Tuan Kechik, Tuan Siti Mastazliha Long
(2018)
Isolation of human salivary exosome on the morphology and gene expression of human periodontal ligament fibroblast cell line.
Masters thesis, Universiti Sains Malaysia.
Abstract
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.
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