Cargando…

Effects of amniotic membrane suspension in human corneal wound healing in vitro

PURPOSE: To investigate the biochemical mechanism of amniotic membrane (AM) suspension on corneal wound healing, particularly on epithelial proliferation and migration. METHODS: Human corneal epithelial cells (HCECs) were cultured in media with different concentrations of AM suspension (5% and 30%),...

Descripción completa

Detalles Bibliográficos
Autores principales: Choi, Jin A, Jin, Hyun-Jin, Jung, Samhyun, Yang, Eunkyung, Choi, Jun-Sub, Chung, So-Hyang, Joo, Choun-Ki
Formato: Texto
Lenguaje:English
Publicado: Molecular Vision 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774451/
https://www.ncbi.nlm.nih.gov/pubmed/19907665
_version_ 1782173936571645952
author Choi, Jin A
Jin, Hyun-Jin
Jung, Samhyun
Yang, Eunkyung
Choi, Jun-Sub
Chung, So-Hyang
Joo, Choun-Ki
author_facet Choi, Jin A
Jin, Hyun-Jin
Jung, Samhyun
Yang, Eunkyung
Choi, Jun-Sub
Chung, So-Hyang
Joo, Choun-Ki
author_sort Choi, Jin A
collection PubMed
description PURPOSE: To investigate the biochemical mechanism of amniotic membrane (AM) suspension on corneal wound healing, particularly on epithelial proliferation and migration. METHODS: Human corneal epithelial cells (HCECs) were cultured in media with different concentrations of AM suspension (5% and 30%), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (negative control), and serum containing Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (positive control). In an effort to evaluate the migratory potential of AM, migration assays were conducted via the manual scraping of HCECs and immunocytochemical staining of cell adhesion molecules (E-cadherin). The relative expression of matrix metallopeptidase 9 (MMP9) and adhesion molecules (E-cadherin, fibronectin) was determined via reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The proliferative potential of AM was evaluated via a proliferation assay using 5-Bromo-2-deoxyuridine (BrdU) incorporation and western blot analysis for proliferating cell nuclear antigen (PCNA). In addition, enzyme-linked immunosorbent assay (ELISA) was used to measure the protein concentrations of mitogenic growth factors (epidermal growth factor [EGF],keratinocyte growth factor [KGF], hepatocyte growth factor [HGF], and basic fibroblast growth factor [bFGF]) in AM suspensions. RESULTS: Migration assay rates were enhanced as AM concentrations increased, with statistically significant changes seen in 30% AM-treated and positive control cells, compared to negative control cells (p<0.05). RT-PCRs revealed that the expression of the MMP9 gene was upregulated by AM, and the expressions of E-cadherin and fibronectin genes were downregulated by AM. Western blot analysis demonstrated significantly higher MMP9 expression in AM-treated groups, versus significantly lower levels of E-cadherin and fibronectin expression in AM-treated groups. Immunocytochemistry showed large quantities of E-cadherin near the wound edges after 24 h of injury in the AM-treated groups. The proliferation assay showed that the BrdU positive cell counts/total cell counts (labeling index) were augmented by AM to a statistically significant degree (p<0.05 in the 30% AM and positive control groups). Western blot analysis showed that the expression cell cycle-associated protein, PCNA, increased gradually as a result of AM treatment. ELISA showed that our AM suspension contained 4 growth factors (HGF, EGF, KGF, and FGF). The amount of HGF was especially large, followed by that of EGF. CONCLUSIONS: These results demonstrate that the suspension form of AM maintains its beneficial effect on corneal epithelial wound healing in vitro, and that AM suspension leads to significant increases in corneal epithelial migration and proliferation with increasing AM concentrations.
format Text
id pubmed-2774451
institution National Center for Biotechnology Information
language English
publishDate 2009
publisher Molecular Vision
record_format MEDLINE/PubMed
spelling pubmed-27744512009-11-11 Effects of amniotic membrane suspension in human corneal wound healing in vitro Choi, Jin A Jin, Hyun-Jin Jung, Samhyun Yang, Eunkyung Choi, Jun-Sub Chung, So-Hyang Joo, Choun-Ki Mol Vis Research Article PURPOSE: To investigate the biochemical mechanism of amniotic membrane (AM) suspension on corneal wound healing, particularly on epithelial proliferation and migration. METHODS: Human corneal epithelial cells (HCECs) were cultured in media with different concentrations of AM suspension (5% and 30%), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (negative control), and serum containing Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (positive control). In an effort to evaluate the migratory potential of AM, migration assays were conducted via the manual scraping of HCECs and immunocytochemical staining of cell adhesion molecules (E-cadherin). The relative expression of matrix metallopeptidase 9 (MMP9) and adhesion molecules (E-cadherin, fibronectin) was determined via reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The proliferative potential of AM was evaluated via a proliferation assay using 5-Bromo-2-deoxyuridine (BrdU) incorporation and western blot analysis for proliferating cell nuclear antigen (PCNA). In addition, enzyme-linked immunosorbent assay (ELISA) was used to measure the protein concentrations of mitogenic growth factors (epidermal growth factor [EGF],keratinocyte growth factor [KGF], hepatocyte growth factor [HGF], and basic fibroblast growth factor [bFGF]) in AM suspensions. RESULTS: Migration assay rates were enhanced as AM concentrations increased, with statistically significant changes seen in 30% AM-treated and positive control cells, compared to negative control cells (p<0.05). RT-PCRs revealed that the expression of the MMP9 gene was upregulated by AM, and the expressions of E-cadherin and fibronectin genes were downregulated by AM. Western blot analysis demonstrated significantly higher MMP9 expression in AM-treated groups, versus significantly lower levels of E-cadherin and fibronectin expression in AM-treated groups. Immunocytochemistry showed large quantities of E-cadherin near the wound edges after 24 h of injury in the AM-treated groups. The proliferation assay showed that the BrdU positive cell counts/total cell counts (labeling index) were augmented by AM to a statistically significant degree (p<0.05 in the 30% AM and positive control groups). Western blot analysis showed that the expression cell cycle-associated protein, PCNA, increased gradually as a result of AM treatment. ELISA showed that our AM suspension contained 4 growth factors (HGF, EGF, KGF, and FGF). The amount of HGF was especially large, followed by that of EGF. CONCLUSIONS: These results demonstrate that the suspension form of AM maintains its beneficial effect on corneal epithelial wound healing in vitro, and that AM suspension leads to significant increases in corneal epithelial migration and proliferation with increasing AM concentrations. Molecular Vision 2009-11-05 /pmc/articles/PMC2774451/ /pubmed/19907665 Text en http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Choi, Jin A
Jin, Hyun-Jin
Jung, Samhyun
Yang, Eunkyung
Choi, Jun-Sub
Chung, So-Hyang
Joo, Choun-Ki
Effects of amniotic membrane suspension in human corneal wound healing in vitro
title Effects of amniotic membrane suspension in human corneal wound healing in vitro
title_full Effects of amniotic membrane suspension in human corneal wound healing in vitro
title_fullStr Effects of amniotic membrane suspension in human corneal wound healing in vitro
title_full_unstemmed Effects of amniotic membrane suspension in human corneal wound healing in vitro
title_short Effects of amniotic membrane suspension in human corneal wound healing in vitro
title_sort effects of amniotic membrane suspension in human corneal wound healing in vitro
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774451/
https://www.ncbi.nlm.nih.gov/pubmed/19907665
work_keys_str_mv AT choijina effectsofamnioticmembranesuspensioninhumancornealwoundhealinginvitro
AT jinhyunjin effectsofamnioticmembranesuspensioninhumancornealwoundhealinginvitro
AT jungsamhyun effectsofamnioticmembranesuspensioninhumancornealwoundhealinginvitro
AT yangeunkyung effectsofamnioticmembranesuspensioninhumancornealwoundhealinginvitro
AT choijunsub effectsofamnioticmembranesuspensioninhumancornealwoundhealinginvitro
AT chungsohyang effectsofamnioticmembranesuspensioninhumancornealwoundhealinginvitro
AT joochounki effectsofamnioticmembranesuspensioninhumancornealwoundhealinginvitro