The expression of the BDNF family of neurotrophic factors in dental pulp stem cells within adult teeth

Authors

Patrick Suezaki, University of the Pacific
Der Thor, University of the PacificFollow
Nan (Tori) Xiao, University of the PacificFollow

Lead Author Affiliation

Dental Surgery Program

Introduction/Context/Diagnosis

The brain derived neurotrophic factor (BDNF) family is one of the many neurotrophic factors implicated in human Neurogenesis. BDNF expression from DPSC cells may have an effect on differentiation and survival of the pulp tissues found in teeth after trauma. Through this experiment, we show that DPSC cells express and release BDNF to its surrounding tissues and show the presence of other ligands important to the BDNF signaling pathways.

Methods/Treatment Plan

Human DPSCs cells were gift from Dr. Songtao Shi, Chair and Professor of Department of Anatomy & Cell Biology, School of Dental Medicine University of Pennsylvania. PCR: RNA was isolated from the stem cells using TRIzol Reagent [Life Technologies, California]. cDNA was transcribed from the purified RNA via reverse transcription. DNA was amplified from cDNA through polymerase chain reaction (PCR). Western Blot: Cells were collected and processed for protein. 50 μg total protein was resolved by SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked and probed with TrkB antibody (abcam). Image was captured with Genesnap. Immunofluorescent staining: DPSC were cultured on coated coverslips, fixed and stained. The images were acquired using Leica TSC SPE confocal microscope. ELISA was performed following the manufacturer’s instruction (Abcam). Serum free culture medium was concentrated and incubated overnight in the ELISA plate. Signals were read at 450nm and the concentration of each sample was calculated based on the standard curve.

Results/Outcome

The following genes were present on the gel at respective base pair (bp) measurements for the DPSC tooth samples: actin at 150 bp, Ngf at 150 bp, Bdnf at 100 bp, Ntf3 at 300 bp, Ntf4/5 at 200 bp, TrkC at 125 bp, P75 at 150bp. We did not see bands present for TrkA and TrkB in the PCR. In our ELISA, we saw a significant amount of BDNF present in the serum in the overnight secretion study. We further confirmed that DPSC express BDNF through immunofluorescent staining. The western blot showed positive bands for TrkB.

Significance/Conclusions

Through this experiment, we were able to confirm through PCR the presence of RNA for Ngf, Bdnf, Ntf3, Ntf4/5, TrkC and P75. We further confirmed the presence of BDNF through the ELISA and the Immunofluorescent staining. In the western blot, we found positive bands for Trk B receptor protein across all of the DPSCs from different donors. Future experiments should include utilizing different primer pairs for the PCR for Trk A/B due to the initial primers not being optimal for the PCR. We should also include Western blots data for BDNF to further confirm the presence of BDNF. Additionally, we can further investigate the changes in neurotrophic factors regulating DPSC under normal physiological and stressful conditions, to see the effect of exogenous neurotrophic factors on both stressed and non-stressed DPSC.

Comments/Acknowledgements

This work is supported by Research Enhancement Award 03-Activity 108 from the University of the Pacific, Arthur A. Dugoni School of Dentistry

Location

University of the Pacific, Dugoni Dental School, San Francisco, CA

Format

Poster

Poster Session

1st and 2nd Year Student Research Presentations