Title Page
Contents
Abbreviations 10
Abstract 11
1. Introduction 13
2. Literature review 16
2.1. Anatomy and physiology of salivary glands 16
2.2. The classification of stem cells 18
2.3. The definition of salivary gland stem cell and their prospection in the salivary gland regeneration 20
2.4. The concept of microgravity and how microgravity affects the growth of cells and stem cells 22
2.5. History of hypergravity experiment on living organism and the effects of hypergravity on stem cells 25
3. Methodology 28
3.1. Isolation of human minor salivary gland stem cells 28
3.2. Cell proliferation evaluation 29
3.3. In vitro differentiation 30
3.4. Flow cytometric analysis 32
3.5. Creation of huMSGSC sphere form 33
3.6. Gene expression analysis 34
3.7. Protein analysis by Western blot 35
3.8. Immunoflurorescence stainning 36
3.9. Periodic acid-Schiff (PAS) staining 36
3.10. Using "Gravite" instrument to create the nearly zero stimulate gravity on earth 37
3.11. Creating the hyper gravity environment by centrifuge 37
3.12. Statistical analysis 38
4. Results 41
4.1. The characteristics of the huMSGSC isolated 41
4.1.1. The morphology and proliferation of huMSGSC 41
4.1.2. Potential differentiation of human minor salivary gland stem cells 42
4.1.3. The characteristics of huMSGSC 44
4.2. The effects of Gravity on huMSGSC 46
4.2.1. The proliferation of human minor salivary gland stem cells under different gravity 46
4.2.2. The ability of sphere form creation of huMSGSC after treatment with different gravity environments 48
4.2.3. Expression of stem cell markers of huMSGSC in different gravity environments 51
4.2.4. Cell-cell junctions were changed after exposing under different gravity environments 55
4.3. Hyper gravity support for human minor salivary gland stem cells differentiate 59
4.3.1. The morphology of huMSGSC under induced environment 59
4.3.2. PAS staining of huMSGSC under induced environment 60
4.3.3. Gene expression of huMSGSC under induced environment 61
4.3.4. Gene structure related to cell association of huMSGSC under nduced environment 62
5. Discussion 64
5.1. The characteristics of the huMSGSC isolated 64
5.2. The effects of Gravity on huMSGSC 66
5.2.1. Impact of Gravity on the development of huMSGSC 66
5.2.2. Effect of Gravity on stemness expression and cell association of huMSGSC 67
5.3. Hyper gravity support for human minor salivary gland stem cells differentiate 69
6. Conclusion 70
References 72
Figure 1. Salivary gland. (a) Major salivary gland117, (b) Minor salivary gland (purple area) 17
Figure 2. The classification of stem cell 19
Figure 3. Gravite machine 38
Figure 4. HuMSGSC isolation and effects of Gravity 39
Figure 5. Effects of hyper gravity on huMSGSC differentiation 40
Figure 6. The morphology and proliferation of huMSGSC. (a) Cell morphology of huMSGSC passage 3. (b) the cell growth of huMSGSC... 41
Figure 7. Potential differentiation of huMSGSC. The multi-lineage differentiation of the huMSGSC (d,e,f,i,j,g). Microscope view for... 42
Figure 8. The characteristics of huMSGSC. Flow cyctometry of huMSGSC. The average of the positive cells for specific stem cell markers... 44
Figure 9. Proliferation of human minor salivary gland stem cells under different gravity. Using CCK8 kit test. Control (1 gravity group), HP-G... 46
Figure 10. The ability of sphere form creation of huMSGSC after treatment with different gravity environments. The morphology of... 49
Figure 11. Expression of stem cell markers of huMSGSC in different gravity enviroments. Stem cell marker analysis by real time PCR, *p... 51
Figure 12. Expression of stem cell marker of protein detected by Western-blot test, **p 〈0.01, ***p 〈0.001, #p〉0.05. 52
Figure 13. Immunoflurocence staining for huMSGSC at different gravity Stem cell markers (a) LGR5, (b) CD90, (c) SOX2. (1) control group: 1... 54
Figure 14. Expression of cell-cell junction primer with real time PCR test. 1G: 1 gravity, Hyper-G: 40 gravity, Micro-G: nearly zero gravity. **p... 55
Figure 15. Protein expression of huMSGSC with Western-blot test, **p〈0.01, ***p 〈0.001 56
Figure 16. Immunoflurocent of cell-cell junction protein of huMSGSC under different gravity enviroment. (a) Zo-1, (b) Zo-2, (1) Control group:... 58
Figure 17. The morphology of huMSGSC under induced environment. 20x magnification microscope image, control group (using normal... 59
Figure 18. PAS staining of huMSGSC under induced environment. PAS staining of huMSGSC for two control groups (normal culture... 60
Figure 19. Gene expression of huMSGSC under induced environment, *p 〈0.05, **p 〈0.01, ***p 〈0.001, #p〉0.05. 61
Figure 20. Gene structure related to cell association of huMSGSC under induced environment, *p 〈0.05, ***p 〈0.001, #p〉0.05. 62