Detection of Salivary Small Extracellular Vesicles Associated Inflammatory Cytokines Gene Methylation in Gingivitis
Abstract
:1. Introduction
2. Results
2.1. Periodontal Status of Participants
2.2. Particle and Characteristics of Salivary sEV by UC or SEC
2.3. Effect of UC and SEC Isolation Methods on Healthy and Gingivitis-Derived sEV Characteristics
2.4. UC and SEC on sEV Inflammatory Cytokines Genes DNA Methylation
2.5. Comparison of Pooled UC- and SEC-sEV on hGFs DNA Methylation
3. Discussion
3.1. Effect of UC and SEC Isolation Method on Salivary sEV Yield and Bacteria Detection
3.2. Utilizing a Commercial sEV Standard as a Control
3.3. Healthy and Gingivitis-Derived sEV Characteristics—A Pilot Study
3.4. UC and SEC on Five Inflammation Genes DNA Methylation in Salivary sEV
4. Materials and Methods
4.1. Participant Recruitment and Saliva Sample Collection
4.2. Salivary sEV Isolation
4.2.1. Salivary sEV Isolation Using the Differential Ultracentrifugation Method
4.2.2. Salivary sEV Isolation Using SEC Method
4.3. Salivary sEV Characterization
4.3.1. Transmission Electron Microscopy (TEM)
4.3.2. Nanoparticle Tracking Analysis (NTA)
4.3.3. Protein Content and Western Blot
4.4. sEV DNA and RNA Isolation by Trizol Method
4.5. DNA Bisulfite Conversion and Quantitative Methylation-Specific Polymerase Chain Reaction (qMSP)
4.6. Salivary sEV Labelling and Uptake by Human Primary Gingival Fibroblasts
4.6.1. Human Primary Gingival Fibroblasts Cell Culture
4.6.2. sEV Labelling, hGFs Uptake, and Cellular qMSP
4.7. Statistical Analysis
4.8. EV-TRACK
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gender | Age | PPD | BOP | Race |
---|---|---|---|---|
F | 33 | <4 mm | 27% | Asian |
M | 28 | <4 mm | 41% | Asian |
M | 39 | <4 mm | 14% | Other |
F | 24 | <4 mm | 39% | Caucasian |
M | 40 | <4 mm | 39% | Asian |
F | 26 | <4 mm | 26% | Caucasian |
F | 38 | <4 mm | 10% | Asian |
M | 24 | <4 mm | 6% | Caucasian |
M | 34 | <4 mm | 49% | Caucasian |
M | 36 | <4 mm | 14% | Asian |
M | 34 | <4 mm | 70% | Caucasian |
M | 48 | <4 mm | 14% | Caucasian |
UC-sEV | SEC-sEV | |
---|---|---|
sEV particles/mL saliva | 5.07 × 109 ± 2.9 × 109 | 2.7 × 1010 ± 2.2 × 1010 |
sEV particles/mL saliva: <50 nm | 2.8 × 105 ± 5.7 × 105 | 1.1 × 105 ± 2.8 × 105 |
sEV particles/mL saliva: 50–150 nm | 1.1 × 107 ± 9.9 × 106 | 6.6 × 108 ± 6.9 × 108 |
sEV particles/mL saliva: 150–200 nm | 1.2 × 107± 8.3 × 106 | 1.3 × 109 ± 9.9 × 108 |
sEV particles/mL saliva: >200 nm | 1.9 × 106 ± 1.1 × 106 | 1.9 × 108 ± 1.6 × 108 |
sEV particles per μg protein | 1.6 × 107 ± 9.4 × 107 | 2.6 × 108 ± 1.5 × 108 |
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Han, P.; Lai, A.; Salomon, C.; Ivanovski, S. Detection of Salivary Small Extracellular Vesicles Associated Inflammatory Cytokines Gene Methylation in Gingivitis. Int. J. Mol. Sci. 2020, 21, 5273. https://doi.org/10.3390/ijms21155273
Han P, Lai A, Salomon C, Ivanovski S. Detection of Salivary Small Extracellular Vesicles Associated Inflammatory Cytokines Gene Methylation in Gingivitis. International Journal of Molecular Sciences. 2020; 21(15):5273. https://doi.org/10.3390/ijms21155273
Chicago/Turabian StyleHan, Pingping, Andrew Lai, Carlos Salomon, and Sašo Ivanovski. 2020. "Detection of Salivary Small Extracellular Vesicles Associated Inflammatory Cytokines Gene Methylation in Gingivitis" International Journal of Molecular Sciences 21, no. 15: 5273. https://doi.org/10.3390/ijms21155273