Isolation of human salivary exosome on the morphology and gene expression of human periodontal ligament fibroblast cell line

The increasing incidence of periodontal diseases has led to the advancement in periodontal therapy including periodontal tissue regeneration. The development of human salivary-derived exosomes has become one of the promising researches to improve cell-based tissue engineering. Due to established...

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Main Author:	Tuan Kechik, Tuan Siti Mastazliha Long
Format:	Thesis
Language:	English
Published:	2018
Subjects:	RK Dentistry
Online Access:	http://eprints.usm.my/56503/1/Dr.%20Tuan%20Siti%20Mastazliha-OCR.pdf
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spelling	my-usm-ep.565032023-01-29T01:05:07Z Isolation of human salivary exosome on the morphology and gene expression of human periodontal ligament fibroblast cell line 2018 Tuan Kechik, Tuan Siti Mastazliha Long RK Dentistry The increasing incidence of periodontal diseases has led to the advancement in periodontal therapy including periodontal tissue regeneration. The development of human salivary-derived exosomes has become one of the promising researches to improve cell-based tissue engineering. Due to established functions showed by exosomes, human salivary exosomes were isolated and its effect on the morphology and gene expression of basic fibroblast growth factor (bFGF) and collagen type 1 (COL1) in human periodontal ligament fibroblast (HPdLF) cell line was studied. Unstimulated saliva samples collected from healthy male subjects were used. Exosomes were isolated by ultracentrifugation while the confirmation and establishment of its storage condition were carried out by Scanning Electron Microscopy (SEM), Western blot assay and Nanoparticle Tracking Analysis (NTA) in the presence and absence of protease inhibitor. Morphology and number of HPdLF cells treated with exosomes were viewed under inverted microscope and calculated by using trypan blue respectively. Determination of the gene expression level of bFGF and COL1 in the presence and absence of human salivary exosomes in HPdLF cells was performed using quantitative reverse transcriptase polymerase chain reaction (RTqPCR). This study showed that salivary exosomes were stable at several temperatures tested with and without protease inhibitor. SEM analysis demonstrated the round shape of exosomes, ranged between 10 nm to 100 nm in diameter. Western blot result confirmed that isolated exosomes expressed exosomal marker CD63. NTA estimated the concentration and individual size of exosomes. There was no significant difference in the morphology and number of HPdLF cells for both exosome treated and untreated samples, however, exosomes upregulated bFGF gene expression. This study concluded that human salivary exosomes are stable biomaterial and able to upregulate bFGF gene that was expressed by HPdLF cells. Thus, they might have potential to be used as alternative biomaterial in tissue engineering for periodontal regeneration. 2018 Thesis http://eprints.usm.my/56503/ http://eprints.usm.my/56503/1/Dr.%20Tuan%20Siti%20Mastazliha-OCR.pdf application/pdf en public masters Universiti Sains Malaysia Pusat Pengajian Sains Pergigian
institution	Universiti Sains Malaysia
collection	USM Institutional Repository
language	English
topic	RK Dentistry
spellingShingle	RK Dentistry Tuan Kechik, Tuan Siti Mastazliha Long Isolation of human salivary exosome on the morphology and gene expression of human periodontal ligament fibroblast cell line
description	The increasing incidence of periodontal diseases has led to the advancement in periodontal therapy including periodontal tissue regeneration. The development of human salivary-derived exosomes has become one of the promising researches to improve cell-based tissue engineering. Due to established functions showed by exosomes, human salivary exosomes were isolated and its effect on the morphology and gene expression of basic fibroblast growth factor (bFGF) and collagen type 1 (COL1) in human periodontal ligament fibroblast (HPdLF) cell line was studied. Unstimulated saliva samples collected from healthy male subjects were used. Exosomes were isolated by ultracentrifugation while the confirmation and establishment of its storage condition were carried out by Scanning Electron Microscopy (SEM), Western blot assay and Nanoparticle Tracking Analysis (NTA) in the presence and absence of protease inhibitor. Morphology and number of HPdLF cells treated with exosomes were viewed under inverted microscope and calculated by using trypan blue respectively. Determination of the gene expression level of bFGF and COL1 in the presence and absence of human salivary exosomes in HPdLF cells was performed using quantitative reverse transcriptase polymerase chain reaction (RTqPCR). This study showed that salivary exosomes were stable at several temperatures tested with and without protease inhibitor. SEM analysis demonstrated the round shape of exosomes, ranged between 10 nm to 100 nm in diameter. Western blot result confirmed that isolated exosomes expressed exosomal marker CD63. NTA estimated the concentration and individual size of exosomes. There was no significant difference in the morphology and number of HPdLF cells for both exosome treated and untreated samples, however, exosomes upregulated bFGF gene expression. This study concluded that human salivary exosomes are stable biomaterial and able to upregulate bFGF gene that was expressed by HPdLF cells. Thus, they might have potential to be used as alternative biomaterial in tissue engineering for periodontal regeneration.
format	Thesis
qualification_level	Master's degree
author	Tuan Kechik, Tuan Siti Mastazliha Long
author_facet	Tuan Kechik, Tuan Siti Mastazliha Long
author_sort	Tuan Kechik, Tuan Siti Mastazliha Long
title	Isolation of human salivary exosome on the morphology and gene expression of human periodontal ligament fibroblast cell line
title_short	Isolation of human salivary exosome on the morphology and gene expression of human periodontal ligament fibroblast cell line
title_full	Isolation of human salivary exosome on the morphology and gene expression of human periodontal ligament fibroblast cell line
title_fullStr	Isolation of human salivary exosome on the morphology and gene expression of human periodontal ligament fibroblast cell line
title_full_unstemmed	Isolation of human salivary exosome on the morphology and gene expression of human periodontal ligament fibroblast cell line
title_sort	isolation of human salivary exosome on the morphology and gene expression of human periodontal ligament fibroblast cell line
granting_institution	Universiti Sains Malaysia
granting_department	Pusat Pengajian Sains Pergigian
publishDate	2018
url	http://eprints.usm.my/56503/1/Dr.%20Tuan%20Siti%20Mastazliha-OCR.pdf
_version_	1776101164956254208

Isolation of human salivary exosome on the morphology and gene expression of human periodontal ligament fibroblast cell line

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