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Expression of fibronectin isoforms in rat cornea after an epithelial-scrape wound
A Espaillat1, S J Lee, V Arrunategui-Correa
1Rhoads Molecular Immunology Laboratory, Harvard Medical School, Boston, MA.
Abstract:
The polymerase chain reaction (PCR) technique was used to analyze the presence or absence of the EIIIB and V region fibronectin (FN) mRNA isoforms, generated by alternative splicing of the FN primary mRNA transcript during epithelial wound healing, in a rat cornea wound model. A central 4mm area of the cornea was denuded of epithelium. At 5, 15, 30, 45 minutes, 1, 2, 4, 8, 24, 48 hours, and 4 days post-wounding, the rats were euthanized and a 6mm diameter full thickness corneal biopsy was performed. cDNA was synthesized from extracted RNA, and specific FN sequences were amplified by PCR as directed by different sets of 5' and 3' primers specific for the alternatively spliced EIIIB and V region domains. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was used to normalize the data. PCR products were analyzed by agarose gel electrophoresis and the identity of the bands were confirmed by direct nucleotide sequencing. The alternatively-spliced EIIIB and all three alternatively spliced isoforms of V domain FN mRNA were observed post-wounding. Similarly, in normal tissue, EIIIB and all three V region alternatively spliced FN isoforms were expressed. These findings suggest that in situ synthesis of cellular associated EIIIB FN and FN V domain may play a role in corneal wound healing.
Insights
Fibronectin (FN) mRNA isoforms, including EIIIB and V regions, are present in normal rat corneas and during wound healing. Their synthesis suggests a role in the corneal wound healing process.
Area of Science:
- Ophthalmology
- Molecular Biology
- Biochemistry
Background:
- Epithelial wound healing involves complex molecular changes.
- Fibronectin (FN) is a key extracellular matrix protein implicated in cell adhesion and tissue repair.
- Alternative splicing of FN mRNA generates various isoforms with potentially distinct functions.
Purpose of the Study:
- To investigate the expression of EIIIB and V region fibronectin (FN) mRNA isoforms during rat corneal epithelial wound healing.
- To determine if these alternatively spliced FN isoforms are present in normal corneal tissue.
Main Methods:
- Utilized the polymerase chain reaction (PCR) technique to detect specific FN mRNA isoforms.
- Analyzed corneal tissue from a rat wound model at various time points post-wounding.
- Confirmed PCR product identity through nucleotide sequencing and normalized data using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression.
Main Results:
- Alternatively spliced EIIIB and all three V region FN mRNA isoforms were detected in normal corneal tissue.
- These specific FN mRNA isoforms were also observed during the corneal wound healing process.
- Expression levels were analyzed and normalized to GAPDH mRNA.
Conclusions:
- The presence of EIIIB and V region FN mRNA isoforms in normal corneas suggests their constitutive role.
- In situ synthesis of cellular-associated EIIIB FN and FN V domain isoforms likely contributes to corneal wound healing.
- This study highlights the dynamic expression of FN isoforms in ocular tissue repair.