Molecular characterisation of odontoblast during primary, secondary ...

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Figure 20: Post-array Sq-RT-PCR analysis showing differential expression in Early stage (ES) and Late Stage (LS) odontoblasts. This sq-RT-PCR was performed to validate the microarray results and to characterize transcript levels of genes involved in the p38 MAP Kinase Pathway (PTPRR, NTRKK2, MAPK13, MAP2K6, MKK3. .......................... 88 Figure 21: Ontological classification of genes up- and down-regulated between young and mature odontoblasts. Only processes involving more than 20 genes are shown. .. 90 Figure 22: The p38-MAPKinase pathway (Pathway-Express software http://vortex.cs.wayne.edu). Differentially egulated genes as identified by microarray analysis are highlighted in yellow. .............................................................. 92 Figure 23: (A) Histology of a bovine unerupted first upper molar cusp, 5μm thickness section, haematoxylin and eosin staining, bar=500μm. (B-C) Immunohistochemistry with anti-DSP antibody on odontoblasts in the coronal area (B) and apical area (C). Expression of protein is visibly unchanged in both sections highlighting the secretory activity of the cells. (D-E) Immunohistochemistry using the anti-DMP1 antibody in odontoblasts in the coronal (D) and apical areas (E). The differential expression of DMP1 protein between both populations of cells corroborates the reported gene expression data (B,C,D,E : Bar=100μm). ....................................................... 94 Figure 24: Mouse first upper incisor (A) 5μm thickness, haematoxylin and eosin staining. (B) Immunohistochemical analysis using anti-mouse phosphorylated p38 antibody in apical area of the erupted mouse tooth. Nuclear staining of odontoblasts is evident, and phosphorylated-p38 is apparently also present in the cells of the pulpal parenchyma. (C) Immunohistochemistry using the same antibody performed on cells nearer the incisal tip, with mature odontoblasts. Staining is enhanced in the apical area, indicating the higher concentration and activity of p38 protein in the young cells compared to that of the mature ones. Cytoplasm of mature cells is free of staining, and nuclear staining is strong indicating that p38 is potentially acting as a transcription factor. P= Pulp – od= Odontoblasts – D = dentine (A) Bar= 1 mm (B) Bar= 50 μm (C) Bar=75 μm. ..................................................................................... 95 Figure 25: Phosphorylated p38 protein expression at a basal level in untreated MDPC- 23 cells and after cell treatment with anisomycin, SB203580 (p38 phosphorylation inhibitor), TGF-β1, TGF-β1+antiTGF-β1 antibody, ADM, TNF-α, Dentine Matrix Proteins for 15, 60, 180mins and 24hours. Bar = Standard deviation. *: statistically significant difference from control. .......................................................................... 97 Figure 26: Phoshorylated p38 protein expression in cytoplasmic and nuclear localisations of MDPC-23 cells, after stimulation with TNF-α, Streptococcus mutans (SM), ADM, DMPs, TGF-β1, TNFα+TGF-β1, ADM+SM, DMP+SM, TGF-β1+SM for 60 10
minutes. (DMP = dentine matrix protein preparation, SM = Streptococcus mutans). Bar = Standard deviation. *: statistically significant difference from control. ............... 98 Figure 27: Histology of the first upper molar in WT, +/- and -/- mice, at 7, 14 and 21 post-natal days. 7μm thickness sections with haematoxylin and eosin staining. Bar=500µm ......................................................................................... 100 Figure 28: First upper molar in Msx2 -/- 7 days post natal. 7μm thickness, haematoxylin and eosin staining. (A): Bar= 250µm (B)-(C): Higher magnification; Bar= 50µm. ............................................................................................... 100 Figure 29: First upper molar in +/+ (A and D), +/- (B and E), -/- (C and F). 7 days post natal days. 7μm thickness, haematoxylin and eosin staining. (D, E and F) higher magnification of odontoblast layer. Note inverted polarisation of odontoblasts in -/animals (black arrows). (A, B, C), bar=200µm – (D, E, F), bar= 70µm. ................... 101 Figure 30: Comparison of dentine structure on +/+ (A) and Msx2 -/- (B) first upper molar 14 days post natal mice. 7μm thickness with haematoxylin and eosin staining. Bar = 100µm ....................................................................................... 102 Figure 31: Dentine structure in the coronal area of the second upper molar of 14 days post- natal Msx2 -/- mice. A line is clearly visible in the thickness of the coronal dentine, which may represent a landmark of an episode of dysregulation of odontoblast secretory activity. 7μm thickness, haematoxylin and eosin staining. A: Bar= 300µm, B: Bar=100µm, C: Bar=50µm. ................................................... 102 Figure 32: Dentine structure of a first upper molar from a 21 days post natal Msx2 -/mouse showing different areas of the crown of the tooth. Abnormalities of dentine structure were obvious, with the presence of vacuoles in the mineralized tissue thickness, which may be a landmark of cellular inclusions. 7μm thickness, haematoxylin and eosin staining. A: Bar= 200µm, B, C, D: Bar=50. ...................... 103 Figure 33: Dentine structure of first upper molar in a 4 month old Msx2 -/- mouse. (A) The line of disturbed matrix secretion in the dentine is still clearly visible (black arrow) (B and C) : dentine structure at higher magnification. 7μm thickness, haematoxylin and eosin staining. A: Bar= 150µm, B,C: Bar=50µm. ....................... 104 Figure 34: Immunohistochemical analysis using anti-mouse beta-galactosidase antibody on first upper molars of 7 days post natal Msx2 +/-mice. Nuclear staining of a few odontoblasts and pulp cells is evident. Counterstained with haematoxylin. A: Bar=150µm, B,C,D: Bar= 60µm. ................................................................. 105 Figure 35: Immunohistochemical analysis using anti-mouse beta-galactosidase antibody on first upper molar of a 14 days post natal Msx2 -/- mouse. Nuclear staining of a few 11
Page 1 and 2: ECOLE DOCTORALE : Génétique, Cell
Page 3 and 4: ACKNOWLEDGEMENTS I wish to express
Page 5 and 6: Abstract : This research aimed to i
Page 7 and 8: CONTENTS List of Figures ………
Page 9 and 10: 4.5.2 MSX2 and pulp repair ........
Page 11: Figure 11 : Dentine bridge formatio
Page 15 and 16: lipofectamin ® vector only (MDPC v
Page 17 and 18: List of Tables: Table 1: Primer det
Page 19 and 20: 1.1 Towards a biological approach I
Page 21 and 22: Introduction odontoblast processes
Page 23 and 24: Introduction Secondary dentine: thi
Page 25 and 26: Introduction Understanding the dist
Page 27 and 28: Introduction terms of the communica
Page 29 and 30: Figure 5 : There is a differential
Page 31 and 32: Introduction Movement of the dentin
Page 33 and 34: 1.2.3.3 Consequences of new technol
Page 35 and 36: Introduction an apical nucleus, whe
Page 37 and 38: Introduction creates a physical bar
Page 39 and 40: Introduction polarisation and the d
Page 41 and 42: Introduction variations in pressure
Page 43 and 44: Introduction also express neurogeni
Page 45 and 46: 1.4.1 Reactionary dentinogenesis In
Page 47 and 48: Introduction responses, including a
Page 49 and 50: Introduction superficial cells move
Page 51 and 52: Introduction induce dentine bridge
Page 53 and 54: Introduction Because of its highly
Page 55 and 56: Introduction (Hayashi 1982). At 11
Page 57 and 58: Introduction To date, involvement o
Page 59 and 60: Introduction In deeper cavities, wh
Page 61 and 62: Introduction dental papilla cells.
Page 63 and 64:
2 Materials and Methods Material an
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Material and methods containing; 12
Page 67 and 68:
2.1.2.3 Microarray analysis Materia
Page 69 and 70:
Material and methods 2.2 Part Two:
Page 71 and 72:
Material and methods a Modulyo free
Page 73 and 74:
Material and methods Figure 16: Pri
Page 75 and 76:
Material and methods To date, sever
Page 77 and 78:
Material and methods (pH=6.8) with
Page 79 and 80:
Material and methods (P), and calci
Page 81 and 82:
Material and methods (Sigma, la Ver
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Material and methods visualized by
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2.4.2.6 MSX2 over-expression in imm
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Material and methods channel. Data
Page 89 and 90:
Results Amelogenin, IBSP, DCN, Alka
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Results data previously reported fo
Page 93 and 94:
91 MAPK PATHWAY probe set GENE ID b
Page 95 and 96:
Results Alkaline phosphatase and am
Page 97 and 98:
Figure 24: Mouse upper incisor (A)
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Figure 25: Phosphorylated p38 prote
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3.3 MSX2 proteins and dentinogenesi
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Figure 29: First upper molar in +/+
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3.3.1.3 At 21days post natal : Resu
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Results expression, obtained using
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Figure 37: Immunohistochemical anal
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3.3.4 BrdU IHC Results BrdU was inj
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3.3.6 Quantitative RT-PCR (Q-RT-PCR
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Results 3.4 Design and use of a new
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Results Figure 46: Histology at 2 w
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Results cells is also characteristi
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4 Discussion Discussion 4.1 Regulat
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Discussion odontoblast secretory be
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Discussion 4.2 Molecular characteri
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Discussion al. 2004; Magloire, Coub
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Discussion differences. Bone cells
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Discussion we can conclude that the
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Discussion pathway is complex and i
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Discussion odontoblast physiologica
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Discussion dentinogenesis. By perfo
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Discussion cellular and molecular s
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Discussion Notably, our results are
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Discussion contribute to reparative
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Discussion other species (Abedi, To
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Discussion dentinogenesis, some cel
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Discussion morphology. It was not p
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5 Conclusion Discussion Research on
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Discussion Figure 50: 14 months pos
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References during normal and in vit
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References Faraco, I. M., Jr. and R
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References Kidd, E. A., A. Banerjee
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References antigen-expressing cells
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References Smith, A. J. and H. Leso
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References Yamashiro, T., M. Tummer
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References Appendix 1: Full list of
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probe set GENE ID baseline mean (LS
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Appendix 3: Molecular characterizat
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Appendix 5: Evaluation of a new lab
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