轉錄因子Forkhead Box M1可以促進細胞增生、分化以及癌症生成。過去研究指出FOXM1也在腫瘤形成、血管新生以及癌症轉移扮演了重要的角色。但是增加FOXM1在肺臟上皮細胞表現對於修復的功能尚未清楚。我們使用轉殖基因鼠CCSP-rtTA/tetO-FOXM1ΔN在肺臟上皮前驅細胞－Club cell表現高活性的FOXM1ΔN蛋白，再由腹腔注射萘以誘導氣道上皮損傷，經免疫組織染色（IHC）結果顯示表現轉殖FOXM1的小鼠，其細支氣管上皮 CCSP陽性細胞數量在修復前期增多。我們利用CCSP-creER/LSL-LacZ/LSL-rtTA/tetO-GFP-FOXM1ΔN四重基因轉殖小鼠追踪肺損傷後的抗萘幹細胞的生長。其樣本進行X-gal染色後，有LacZ表現的抗萘細胞被標記成藍色。CCSP-creER/Brainbow2.1/LSL-rtTA/tetO-GFP-FOXM1ΔN小鼠的CCSP表現細胞可以螢光標定，單顆抗萘幹細胞增殖得以被追蹤。我們進一步使用共軛焦顯微鏡掃描樣本並建構出2D及3D圖像以顯示幹細胞及其子細胞的立體分布。定量結果顯示在表現FOXM1的組別中，被標記的細胞數量顯著增加，推測FOXM1可以促進幹細胞及其子細胞增生。我們將可誘導表達或是抑制FOXM1的人類肺臟上皮細胞－NL-20透過懸浮培養，結果顯示增加FOXM 1表現可促進細胞球形成以及增生。另外，我們使用染色以及流式細胞儀發現增加FOXM1表現可促進NL-20懸浮細胞球中細支氣管肺泡幹細胞(BASC)數。在本研究中，結果顯示FOXM1促進肺臟上皮損傷修復，抗萘細胞增殖和細支氣管肺泡幹細胞數量及增生來幫助肺臟細支氣管上皮再生。

Forkhead Box M1 (FOXM1) is a transcription factor which plays an important role in cell proliferation, differentiation, and tumorigenesis. However, the role of FOXM1 overexpression in pulmonary injury remains unknown. Previously our lab has generated the CCSP-rtTA/tetO-FOXM1ΔN mice that renders conditionally to express a constitutively active form of FOXM1 in Club cells. Naphthalene-induced lung airway epithelium injury was analyzed by immunohistochemistry staining, we found that transgenic expression of FOXM1 caused increase of the number of the Club cell in bronchiolar epithelium at the early stage of regeneration. Moreover, CCSP-creER/LSL-LacZ/LSL-rtTA/tetO-FOXM1ΔN and CCSP-creER/Brainbow2.1/LSL-rtTA/tetO-FOXM1ΔN mice were used for naphthalene-resistant Club cells lineage tracing after injury. The naphthalene-resistant Club cells which contain stem cells were labelled and the results showed that increased labelled cells were detected in regenerating bronchiolar epithelium. Additionally, inducible FOXM1 gain-of-function and loss-of-function in human lung epithelial NL-20 cells were generated and used for sphere culture, and we found that increased FOXM1 levels promote CCSP/pro-SPC dual expressing BASC population in suspension culture. Intratracheal injected FOXM1 expressing-NL20 cells were able to attach and grow on the injured lung bronchiolar epithelium of nude mouse, suggesting FOXM1 expression increased BASC proliferation and help injury repair. Altogether, we conclude that FOXM1 promotes lung repair by stimulating lung progenitors and stem cell proliferation.

Contents
摘要 1
Abstract 2
Contents 3
Introduction 9
Lung is primary respirational organ 9
Structure of airway epithelium 9
Specific Club cell in lung epithelium 10
Multiple types of stem cells in lung 11
Acute pulmonary injury and injury mouse model 12
Stem cells participate in tissue regeneration 13
Role of FOXM1 in development and cell proliferation 14
Hypothesis 16
Materials and methods 17
Mouse model 17
Gene induction and pulmonary injury 17
Sample processing 18
X-gal staining 19
Tissue embedded in paraffin, OCT or agarose 19
Immunochemistry (IHC) and NFR contrast staining 20
Immunofluorescence (IF) staining 21
Tissue clearing 21
Confocal microscope 22
Image processing 22
Real-time reverse transcription PCR analysis 22
Cell culture 23
Lent-viral gene transduction 23
Sphere-forming culture 24
Immunofluorescence staining of Sphere 24
Flow cytometry 24
Intratracheal injection 25
Statistical analysis 26
Result 27
Inducible system drives FOXM1ΔN expression in CCSP positive Club cells by Doxycycline activation. 27
FOXM1 benefited in injury repair by improving Club cell proliferation. 28
The stem cell marker was highly expressed in CCSP/FOXM1. 29
The Naphthalene-resistant cell could be identified by lineage tracing. 30
FOXM1 promoted Naphthalene-resistant cell proliferation after pulmonary injury. 31
3D image is helpful for proliferation pattern tracing. 32
Sphere culture was used to mimic condition in vivo and specific culture medium was used for stem cell survival. 32
FOXM1 is important in NL-20 sphere growth. 33
BASCs cell marker could be detected in NL-20 cell sphere. 34
FOXM1 improved BASCs cell population. 34
Engraftment of NL-20 sphere cells to injured lung airways of nude mouse facilitated bronchiolar epithelium repair. 35
Conclusion 36
Discussion 37
FOXM1 improved the proliferating cell population to help bronchiolar epithelium regeneration. 37
FOXM1 increased the population of a specific type of stem cell. 39
The BASCs from sphere culture were functional in injury repair 41
Reference 42
Figure 54
Figure 1. Constitutively active form of FOXM1 protein 54
Figure 2. Gene induction and drug treatment of CCSP/FOXM1 mice 55
Figure 3. IHC staining of CC10 show the Club cells in BADJ and bronchiole region. 56
Figure 4. FOXM1 improved the CCSP positive cell number after pulmonary injury. 57
Figure 5. FOXM1 improved the number of proliferating cells after lung injury 59
Figure 6. Transgenic FOXM1 was highly induced and triggered cell proliferation. 61
Figure 7. FOXM1 promoted the stem cell marker of mouse improvement after pulmonary injury. 62
Figure 8. The inducible system and drug treatment processing of CCSP/LacZ/FOXM1. 63
Figure 9. Naphthalene-resistant Club cells were traced by blue color. 64
Figure 10. FOXM1 improved the number of naphthalene-resistant cells after lung injury. 65
Figure 11. Sequence of Brainbow2.1 67
Figure 12. The single cell labeled with Brainbow2.1 was traced by color. 68
Figure 13. FOXM1 expressed in Club cell can be detected by GFP. 69
Figure 14. 3D structure shows the arrangement of Club cells. 71
Figure 15. Procedure of sphere culture. 73
Figure 16. PINDUCER transfection result. 74
Figure 17. FOXM1 is important in NL-20 sphere growth. 76
Figure 18. FOXM1 is important in cell proliferation. 78
Figure 19. CCSP, pro-SPC, BASCs cell marker can be tested in sphere culture. 79
Figure 20. Co-localization of CCSP and pro-SPC 80
Figure 21. FOXM1 increase the population of BASC in NL-20 sphere. 82
Figure 22. The procedure of Nude mice injury and cell injection 83
Figure 23. NL-20 sphere helped nude mice injury repair. 84
Figure 24. Expression of human genes in Nude mice. 85
Appendices 86
Supplemental figure 1. CCSP/pro-SPC dual positive cells in NL-20 cell spheres. 86
Supplemental figure 2. P-H3 positive cell in C/Foxm1-/- mice. 87
Supplemental figure 3. Expression of endogenous mouse Foxm1 in C/Foxm1-/-. 88
Supplemental figure 4. BASC population in CCSP/FOXM1. 90
Supplemental figure 5. Sca-1/EpCAM dual positive cells in NL-20 cell sphere 91
Table 92
Table 1. list of mouse 92
Table 2. list of Antibody 93
Table 3. list of primer for qPCR 94
 

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