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Skin basement membrane and extracellular matrix proteins characterization and quantification by real time RT-PCR

Paper ID Volume ID Publish Year Pages File Format Full-Text
11822 760 2006 10 PDF Available
Title
Skin basement membrane and extracellular matrix proteins characterization and quantification by real time RT-PCR
Abstract

Three-dimensional gelatin-chondroitin 6 sulphate-hyanuronic acid (gelatin-C6S-HA) biomatrices were used as the scaffold to investigate the phenotypic and molecular expression of basement membrane (BM) and extracellular matrix (ECM) proteins in vitro. The cells were cultured in three different culture conditions: keratinocytes (K) monoculture, or dermal fibroblasts (FB) monoculture, or organotypic keratinocytes and dermal fibroblasts (K&FB) coculture model. The deposition of BM proteins and ECM proteins secreted by these two kinds of cells was quantitatively characterized by real time RT-PCR and examined by immunohistochemistry.The results showed that K expressed specific keratin and E-cadherin proteins, while type I collagen was secreted by FB. FB were shown to synthesize and deposit laminin 5, type IV collagen, and type VII collagen, whereas K dominantly produced integrin alpha 6 and integrin beta 4 as well as laminin 5. Interestingly, the integrin beta 4 was expressed neither in K monoculture nor in FB monoculture, but was seen in organotypic K&FB coculture model in the early culture stage.The histology studies revealed numerous features of epidermalization including a well organized basal layer of distinct cylindrical cells, granular and a horny layer, as well as complete BM formation. These results indicated that K and FB not only kept their phenotype when culturing on 3D scaffold, but also worked together to reconstruct dermal–epidermal basement membrane zone. In brief, our results directly provide the quantification in the expression of BM and ECM proteins by using real time RT-PCR in mRNA level and morphological appearance by immunostain in protein level.

Keywords
Keratinocyte; Fibroblast; PCR (polymerase chain reaction); Biomimetic materialBM, Basement membrane; ECM, Extracellular matrix; K, Keratinocytes; FB, Dermal fibroblasts; K&FB, Keratinocytes and dermal fibroblasts; RT-PCR, Reverse transcription-polymerase
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Skin basement membrane and extracellular matrix proteins characterization and quantification by real time RT-PCR
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biomaterials - Volume 27, Issue 29, October 2006, Pages 5059–5068
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us