important function of the skin is to protect and shield the body against heat, light, pathogens and injury as well as infection(institute)The skin is an effective barrier(Chuong. CM et al., 2002), and is the bodies first line of defence (Martini, 2006) It can develop an adaptive immune response to remove so pathogens can be removed efficiently before harming any other part of the body and protect us from disease. The skin can perform its function, by consisting of 3 important layers. The epidermis, dermis, and the subcutaneous layer. The skin can also control the body temperature through insulation or evaporating. It can synthesise nutrients, vitamin D3, and store lipids in the dermis.

The epidermis is mainly dominated by keratinocytes. The epidermis layer is made up of five sub layers that constantly work together to continually re-create the surface of the skin(institute) and provides mechanical protection. The epidermis does not contain blood vessels. The dermis is made of the papillary layer and the reticular layer.

The stratum germinativum is the inner most epidermal layer(Martini, 2006 ) It contains stem cells which are undifferentiated and then proliferate. The stratum germinativum forms epidermal ridges and after mitotic division new cells migrate to the top of the surface and form mature cells called keratinocytes.

The stratum spinosum also contains stem cells which then divide into daughter cells. It contains 8 - 10 layers of keratinocytes. In this layer cells begin to synthesise keratin.

In the stratum granulosum, most cells have stop dividing and have started to make large number of protein Keratin. The stratum granulosum is made of 3 - 5layers of keratinocytes (Martini, 2006). Keratin is a tough, durable protein which is a component present in hair and nails.

Stratum Lucidum is densely packed with keratin (Martini, 2006) This layer covers the stratum granulosum(Martini, 2006)and forms the thickest part present in the palms and soles.

The stratum corneum is made out of both thick and thin skin. It consists of 15 - 30 layers of keratinocytes (Martini, 2006) and is made of dead flat skin cells. Keratinisation occurs and it takes cells 15 - 30 days for cells to move from the stratum germinativium to the stratum corneum (Martini, 2006)

There are many types of keratin genes that are expressed in the skin epithelia. Progenitors basal keratinocytes express genes such as K5- K14, and K15.(Hopkins, 2008-2010)When differentiation takes place, keratinocytes express different genes K1-K10 and switch off K4- K14(Hopkins, 2008-2010). Different genes have a different role in different areas. K6, K16 and K17 are generally activated in the expense of K1- K10. This usually occurs when the epidermis undergoes environmental changes i.e viral infection, tissue injury, UV exposure.

The dermis lies between the epidermis and the subcutaneous layer and has two major components; the papillary layer and the reticular layer. The papillary layer of the dermis, consists of aerolar tissue which contain capillaries, lymphatic and sensory neurons that supply to the surface of the skin(Martini, 2006).It supplies nutrients to selective layers and regulates temperature(institute). The papillary layer functions by an extensive vascular system by constriction and expansion. This can regulate the amount blood that flows through the skin(institute). However the abundance of sensory receptors in the skin can result promote infection and inflammation.

An interwoven meshwork of dense irregular connective tissue is also known as the reticular layer. This is deeper down the papillary layer and produces collagen and elastic fibres(Martini, 2006)It strengthens the skin, providing structure and elasticity(institute)

1.1 Hair Morphology in relation to skin and wound healing

Hairs are non- living structures produced in organs called hair follicles(Martini, 2006). Each hair follicle opens onto the surface of the epidermis but extends deep into the dermis and subcutaneous layer. The follicle is wrapped in dense connective tissue sheath and a root hair plexus of sensory nerve surrounds it. This sensitivity provides an early warning system that may help prevent injury.

Hair follicle contains stem cells, which progenise and contribute to wound healing and regeneration of the epidermis.

If a skin wound has a hair follicle intact, this makes a large contribution to the healed epidermis from the follicle remnants. They act as normal cut epidermal wound edges and hours later epidermal cells grow after migration.(Maibach. Howard and Rovee. David, 1972)If the skin wound is deeper in the dermis, therefore no remnants of hair follicle remain, the repairing epithelium does not regenerate hair, and receives instructive signals that govern the positions and types of other cutaneous appendages that will differentiate from the overlying epidermis(Maibach. Howard and Rovee. David, 1972).

1.2 Wound healing

Wound healing is the repair process of restoring cellular structures and tissue layers.(Michael Mercandetti, 1994-2009).The epidermis and dermis no longer exist in a state of equilibrium. Skin can regenerate from injury because stem cells persist in the epithelial and connective tissue components. Germinative cell divisions restore lost epidermal cells, and mesenchymal cell divisions replace lost dermal cells(Martini, 2006). Under normal state, the epidermis usually serves a barrier to retain water and prevent infection, alongside dendritic and langerhan cells. However, in response to trauma the epidermis depicts a full thickness, and keratinocytes increase proliferation and cytokines are released(Segre, May 2006; Werner. S and Grose. R, 2003). Epidermal appendages such as hair and glands, which project deep in the deep dermal layer become lost at the site of damage, however do not regenerate and result in a connective tissue scar once the wound has healed and the collagen matrix has been re- constructed.

Because the skin acts a protective barrier, any break must be efficiently mended. Important phases include the inflammatory phase, proliferative phase, and the remodelling phase. (Michael Mercandetti, 1994-2009)Haemostasis is another process and has a response to vasoconstriction, platelet aggregation, and the clotting cascade. The repair process is instigated by the release of growth factors, cytokines and low molecular weight compounds from the serum of injured blood vessels and from deregulating platelets(Werner. S and Grose. R, 2003)Important growth factors in the healing process include, platelet derived growth factor, Fibroblast growth factors, and granulocyte- macrophage colony stimulating factor and epidermal growth factors.(Werner. S and Grose. R, 2003)These factors affect migration, mitosis and differentiation. Since the lesions extend into the dermis, epithelial migration must occur to re-establish an epidermal continuum. In wound healing mitotic response ( cell divison)and differentiation of a new stratum corneum is important(Maibach. Howard and Rovee. David, 1972) .When blood vessels become ruptured, the formation of a blood clot acts as a temporary shield from cross link fibrin. This provides a barrier against invading micro- organisms as well as serving as a matrix for invading cells and as a reservoir of growth factors and cytokines.(Werner. S and Grose. R, 2003)Inflammatory cells such as neutrophils, lymphocytes and monocytes invade the cell by a variety of chemotactic signals or mitogens(Maibach. Howard and Rovee. David, 1972; Werner. S and Grose. R, 2003) The neutrophils are a source of pro - inflammatory cytokines and activate local fibroblasts and keratinocytes (Paul Martin, 1997)These cells initiate tissue movements of re- epithelialization, and connective tissue contraction, and stimulate the characteristics wound angiogenesis response(Paul Martin, 1997). A new granulation tissue is then formed. EGF ( epidermal growth factor) HB- EGF(Heparin Binding growth factor), and TGF- a (transforming growth factor) all act as a ligand for the EGF receptor. These are key regulators in keratinocytes proliferation at a wound edge(Paul Martin, 1997)

Table 1 Shows growth factor signals at the wound site

http://www.sciencemag.org

There are different types of wound healing in the skin. There are superficial wounds, primary union and secondary union. Primary union is healing by first intention. This is when healing proceeds without minimal delay, and the surface of the wound dehydrates and forms a scab. Wound tissue hydration is important as it can alter the course of all phases of epidermal healing(Maibach. Howard and Rovee. David, 1972). When hydration occur with occlusion, migration of epithelial cells occur as the same plane as an un involved epidermis thereby leaving no scar(Maibach. Howard and Rovee. David, 1972). Secondary union by secondary intention is when wound margins are not apposed due to tissue loss and therefore it is slow and involves increased scaring. Delayed wound healing can occur by many systemic factors e.g. age, irradiation, steroids, vitamin deficiency etc.

Figure 2 Illustrating healing of a skin wound

SATB1 and SATB2 are implied to be transcription factors and are expressed in epidermal keratinocytes. Therefore they will have an effect up regulating the migrating keratinocytes, enabling them to crawl beyond the cut basal lamina in wound healing skin.

Http://www.sciencemag.org

1.3 Expression of SATB1 and SATB2

SATB1 is a special AT- Rich binding protein. It is a global chromatin organisers and transcription factor(Sanjeev Galande et al., 2007). Studies have shown they are a key aspect in integrating higher order chromatin architecture with gene regulation(Sanjeev Galande et al., 2007). SATB1 are found pre- dominantly in undifferentiated thymocytes(Dag Yasui, 2002). SATB1 forms a three dimensional network structure In the thymocytes nuclei recruiting histone deacetylase in this network(Britanova O et al., 2005)Satb1 has also been postulated as a novel type of regulator gene by folding chromatin into loop domains;(Dag Yasui, 2002) tethering a MARs to nuclear matrix at fixed distance(Sanjeev Galande et al., 2007).MARs has been implicated to do with the regulation of transcription by changing the eukaryotic chromosomes(Sanjeev Galande et al., 2007). SATB1 was shown to regulate genes in MARs which resulted in the formation of heterochromatin.

SATB1 modifies target gene loci upregulating metastasis associated genes whilst down regulating tumour suppressor genes (Hye-Jung Han1, 2008). SATB1 also reprograms chromatin organisation and promotes breast tumours and metastasis. SATB1 is also expressed in epidermal keratinocytes and therefore will have an effect on wounded skin. SATB1 regulates predominantly nanog (Savarese F et al., 2009) Nanog is a key regulator of embryonic stem cells pluripotency and the expression levels correlate with the probability of self- renewal versus differentiation.(Savarese F et al., 2009)Nanog has a role in keeping embryonic stem cells in pluripotent state. SATB1 is required for the differentiation of pluripotent cells and negatively regulates Nanog (Savarese F et al., 2009). Pluripotent stem cells can give rise to various cell lineages; mixed myeloid progenitors, Erythroid progenitors, lymphoid stem cells etc. The cell lines that arise from this are red cells, mono-cytes, neutrophils, lymphocytres etc. These cells play a key role in inflammation, therefore will be present in the basal lamina in the wounded skin. There is a high expression of SATB1 in undifferentiated tissue, than differentiated tissue. Proving to us satb1 has a role in differentiation. SATB1 also regulates the expression of Bcl2.

SATB2 is a close homologue of SATB1 showing 61% amino acid homology to satb1 (Britanova O et al., 2005)SATB2 contains two SUMO acceptors sites that are not present in SATB1.(Savarese F et al., 2009)This changes the transcriptional activation of the SATB1B target gene. SATB2 has also been demonstrated to modulate immunoglobulin gene expression in neuronal differentiation, and is associated with cleft plate and other craniofacial dysmophologies(Neil Patani, July 30th 2009 ).SATB2 can enhance gene expression and that the protein can be modified by somoylation. (Britanova O et al., 2005)

Aims

The aim of this project is the analysis of the expression of SATB1 and SATB2 at different stages of wound healing process using immuno- flourescent technique.

2.0 Materials

See appendix

2.1 Cryosection of mouse skin

Skin samples were obtained from the back of female CH3 mouse skin of 8-9 weeks old. Tissues samples were taken at the teleogen stage.

The tissue samples for single wounds were obtained using a biopsy punch; removing wounds of 5mm thick. Different stages of wound healing were taken e.g. day 1 of wound healing, day 3, and day 5 of wound healing. This allowed us to check the difference in Satb1 expression at different stages of wound healing. The samples were then fixed with OCT (Raymond A Lamb, East Sussex, U.K.) and then kept frozen in nitrogen at -80 ÌŠc. 9- 10 µm sections were cut using a Cryostat machine and then mounted onto a microscopic slide.

Solutions used during the experiment.

PBS and washing solution was made before hand. See appendix for further details.

2.2 Immuno histochemistry

Immuno- histochemistry (IHC) is a technique which usually refers in localising antigens (e.g.) proteins. It is the process when an antigen binds specifically to an antigen in biological tissue. This technique is widely used to detect abnormal cells such as cancer. It can be visualised using markers such as conjugated enzymes peroxidases; or the antibody can be tagged to a flourophore.

2.3 Immunoflourescence technique

This technique was used to find out the expression of Satb1 in mouse skin during wound healing. The slides with cryostat sections were stored at 80 ÌŠc until needed.

Fixation is a chemical process which ceases any ongoing biochemical reactions and also allows any biological tissues to be preserved from decay. It also protects the sample from any extrinsic damage; making it less palatable as well as increasing the strength and stability. It can target and prevent antibody binding.

Before staining, the slides were taken out the freezer and allowed to dry at room temperature for 15minutes.

Sections were not allowed longer than 15minutes air dry before fixation to preserve signalling epitopes.

Whilst the slides were left to dry a PAP pen (Vector Laboratories, H-4000) was used to encircle the tissue section on each slide.

The PAP pen is a special marking pen that provides a thin film- like green tinged hydrophobic barrier which repels from a mass of water, when a circle is drawn around a specimen on a slide.

During the drying process, Paraformaldehyde (PFA 37 % formaldehyde- Sigma, F8775) was used to fix the samples. PFA was placed in copplin jars covering the slides for 10minutes at room temperature. Formalin uses cross linking, and is used for substances such as RNA, DNA, and proteins. The antibody binds to the antigen and alters the 3D tertiary structure which is usually recognised by antibodies therefore affecting the solution.

The slides were then rinsed in PBS (washing solution) once for 10minutes. The blocking solution was made from 200µl 5% BSA/PBS (Sigma A2153), 100µl 1% triton x 100 - (Sigma T8787), 20µl Saponin (Sigma 84510), and 680 µl of PBS. The primary antibody was then diluted at a 1:100 dilution in the blocking solution. Therefore 5µl of SATB1 (L745) rabbit antibody (Cell signaling technology, 3650) was used. The solution was then centrifuged at 1300 rpm before using. The tissue sections on the slides were completely covered with 250 µl of solution. The slides were then placed in a blocking tray with PBS solution and incubated overnight at 4 ÌŠc

The next day, the slides were taken out of the blocking tray and tapped gently removing any primary antibody. The slides were then placed in copplin jars which had 1x PBS solution inside. The slides were then rinsed in PBS in the copplin jars 3 times for 5minute. The TRITC/FIT conjugated secondary antibody GoatαRabbit (Cy3® goat anti-rabbit IgG (H+L) - Invitrogen, A10520) was diluted in blocking solution at a concentration of 1:200 and then placed onto the slides. After this the sections were incubated at 37ÌŠÌŠÌŠÌŠoc for 2 hour. The cryosections were then washed in PBS 3 times for 10minutes.Changing the solution of the PBS with new PBS at each wash.

Each slide was then taken out the copplin jar once at a time, and wiped with tissue. The slides were mounted with Vectashield mounting medium with DAPI - (Vector Laboratories, H-1000). DAPI is a blue fluorescent that binds specifically to the nucleus; making it an ideal counter stain for the immunoflourescence microscope. DAPI has an excitation maximum at 345nm and an emission maximum at 455nm (scientific, 2010) . A cover slip was placed over the tissue samples covered in DAPI. Nail varnish was used around the cover slip to hold the slide in place. The slides were kept at 4 ÌŠc flat and in the fridge protected from light until they were needed also to prevent any degradation.

2.4 Image Preparation

All the slides were examined using a conofocal microscope (Zeiss LSM 510M). The sections were viewed under the TRITC filter and the DAPI filter. The images were than merged using a programme called Image J.

3.0 Results

Key

DAPI

Figure 3.Negative control showing normal skin during anagen stage. (a)No goat- anti rabbit TRITC labelled secondary antibody present.Technique used DAPI immunoflourescence staining(x40).

a)Anagen Epidermis Neg

H:\STUDENTS2010\SATB1.mdb\wound healing\C2-dp 12\wound healing teleogen\C2-dp12satb1 merged.jpg

Epidermal basal layer(a)

Key

SATB1

DAPI

Positive SATB1 expression

Dermis

H:\STUDENTS2010\SATB1.mdb\wound healing\C2-dp 12\C2-dp12satb1-2 merged.jpg

(b)

Basal layer

Outer root sheath

Key

SATB1

DAPI

Figure 4.SATB1 expression in normal skin during telogen stage. (a) SATB1 expression in the epidermis. (b) SATB1 expression in the hair follicle and dermis. Technique used SATB1 (TRITC) immunoflorescence merged with DAPI immunoflourescence staining(x40).

(a)H:\STUDENTS2010\SATB1.mdb\wound healing\wound day satb1\woundday 1\C2-woundday1-2 merged.jpg

Epidermis

Key

SATB1

DAPI

H:\STUDENTS2010\SATB1.mdb\wound healing\wound day satb1\C1-woundday1satb1 merged.jpg

(b)

Epidermis

Wound debris

Key

SATB1

DAPI

Figure 5.SATB1 expression in the skin on day 1 after wounding. (a)SATB1 expression in the epidermis distant to the wound. (b) SATB1 expression in the epidermis close to the wound. Technique used SATB1(TRITC) immunoflorescence merged with DAPI immunoflourescence staining(x40).

H:\STUDENTS2010\SATB1.mdb\wound healing\wound day3\woundhealing day3satb1\C2-woundday3satb1-2 merged.jpg

(a)

Figure 6.SATB1 expression in the skin on day 3 after wounding. (a) SATB1 expression in the epidermis distant to the wound. (b) SATB1 expression in the epidermis close to the wound. Technique used SATB1(TRITC) immunoflorescence merged with DAPI immunoflourescence staining(x40).

Key

SATB1

DAPI

Wound epithelium

Epidermis Key H:\STUDENTS2010\SATB1.mdb\wound healing\wound day3\C1-woundday3 merged.jpg

SATB1

DAPI

Dermis

(b)

Wound formation

(b)

(a) Key

SATB1

DAPI

Key H:\STUDENTS2010\SATB1.mdb\wound healing\wound healing day5satb1\C2-woundday5satb1 merged.jpg

SATB1

DAPI

EpidermisH:\STUDENTS2010\SATB1.mdb\wound healing\wound healing day5satb1\wound day 5\C1-woundday5-merged.jpg

Figure 7.SATB1 expression in the skin on day 5 after wounding. (a)SATB1 expression in the epidermis distant to the wound. (b) SATB1 expression in the epidermis close to the wound. Technique used SATB1 (TRITC) immunoflorescence merged with DAPI immunoflourescence staining(x40).

Wound formation

Figure 7.SATB1 expression in the skin on day 5 after wounding. (a)SATB1 expression in the epidermis distant to the wound. (b) SATB1 expression in the epidermis close to the wound. Technique used SATB1 (TRITC) immunoflorescence merged with DAPI immunoflourescence staining(x40).

3. 1 Results write up

To achieve the goals of the project, skin cryosections were immuno stained with primary antibody against SATB1 and goat- anti - rabbit TRITC labelled secondary antibody.

Figure 3(a) shows normal epidermis of the skin at the anagen stage. This was used as a negative control so no addition of the secondary antibody - goat - anti- rabbit TRITC labelled was used. The DAPI stained positive for cell nuclei.

Figure 4(a) shows normal epidermis of the skin at the telogen stage. Here SATB1 is stained positive and the merged colour showing DAPI staining overlapping SATB1/TRITC shows specific nuclei staining in the basal layer. The weak red fluorescence above that SATB1 / TRITC stained cell nuclei which showed non- specific staining above the basal layer. Figure 4(b) shows SATB1 expression in the hair follicle and the dermis. SATB1 is positively expressed in the basal layer shown by the merging of colours. However the staining is limited to the basal layer, as there is decreased staining in the outer root of the hair follicle.

Figure 5(a) shows SATB1 expression on day 1 of wound healing where the epidermis is distant to the wound. Here we can see again that the epidermis is positively stained shows SATB1 expression. This is represented by the merging of colours. Figure 5(b) shows SATB1 expression in day 1 of wound healing where the epidermis is close to the wound. Again the image is showing distinct SATB1 expression in the epidermis however, in the wound debris there is only DAPI staining represented by the blue fluorescence and no staining by SATB1/ TRITC thus showing that there no SATB1 expression in the wound debris.

Figure 6(a) shows SATB1 expression on day 3 of wound healing where the epidermis is distant to the wound. The epidermis is showing positive SATB1 expression represented by the merged of colours. Yet above the epidermis, there is SATB1/TRITC staining; this showing non- specific staining. Figure 6(b) shows SATB1 expression in day 3 of wound healing where the epidermis is close to the wound. In the epidermis, there is a down regulation expression of SATB1 as there is no positive staining, but DAPI staining. There is also a slight non- specific staining of SATB1/TRITC.

Figure7 (a) shows SATB1 expression on day 5 of wound healing where the epidermis is distant to the wound. There is positive SATB1 expression in the epidermis shown by the merged of colours. This is when DAPI overlaps SATB1/TRITC. There is non- specific SATB1/TRITC staining shown in the hair shaft. Figure7 (b) shows SATB1 expression on day 5 of wound healing where the epidermis is close to the wound. There is no positive staining in the area of wound formation thus showing no expression of SATB1. This illustrates to us that SATB1 expression is strongly decreased in the epidermis closely adjacent to the wound, as well as in the wound epithelium versus normal epidermis.

4. 0 Discussion

The problem investigated in this study was to perform the analysis of SATB1 expression in mouse skin during wound healing. To confirm the presence of SATB1 in mouse skin during wound healing, we used immunoflourescence approach to detect SATB1 expression in cryosections of normal mouse skin during telogen as well as in the wound skin on days 1, 3, and 5 of the experiment. The expression of SATB1 was examined in parallel to DAPI staining. DAPI can bind specifically to DNA in the nucleus and also show little background from the cytoplasm.

SATB1 is a nuclear AT- rich protein that recruits higher -order chromatin remodelling factors and regulates expression of several genes during thymocytes differentiation(S et al., 2003). SATB1 also plays a role to regulate region- specific histone modification(S et al., 2003). SATB1 tethers to matrix attachment regions (MARs) of the DNA. This nuclear matrix has been implicated in many cellular processes including DNA replication, transcription and RNA processing(Liu J et al., 1997). SATB1 is a single major protein of 115KD which is a negatively regulatory element present in cellular protein such as NBP(Liu J et al., 1997). Experiment showed SATB1- binding site radically elevates MMTV(mouse mammary tumour virus) transcription in the lymphoid tissues(Liu J et al., 1997). This shows to us that SATB1 has a modulatory effect in tissue- specific gene regulation. Recent studies have shown that knock down of SATB1 has a substantial effect on MHC class- 1 regulation in the manner by mimicing IFN- gamma induction(Galande et al., 2007). SATB1- knockout mice merely lived up to 3weeks after birth as their development is blocked mainly due to the CD4+CD8+ double positive cell leading to apoptosis(S et al., 2003).

Normal skin during anagen stage, was used a negative control. A negative control meant no addition of the primary antibody, with only the (goat - anti- rabbit TRITC labelled) secondary antibody was to be used. The DAPI stained positive for cell nuclei.

In normal skin during the telogen stage, SATB1 is present in the epidermal basal layer. This is represented by the results shown in figure 4(a), as there is a great deal of positive merged staining. This verifies that SATB1 must have a role in the control of gene expression in the epidermis. The transition from basal cells to corneocytes is a complex process and entails a multiplicity of genes being activated and inactivated. Normal epidermis is regenerated from un-differentiated keratinocytes to differentiated keratinocytes; and genes must be expressed at the correct time and location. As we know, SATB1 has an importance in higher- order chromatin structure in gene regulation(S et al., 2003)and is involved in establishing specific conformations of locus by targeting chromatin remodelling enzymes and transcription factors. Transcription factors present in the epidermis include API, AP2, Sp1 and STAT factors(Eckert, 1996). Klf4 is another transcription factor present in epidermal keratinocytes and plays the important role in epidermal barrier formation and regulation(Segre, May 2006). Therefore it can be concluded as SATB1 is known to be present in the epidermal keratinocytes it has an underlying mechanism to regulate transcription factors and pathways in the epidermis resulting in chemo-taxis, migration and differentiation of cells leading to the formation of a normal epidermis.

In the hair follicle figure 4(b) SATB1 staining is limited to single cells in the distal outer root of the hair follicle. Minimal staining of SATB1in the outer root sheath suggests that SATB1 may not be needed to control any genes leading up to the outer root sheath and controlling the growth of hair follicle up to a certain point.

SATB1 expression in the epidermis on day 1 of wound healing show a lack of SATB1 in the wound epithelium. However, SATB1 staining is present in the epidermis closely adjacent to the wound.

SATB1 mimics IFN- gamma(Galande et al., 2007). However over expression of IFN- gamma (MIG/CXCL9) with an up regulation of chemokine (IP10/CXCL10); after full thickness of injury, showed disruption of normal development of the granulation tissue. This stimulated inflammation but inhibited re- epithelialization (Werner. S and Grose. R, 2003)

There is a down regulation of SATB1 in the wound region on day 3 of wound healing.

However, there is some positive SATB1 expression outside the wound. This again proving SATB1 does not have a role in the formation of a scar, but SATB1 has a role in regulating transcription factors in the formation of a normal epidermis.

SATB1 is expressed positively in the epidermis on day 5 after wounding. The specific nuclei staining was shown the epidermis, by the merging of colours showing DAPI staining overlapping SATB1/TRITC. The hair shaft shows non-specific expression. This expression is showing the affinity to other antibodies which may be present in the hair shaft therefore the staining is shown red.

In day 5 of wounding, it has been observed epidermal keratinocytes cells are migrating to cover the wound. Keratinocytes have been recognised to play an important role in regulating inflammation as they express cytokines that endorse chemo- taxis, and induce lymphocyte homing(L et al., 1992).

There is a slight expression of SATB1 outside the wound; this is because the epidermis is starting to regenerate therefore SATB1 can perform its function as global chromatin organiser and transcription factor.

In normal skin, there is normal SATB1 expression as there is a normal epidermis created therefore SATB1 can perform its normal function as a genome organiser. However in a wound; there is damage to the normal anatomical structure(Velnar T et al., 2009)where the dermis and epidermis no longer exist in a state of equilibrium resulting in a formation of a scar instead of an epidermis. Therefore SATB1 is not present in the wound and is shown as there is no positive staining in the wound. Wound healing is dived into four phases; coagulation, haemostasis, inflammation and proliferation. The mechanisms that underlie these processes are the presence of inflammatory mediators and growth factors(Velnar T et al., 2009).Important growth factors present in the wound, include VPF which is an important cytokine in wound healing(L et al., 1992)platelet derived growth factor, epidermal growth factor etc(Werner. S and Grose. R, 2003).These are vital as with time, they stop blood flow to the area and result in a fully matured scar(Velnar T et al., 2009).

In conclusion, it has been deduced that SATB1 does not have a role in the formation of a scar in wound healing but has a function as a high order chromatin organiser in the normal formation of an epidermis. Recent studies have also showed SATB1 can re- programme gene expression to promote breast tumour growth and metastasis.

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