TGF-β and Physiological Root Resorption of Deciduous Teeth
Abstract
:1. Introduction
2. Results
2.1. Sequential Extraction of Proteins from Root-Surrounding Tissues
2.2. Detection of TGF-β and Tartrate-Resistant Acid Phosphatase (TRAP) Activities in Root-Surrounding Tissues and Identification of TGF-β Isoforms
2.3. In Vitro Experiment for RANKL-Mediated Osteoclast Differentiation
2.4. Gene Expression in Root-Surrounding Tissues
3. Discussion
3.1. TGF-β in Root-Surrounding Tissues of Deciduous Teeth
3.2. TGF-β Activity in Root-Surrounding Tissues of Deciduous Teeth and In Vitro Experiment for RANKL-Mediated Osteoclast Differentiation
3.3. Potential Roles of TGF-β in Root-Surrounding Tissues of Deciduous Teeth
4. Materials and Methods
4.1. Tissue Preparation
4.2. Sequential Extraction of Proteins from Root-Surrounding Tissues
4.3. Alkaline Phosphatase (ALP) Activity Assay (ALP-HPDL System)
4.4. SDS-PAGE
4.5. Endogenous and Induced Tartrate-Resistant Acid Phosphatase (TRAP) Assays and TRAP Staining
4.6. Enzyme-Linked Immunosorbent Assay (ELISA)
4.7. Quantitative Real-Time PCR (qPCR)
4.8. Statistical Analysis
Acknowledgments
Author Contributions
Conflicts of Interest
Appendix A
Triton X | Tissue (mg) | 660 Assay (μg/mL) | Total (μg/μL) | μg/mg Tissue |
---|---|---|---|---|
N1 | 106.3 | 5450 | 2779.5/510 | 26.1 |
N2 | 99.45 | 4700 | 3543.8/754 | 35.6 |
N3 | 109.2 | 6500 | 4680/720 | 42.9 |
R1 | 109.36 | 3875 | 2828.8/730 | 25.9 |
R2 | 102.8 | 6125 | 3711.8/606 | 36.1 |
R3 | 156.7 | 5450 | 2550.6/468 | 16.3 |
NP40 | Tissue (mg) | 660 Assay (μg/mL) | Total (μg/µL) | μg/mg Tissue |
N1 | 106.3 | 1210 | 750.2/620 | 7.06 |
N2 | 99.45 | 1330 | 771.4/580 | 7.76 |
N3 | 109.2 | 1630 | 717.2/440 | 6.57 |
R1 | 109.36 | 1200 | 648/540 | 5.93 |
R2 | 102.8 | 1190 | 747.3/628 | 7.27 |
R3 | 156.7 | 960 | 604.8/630 | 3.86 |
SDS | Tissue (mg) | 660 Assay (μg/mL) | Total (μg/μL) | μg/mg Tissue |
N1 | 106.3 | 135 | 82.4/610 | 0.78 |
N2 | 99.45 | 120 | 70.8/590 | 0.71 |
N3 | 109.2 | 100 | 56.2/562 | 0.51 |
R1 | 109.36 | 135 | 79.7/590 | 0.73 |
R2 | 102.8 | 145 | 67.9/468 | 0.66 |
R3 | 156.7 | 85 | 39.3/462 | 0.25 |
Gene | Sequence (5′–3′) | Size (bp) | qPCR Protocol (45 Cycles) | ||||||
---|---|---|---|---|---|---|---|---|---|
Denaturation | Annealing | Extension | |||||||
°C | Second | °C | Second | °C | Second | ||||
TGF-β1 | Forward | TTCATGAACCCAAGGGCTACC | 104 | 95 | 10 | 65 | 10 | 72 | 15 |
Reverse | CTGGTTGTACAGAGCCAGGAC | ||||||||
TGF-β2 | Forward | CAGTGGGAAGACCCCACATC | 150 | 60 | |||||
Reverse | ATGTAAAGTGGACGCAGGCA | ||||||||
TGF-β3 | Forward | CTGTGCGTGAATGGCTCTTG | 93 | ||||||
Reverse | TATCCCCGTTGGGCTGAAAG | ||||||||
RANKL | Forward | ACGATCAACGCCACAGACAT | 89 | 65 | |||||
Reverse | TTGGAGATCTTGGCCCAACC | ||||||||
RANK | Forward | GAAGCACGTCATGGGACTGA | 95 | 60 | |||||
Reverse | CTGGGCAAGTAAGTCTGGGG | ||||||||
TRAP | Forward | AACGTCTCGGCACAGATAGC | 101 | 65 | |||||
Reverse | GACACATTGGACCGTGGGAT | ||||||||
CALCR | Forward | CGCCTGTGGTGGTATCATGT | 142 | ||||||
Reverse | CAGGGCCGTGGATGATGTAA | ||||||||
NFATc1 | Forward | TCGTGGAGAAAGCACCAGAC | 91 | ||||||
Reverse | TCGACCACCAAGGAATTCGG | ||||||||
OPG | Forward | TGTTCTGGAAACAGTGAATCGAC | 80 | ||||||
Reverse | ACAGCAAACCTGAAGAACGC | ||||||||
GAPDH | Forward | CCATCACCATCTTCCAGGAG | 346 | ||||||
Reverse | ACAGTCTTCTGGGTGGCAGT |
Abbreviations
SDS | Sodium dodecyl sulfate |
PAGE | Polyacrylamide gel electrophoresis |
NP-40 | Nonidet P-40 |
kDa | Killodalton |
ACP5 | Acid phosphatase 5 |
PCR | Polymerase chain reaction |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
GST | Glutathione S-transferase |
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Root-Surrounding Tissue | Non-Resorption | Resorption | |||||
---|---|---|---|---|---|---|---|
Cervical | Center | Apical | Cervical | Center | Apical | ||
Region | N1 | N2 | N3 | R1 | R2 | R3 | |
Protein study | Collagen | ↑ | ↑↑ | ↑↑↑ | ↑↑↑ | ↑↑ | trace |
Decorin | ↑ | ↑↑↑ | ↑↑ | ↑↑ | ↑ | trace | |
ALP activity | ↑ | ↑↑ | ↑↑ | ↑↑ | ↑ | trace | |
eTRAP activity | ↑ | ↑ | ↑↑ | ↑↑ | ↑↑↑↑ | ↑↑↑ | |
In vitro study | iTRAP activity | ↑ | ↑↑ | ↑ | ↑ | trace | trace |
Genetic study (mRNA level) | TGF-β1 | ↑↑↑↑ | ↑↑↑↑ | ↑↑ | ↑↑ | ↑ | ↑ |
TGF-β2 | ↑ | ↑↑ | ↑↑↑↑ | ↑ | ↑ | ↑↑ | |
TGF-β3 | ↑ | ↑↑ | ↑↑↑ | ↑↑↑ | ↑ | ↑↑ | |
RANKL | trace | ↑↑↑ | ↑ | ↑ | ↑↑↑ | ↑↑↑↑ | |
RANK | trace | ↑↑ | ↑ | ↑ | ↑↑↑ | ↑↑↑↑ | |
TRAP | N.D | ↑ | trace | ↑ | ↑↑↑ | ↑↑↑↑ | |
CALCR | N.D | N.D | N.D | ↑ | ↑↑↑ | ↑↑↑↑ | |
NFATc1 | trace | ↑↑ | ↑ | ↑↑ | ↑↑↑ | ↑↑↑↑ | |
OPG | ↑↑↑↑ | ↑↑ | trace | trace | trace | trace | |
RANKL/OPG ratio | 1.34 | 63.8 | 157 | 393 | 5192 | 3092 |
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Shimazaki, E.; Karakida, T.; Yamamoto, R.; Kobayashi, S.; Fukae, M.; Yamakoshi, Y.; Asada, Y. TGF-β and Physiological Root Resorption of Deciduous Teeth. Int. J. Mol. Sci. 2017, 18, 49. https://doi.org/10.3390/ijms18010049
Shimazaki E, Karakida T, Yamamoto R, Kobayashi S, Fukae M, Yamakoshi Y, Asada Y. TGF-β and Physiological Root Resorption of Deciduous Teeth. International Journal of Molecular Sciences. 2017; 18(1):49. https://doi.org/10.3390/ijms18010049
Chicago/Turabian StyleShimazaki, Emi, Takeo Karakida, Ryuji Yamamoto, Saeko Kobayashi, Makoto Fukae, Yasuo Yamakoshi, and Yoshinobu Asada. 2017. "TGF-β and Physiological Root Resorption of Deciduous Teeth" International Journal of Molecular Sciences 18, no. 1: 49. https://doi.org/10.3390/ijms18010049