Role of COMP in skin connective tissue architecture and fibrotic diseases:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. und engl. Sprache |
| Beschreibung: | X, 99 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Role of COMP in skin connective tissue architecture and fibrotic diseases |c vorgelegt von Pallavi Agarwal |
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Datensatz im Suchindex
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| adam_text | Titel: Role of COMP in skin connective tissue architecture and fibrotic diseases
Autor: Agarwal, Pallavi
Jahr: 2012
TABLE OF CONTENTS
ABSTRACT.............................................................................................................................Ill
ZUSAMMENFASSUNG..........................................................................................................V
1. INTRODUCTION...............................................................................................................1
1.1 Cartilage Oligomeric Matrix Protein - structure, localization, and function................1
1.2 The skin.....................................................................................................................4
1.2.1 The epidermis.....................................................................................................5
1.2.2 The dermo-epidermal junction............................................................................6
1.2.3 The dermis and its extracellular components.....................................................7
1.2.3.1 Fibrillar collagens and fibrillogenesis...........................................................9
1.2.3.2 Modulators of collagen fibrillogenesis..........................................................9
1.2.3.3 Fibril-associated collagens XII and XIV and the role of COMP.................10
1.3 Fibrotic/desmoplastic reactions in skin....................................................................11
1.3.1 Scleroderma.....................................................................................................13
1.3.2 Wound healing..................................................................................................14
1.3.3 Skin carcinoma.................................................................................................15
1.4 Aim of the study.......................................................................................................16
2. RESULTS........................................................................................................................17
2.1 COMP is deposited in a characteristic pattern in human skin by dermal
fibroblasts.................................................................................................................17
2.1.1 COMP localizes subepidermal^ in the papillary dermis of human skin............17
2.1.2 COMP is produced by dermal fibroblasts but not epidermal keratinocytes......18
2.2 Potential function of COMP as a matrix adapter molecule in skin suprastructure ...21
2.2.1 FACIT collagens XII and XIV are novel binding partners of COMP..................21
2.2.1.1 Expression and purification of recombinant full-length
COMP, collagen XII, collagen XIV and fragments thereof.........................21
2.2.1.2 Interaction of COMP with collagens XII and XIV.......................................23
2.2.1.3 COMP binds to the collagenous domain of collagen XII but not to
NC3 domain...............................................................................................24
2.2. i .4 COMP binds to collagenous domain of collagen XIV but not to
its NC3 domain..........................................................................................26
2.2.2 COMP co-localizes with FACIT collagens XII and XIV in human
papillary dermis and is deposited in anchoring plaques...................................28
2.2.2.1 COMP partially co-distributes with FACIT collagens XII and XIV
in papillary dermis......................................................................................28
2.2.2.2 COMP, collagen XII and collagen XIV are deposited in
anchoring plaques.....................................................................................29
2.2.2.3 COMP co-localizes with collagens XII and XIV, that decorate
skin major collagen I fibrils........................................................................30
2.3 COMP expression is enhanced in human skin pathologies associated
with fibrotic or desmoplastic reactions........................................................................31
2.3.1 COMP is overexpressed in fibrotic lesions of patients with localized
scleroderma in comparison to healthy human skin...........................................31
2.3.2 COMP is not produced in the granulation tissue of human healing wounds ....33
2.3.3 COMP deposition is enhanced in non-healing venous leg ulcers.....................33
2.3.4 COMP expression is strongly increased in fibrotic stroma of basal cell
carcinoma.........................................................................................................35
2.4 TGFB1 induces COMP expression in normal and scleroderma fibroblasts in vitro..36
2.4.1 COMP levels are markedly induced by exogenous TGFB1..............................36
2.4.2 COMP is overexpressed in scleroderma fibroblasts in vitro and further
induced by TGFB1............................................................................................37
2.5 Silencing COMP in scleroderma fibroblasts does not alter secreted
levels of collagen I...................................................................................................39
2.6 Keratinocyte-fibroblast crosstalk, a potential mechanism leading to increased
COMP expression in tumor stroma..........................................................................40
2.6.1 COMP expression remains unaltered in primary dermal fibroblasts upon
treatment with conditioned media of either squamous cell carcinoma
(A431) or normal keratinocyte (HaCaT) cell lines.............................................40
2.6.2 Continuous crosstalk of primary dermal fibroblasts either with
malignant HaCaT (Il4rt) or normal keratinocyte (HaCaT) cell lines
does not modulate COMP gene expression.....................................................43
2.6.3 COMP protein levels are significantly increased upon direct
coculture of dermal fibroblasts with SCC cells (A431) but not
with normal keratinocyte (HaCaT) cell line.......................................................44
. DISCUSSION....................................................................................................47
3.1 COMP is a matrix component of healthy human skin and might function
as a matrix adapter molecule by interacting with collagens XII and XIV..................47
3.2 Enhanced COMP deposition is a common feature in fibrotic skin diseases............50
3.3 COMP is a downstream target of TGFS1 signaling in normal as well as
scleroderma fibroblasts............................................................................................52
3.4 Role of fibroblast-tumor cell contact in regulating COMP levels..............................54
4. MATERIALS AND METHODS........................................................................................59
4.1 Materials..................................................................................................................59
4.1.1 Chemicals.........................................................................................................59
4.1.2 Antibodies.........................................................................................................59
4.1.3 Enzymes...........................................................................................................60
4.1.4 Antibiotics.........................................................................................................60
4.1.5 Primers and PCR program...............................................................................61
4.1.6 siRNA sequences.............................................................................................62
4.1.7 Buffers and solutions........................................................................................62
4.1.8 Equipments.......................................................................................................64
4.2 Methods...................................................................................................................64
4.2.1 Collection of human skin biopsies (healthy skin, wounds,
tumors) and wound exudates...........................................................................64
4.2.2 Histological methods.........................................................................................65
4.2.2.1 Immunohistochemistry and immunofluorescence.....................................65
4.2.2.2 Immuno-electron microscopy....................................................................65
4.2.3 Cell culture........................................................................................................66
4.2.3.1 Isolation and culture of primary fibroblasts and cell lines..........................66
4.2.3.2 Stimulation of cells with TGFB1.................................................................66
4.2.3.3 Silencing of COMP in fibroblasts...............................................................66
4.2.3.4 Stimulation of fibroblasts with conditioned media......................................67
4.2.3.5 Coculture of cells in transwell chambers...................................................67
4.2.3.6 Direct coculture experiment.......................................................................68
4.2.4 Molecular biological methods...........................................................................68
4.2.4.1 RNA extraction from cells..........................................................................68
4.2.4.2 RNA extraction from separated dermis and epidermis..............................68
4.2.4.3 Semi-quantitative and real time quantitative PCR.....................................69
4.2.4.4 Extraction of proteins from cells and skin biopsies....................................69
4.2.4.5 Precipitation of culture supernatants.........................................................69
4.2.4.6 SDS polyacrylamide gel electrophoresis and Western blot analysis.........70
4.2.4.7 Production of recombinant proteins...........................................................70
4.2.4.7.1 Cloning of full-length mouse COMP.......................................................70
4.2.4.7.2 Transfection of HEK293 cells with mCOMP cDNA................................71
4.2.4.7.3 Purification of recombinant mCOMP by column chromatography..........71
4.2.4.7.4 Production of recombinant collagen XII, XIV and fragments..................72
4.2.4.8 Solid phase binding assays.......................................................................72
4.2.4.9 Surface plasmon resonance spectroscopy...............................................73
4.2.4.10 Enzyme linked immunosorbent Assay for COMP in wound exudates.......73
4.2.5 Production of primary antibodies......................................................................74
4.2.6 Statistical analysis............................................................................................74
5. REFERENCES................................................................................................................75
6. APPENDIX......................................................................................................................89
6.1 Vector map...............................................................................................................89
6.2 Abbreviations...........................................................................................................91
ERKLARUNG.........................................................................................................................93
TEILPUBLIKATIONEN...........................................................................................................95
ACKNOWLEDGMENTS.........................................................................................................97
LEBENSLAUF........................................................................................................................99
|
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| discipline | Medizin |
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| spelling | Agarwal, Pallavi 1980- Verfasser (DE-588)102835486X aut Role of COMP in skin connective tissue architecture and fibrotic diseases vorgelegt von Pallavi Agarwal 2012 X, 99 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache Köln, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025432565&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Agarwal, Pallavi 1980- Role of COMP in skin connective tissue architecture and fibrotic diseases |
| subject_GND | (DE-588)4113937-9 |
| title | Role of COMP in skin connective tissue architecture and fibrotic diseases |
| title_auth | Role of COMP in skin connective tissue architecture and fibrotic diseases |
| title_exact_search | Role of COMP in skin connective tissue architecture and fibrotic diseases |
| title_full | Role of COMP in skin connective tissue architecture and fibrotic diseases vorgelegt von Pallavi Agarwal |
| title_fullStr | Role of COMP in skin connective tissue architecture and fibrotic diseases vorgelegt von Pallavi Agarwal |
| title_full_unstemmed | Role of COMP in skin connective tissue architecture and fibrotic diseases vorgelegt von Pallavi Agarwal |
| title_short | Role of COMP in skin connective tissue architecture and fibrotic diseases |
| title_sort | role of comp in skin connective tissue architecture and fibrotic diseases |
| topic_facet | Hochschulschrift |
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