The role of the Met tyrosine kinase receptor in skin maintenance and regenerationJolanta Chmielowiec1Zusammenfassung2Introduction Wound healing in the skin 23534#4274551#2335Mammalian skin6534#328The tyrosine kinase receptor Met7Met signal transduction8534#372534#6089550#226Met signalling during development10534#20111Met function in the adult The aim of the study12ResultsExpression of Met and HGF/SF in the skin and during skin wound healing12538#18913451#190Generation of mice deficient in Met in keratinocytes 14476#38215393#294Met signaling during generation and maintenance of the skin16382#658507#43417Wound closure in conditional Met mutant mice18380#672390#14819534#69620584#554Contribution of cells in the hyperproliferative epithelium21436#419481#30022Scratch-wound healing of Met mutant keratinocytes in cell culture585#21323587#47424612#376Cytoskeleton rearrangement in cultured scratchwounded keratinocytes 476#94125Signal transduction in primary keratinocytes26586#731Discussion26Conditional mutagenesis to investigate Met function in the skin27The role of the tyrosine kinase receptor Met in the skin28Only non-recombined cells contribute to wound healing29The role of HGF/SF and Met in development and regeneration30Only Met-positive keratinocytes contribute to healing of scratch-wounds in vitro31The Met receptor as a therapeutically targetMaterials and Methods32Extraction and purification of DNA3334Polymerase chain reaction (PCR)Southern blotting35Cell culture36Wounding of skinImmunhistochemical techniques37383940414243Protein biochemistry 4445534#216464748AbbreviationsReferencesErklärungPublicationsenTable of contentsHelp Abbreviations

ATP

adenosine triphosphate

bp

base pair(s)

BrdU

5bromo2’deoxyuridine

BSA

bovine serum albumin

d

day

DAPI

4’,6-Diamidin-2-phenylindoldihydrochloride

DMSO

dimethylsulfoxide

DNA

deoxyribonucleic acid

dNTP

deoxyribonucleoside triphosphate

DNase

deoxyribonuclease

DTT

dithiothreitol

EGTA

Ethylene-glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid

et al.

et altera

EDTA

ethylene-diaminetetraacetic acid

FCS

fetal calf serum

g

gram

G418

geneticin

HEPES

4-(2-Hydroxyethyl)-piperazin-1-ethansulfonic acid

h

hour

HRP

horseradish peroxidase

HS

heat inactivated horse serum

kDa

kilodalton

kb

kilobase pairs

l

liter

M

molar

mA

milliampere

min

minute

ml

milliliter

mM

millimolar

µM

micromolar

MEM

Modified Eagle Medium

NaAc

sodium acetate

ON

over night

PBS

phosphate-buffered saline

PBT

PBS containing 0.05% Tween-20

PCR

polymerase chain reaction

PFA

paraformaldehyde

pH

potentium hydrogenii

rpm

rotations per minute

RT

room temperature

SDS

sodium dodecyl sulphate

SSC

standard saline citrate

TEMED

N, N, N’, N’-Tetramethylethylenediamine

Tris

Tris-(hydroxymethyl)aminoethane

U

unit (enzymatic activity)

V

Volt

Vol.

Volume

W

Watt