Effects of metal-induced oxidative stress on endothelial cells in vitro [Elektronische Ressource] = Auswirkung von Metall-induziertem oxidativen Stress auf Endothelzellen in vitro / Roman Tsaryk

Tsarikoff

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2009
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Effects of metal-induced oxidative stress on
endothelial cells in vitro

Auswirkung von Metall induziertem
oxidativen Stress auf Endothelzellen in vitro


Dissertation
Zur Erlangung des Grades
Doktor der Naturwissenschaften
Am Fachbereich Biologie
der Johannes Gutenberg-Universität Mainz


Roman Tsaryk
geb. am 09.06.1981 in Lviv
Mainz, 2009ii


Tag der mündliche Prüfung:12.03.2010
iii

Contents

1. Introduction ......................................................................................................................................... 1
1.1. Metallic materials, implant integration and aseptic loosening of implants .......................... 1
1.1.1. Metallic materials for implantation ................................................................................. 1
1.1.2. Integration of metal implants and the role of endothelial cells ...... 2
1.1.3. Aseptic loosening and metal corrosion ........... 5
1.2. Oxidative stress ................................................................................................................... 8
1.2.1. ROS formation and antioxidant defence mechanisms .......................... 8
1.2.2. Oxidative DNA damage and repair ...................... 11
1.2.3. p53 pathway and cell cycle regulation ................................................................................ 13
1.3. HIF-1 pathway......................................................... 17
1.4. Aim of the study ................................................................ 20
2. Materials and methods ..................... 21
2.1. Materials ..................................................................................................................................... 21
2.1.1. Chemicals ............................. 21
2.1.2. Buffers . 23
2.1.3. Instruments ......................................................................................................................... 25
2.1.4. Consumables ....................... 26
2.1.5. Kits ....................................................................................................................................... 27
2.1.6. Antibodies............................ 27
2.1.7. Oligonucleotides .................................................................................................................. 28
2.1.7.1. Primers ............ 28
2.1.7.2. siRNA .............. 29
2.1.8. Biomaterials ......................................................................................................................... 29
2.1.8.1. Ti6Al4V preparation ....................... 29
2.1.8.2. CoCl solution ................................................................................................. 29 2
2.1.8.3. TiO -coating of Co28Cr6Mo alloy . 29 2
2.1.9. Software .............................................................................................. 30
2.2. Methods ..................................................................... 30
2.2.1. Isolation and culture of endothelial cells ............ 30
2.2.2. Cell seeding .......................................................................................................................... 31
2.2.3. Cell treatment with various compounds ............. 31
2.2.4. Scanning electron microscopy (SEM) .................................................................................. 32 iv

2.2.5. Immunofluorescent staining ............................................................................................... 32
2.2.6. Cytotoxicity assays ............................................................................................................... 32
2.2.6.1. LDH activity assay .......................... 32
2.2.6.2. MTS conversion assay .................................................................................... 33
2.2.6.3. Crystal violet staining ...................... 33
2.2.6.4. Cell number quantification .............. 34
2.2.7. Oxidative stress evaluation ................................................................................................. 34
2.2.7.1. H O quantification assay ................ 34 2 2
2.2.7.2. DCF assay ....................................................................................................... 34
2.2.7.3. Determination of GSH amount ....... 35
2.2.7.4. Catalase activity assay ..................................................................................... 35
2.2.7.5. SOD activity assay .......................... 35
2.2.7.6. Cathodic corrosion model ............... 36
2.2.8. DNA damage assessment .................................................................................................... 37
2.2.8.1. 8-oxoG staining ............................... 37
2.2.8.2. DNA isolation . 38
2.2.8.3. AP sites quantification .................................................................................... 38
2.2.9. siRNA technique .................................................. 39
2.2.10. Analysis of gene expression .............................. 39
2.2.10.1. RNA Isolation ............................................................................................... 40
2.2.10.2. Reverse transcription ..................... 40
2.2.10.3. Quantitative RT-PCR .................................................................................... 40
2.2.10.4. Microarrays ................................................................................................... 41
2.2.11. Analysis of protein expression........................... 42
2.2.11.1. ELISA ............ 42
2.2.11.2. Detection of protein expression in fixed cells ............................................... 43
2.2.11.3. Whole cell protein extraction ........................................ 43
2.2.11.4. Nuclear protein extraction ............. 44
2.2.11.5. Protein quantification assays ......................................... 44
2.2.11.5.1. BCA assay .............................................................................................. 44
2.2.11.5.2. Bradford assay ........................ 44
2.2.11.5.3. NanoOrange assay .................................................................................. 45
2.2.11.6. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ............................... 45
2.2.11.8. Western blot .................................................................................................. 45
2.2.12. Angiogenesis assay ............ 46
2.2.13. Statistical analysis .............. 46
3. Results ............................................................................................................................................... 47 v

3.1. Oxidative stress in endothelial cells on Ti6Al4V alloy ................................................................ 47
3.1.1. Response of endothelial cells to H O ................................................................................. 47 2 2
3.1.2. Phenotype of endothelial cells growing on Ti6Al4V alloy ................... 49
3.1.3. Evaluation of cytotoxicity of H O on endothelial cells grown on Ti6Al4V ......................... 51 2 2
3.1.4. Oxidative stress in endothelial cells exposed to Ti6Al4V surface ....................................... 54
3.1.5. Effects of cathodic partial reaction of Ti6Al4V corrosion on HDMEC . 58
3.1.6. Effects of oxidative stress on Ti6Al4V alloy on pro-inflammatory activity of HDMEC ........ 62
3.1.7. Angiogenic potential of HDMEC on Ti6Al4V ........................................................................ 71
3.2. The effect of TiO -coating of Co28Cr6Mo alloy on endothelial cells ......... 73 2
3.3. Response of endothelial cells to CoCl ....................................................................................... 80 2
3.3.1. CoCl induces cytotoxicity and oxidative stress in HDMEC ................. 80 2
3.3.2. CoCl causes DNA damage and p53 up-regulation in HDMEC ............................................ 83 2
3.3.3. HIF-1α dependency of CoCl -induced response in HDMEC ................. 87 2
4. Discussion .......................................................................................................... 95
4.1. Response of endothelial cells to Ti6Al4V alloy ........................................... 95
4.1.1. Ti6Al4V-induced oxidative stress ........................................................ 95
4.1.2. Cathodic half-reaction of corrosion as the possible source of oxidative stress on Ti6Al4V
alloy ............................................................................................................. 100
4.1.3. Oxidative stress on Ti6Al4V alloy and pro-inflammatory response of endothelial cells ... 102
4.1.4. Angiogenesis on Ti6Al4V alloy ........................................................................................... 109
4.2. Effects of Co28Cr6Mo alloy and Co-ions on endothelial cells .................. 110
4.2.1. TiO coating improves biocompatibility of Co28Cr6Mo alloy to endothelial cells ............ 111 2
4.2.1.1. Effects of TiO -coating on Co28Cr6Mo-induced changes in proliferation .. 112 2
4.2.1.2. TiO -coating reduces the induction of stress response to Co28Cr6Mo alloy 113 2
4.2.2. Cobalt ion effects on the functions of endothelial cells .............................