Role of interleukin-8 in odontogeneic differentiation of stem cells from human exfoliated deciduous teeth cultured on human amniotic membrane
Interleukin-8 (IL-8), an inflammatory cytokine with pleiotropic biological effects, has been reported to be implicated in odontogenesis and involved in odontoblast-mediated immune responses. However, the exact mechanism on how IL- 8 influences odontogenesis remains unclear. Hence, this study was...
محفوظ في:
| المؤلف الرئيسي: | |
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| التنسيق: | أطروحة |
| اللغة: | English |
| منشور في: |
2018
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| الموضوعات: | |
| الوصول للمادة أونلاين: | http://eprints.usm.my/56548/1/Dr.%20Wafa%27%20Zahari-24%20pages.pdf |
| الوسوم: |
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| الملخص: | Interleukin-8 (IL-8), an inflammatory cytokine with pleiotropic biological
effects, has been reported to be implicated in odontogenesis and involved in
odontoblast-mediated immune responses. However, the exact mechanism on how IL-
8 influences odontogenesis remains unclear. Hence, this study was conducted to
investigate the role and mechanism of IL-8-immunomodulatory pathway in the
differentiation of stem cells from human exfoliated deciduous teeth (SHED) into
odontoblast-like cells. SHED were seeded on human amniotic membrane (HAM) and
treated with bone morphogenetic protein-2 (BMP-2). Four groups were assigned:
SHED only (S), SHED with BMP-2 (SB), SHED on amniotic membrane without
BMP-2 (SA), SHED on amniotic membrane with BMP-2 (SAB). Following
treatment, SHED were harvested on day 1, 7, 10 and 14, and odontogeneic
differentiation potential was assessed by the expression of odontogeneic markers
using reverse transcriptase polymerase chain reaction and calcium deposition by
Alizarin Red S staining. Thereafter, the optimal concentration of reparixin and rhIL-8
were determined using Real Time RT-PCR. For IL-8 study, another four groups were
assigned; SHED on amniotic membrane with BMP-2 (SAB), SHED on amniotic
membrane with BMP-2 and reparixin (SABR), SHED on amniotic membrane with
BMP-2 and rhIL-8 (SAB8), SHED on amniotic membrane with BMP-2, reparixin
and rhIL-8 (SABR8). SHED were treated with reparixin as IL-8 inhibitor, while recombinant human IL-8 (rhIL-8) as IL-8 inductor, and downstream analysis were
conducted using Real Time RT-PCR. The levels of IL-8 protein secretion of SHED
with and without IL-8 induction and inhibition during odontogeneic differentiation
was analysed using ELISA. The effect of IL-8 in calcium deposition of SHED was
determined using Alizarin Red S staining. Results of the present study showed that
odontoblast specific gene markers DSPP, DMP1, OPN were highly expressed on day
7 onwards as odontogeneic differentiation occurred. On the other hand, the BMP-2
and COL-1 expressions remain lowly expressed. The optimal concentration for
reparixin and rhIL-8 were found to be 50 nM and 0.01 ng/ml, respectively. For IL-8
downstream pathway analysis, PI3K/AKT/mTOR and JAK2/STAT3 signalling
pathways were suggested to be involved in odontogeneic differentiation as the
expression of all the markers were high, whereas inhibition of IL-8 using reparixin
caused significant reduction of their expression. NF-κB expression showed relatively
low expression in all samples when compared to other genes suggesting that NF-κB
signalling plays a minor role in odontogeneic differentiation. In addition, upregulation
of DSPP, DMP1 and OPN expression, and higher intensity of alizarin
staining were observed when SHED were inhibited with reparixin (SABR) suggested
odontogeneic differentiation has occurred. However, no significant changes were
observed in the expression of odontoblast markers and SHED mineralisation when
SHED were induced with rhIL-8. In conclusion, this study suggests that inhibition of
IL-8 receptor by reparixin promotes odontogeneic differentiation of SHED when
cultured on HAM treated with BMP-2. |
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