臺灣博碩士論文加值系統

中文摘要
背景：皮膚長期受到紫外線照射，會造成光老化 (photoageing) 的現象。脈衝光是一種非侵襲性臉部皮膚光回春術(photorejuvenating)，在臨床研究上，已經被證實會使老化皮膚回春。然而，目前脈衝光影響機制卻尚未研究清楚，我們的研究目的在探討脈衝光照射對於培養在膠原蛋白三度空間培養系統之皮膚纖維母細胞的影響。並藉此來研究皮膚纖維母細胞年輕化過程中的基因變化。
材料&方法：我們將人類真皮纖維母細胞培養在膠原蛋白三度培養空間系統中，利用脈衝光照射24小時後研究脈衝光所造成的影響。藉由色素排除法來分析細胞的存活率；zymography分析間質金屬蛋白脢-2 (MMP-2)的活性；以即時定量聚合酶鏈鎖反應分析基因表現；蛋白質的表現以西方墨點及ELISA來分析。為了進一步闡明脈衝光影響的機制，我們利用cDNA微陣列的技術來分析經由脈衝光照射後人類纖維母細胞的整體基因表現，並尋找有差異表現之基因。在這些表現不同的基因當中，其中干擾素誘發蛋白 IFI27 （Interferon-induced protein 27）基因有明顯增加的情形。我們利用免疫組織化學染色與即時定量聚合酶鏈鎖反應來分析IFI27在年輕與老化纖維母細胞中的表現。我們以血小板衍生性生長因子(PDGF)刺激來確認IFI27表現量的提高是否與細胞生長有關。接著以IFI27 shRNA來降低IFI27表現量，並檢視細胞週期的改變情況。除研究IFI27、PCNA、cyclin A、D、E等基因的表現量高低，並以MTT assay來評估降低IFI27的表現量之後纖維母細胞增生的情形，細胞週期的分布則以流式細胞儀進行分析。
結果：細胞存活率證實脈衝光照射後會呈現劑量相依性(dose-dependent)增加。脈衝光會減少間質金屬蛋白脢-2的活性及蛋白質與mRNA的表現量，而間質金屬蛋白脢-14 (MMP-14) 及間質金屬蛋白脢抑制素-2 (TIMP-2)的mRNA的表現量也會降低。相反地，纖維母細胞接受脈衝光24小時照射後膠原蛋白III(collagen III)和轉化生長因子-β(TGF-β)的表現量則有增加的情形。cDNA微陣列分析，顯示一組干擾素誘導蛋白的基因表現在照射脈衝光後會受到向上調控，其中包括IFI27基因。在老化的皮膚中，表現IFI27基因的纖維母細胞數明顯降低。我們的研究也顯示IFI27在細胞增生過程中扮演重要角色， IFI27的表現會隨受到PFGF刺激而誘發。IFI27 shRNA轉殖細胞的細胞存活率會降低且細胞週期會停滯在G1期。IFI27 shRNA轉殖細胞中PCNA 、cyclin A與 cyclin E的基因表現量也下降，但對cyclin D則無顯著影響。 流式細胞儀分析結果也顯示S期DNA含量分佈會有降低的情況。
結論：我們的研究結果顯示脈衝光光回春術的可能分子機轉，在於減少間質金屬蛋白脢-2的表現以抑制胞外基質的破壞，加上向上調控collagen III和TGF-β的表現，而促使胞外基質重建。另外藉由脈衝光照射後會造成IFI27基因表現增加。IFI27的表現量會隨著纖維母細胞的老化而降低，在重新進入細胞週期時則可見IFI27的表現量增加，除此之外，在IFI27表現降低的纖維母細胞中，由於cyclin A與cyclin E表現量降低，導致細胞週期停滯在G1/S期。綜合以上結果， IFI27可以作為皮膚纖維母細胞中細胞生長與年輕化的標識。

Abstract
Background: Chronic exposure of skin to solar ultraviolet radiation is known to cause photoaging. Intense pulsed light (IPL), one of the non-ablative facial skin photorejuvenation techniques, had been clinically demonstrated to rejuvenize aged skin. However, mechanisms underlying this IPL effect have not yet been investigated. Our goal was to investigate the effects of IPL upon skin fibroblasts cultured in collagen lattices. In addition, the molecular alterations of cells during rejuvenation process were also studied.
Materials & Methods: Human dermal fibroblasts grown in contracted collagen lattices were treated with IPL and potential effects of IPL were examined 24 hours after the irradiation. Cell viability, MMP-2 activity, mRNA levels of MMPs, cytokines and ECM proteins, and protein levels of cytokines were analyzed by MTT assay, zymography, RT-PCR, Western blotting, and ELISA, respectively. To further elucidate the mechanisms for IPL effects, we used cDNA microarray technique to investigate the gene expression profiles of human fibroblasts irradiated by IPL. Among these differential expressed genes, interferon-induced protein 27 (IFI27) was significantly increased. We studied by immunohistochemistry and RT/real-time PCR to compare the IFI27 expression in young and old skin fibroblasts. The effect of platelet-derived growth factor (PDGF) on stimulating serum deprived skin fibroblast was examined to clarify whether the elevation of IFI27 is growth related. IFI27 shRNA construct was then used to knock down the IFI27 expression and the changes of cell cycle progression and expressions of IFI27, PCNA, cyclin A, D, and E werestudied. MTT assay was also carried out to evaluate the cell proliferation after IFI27 knock-down. Cell cycle distribution after knock-down of IFI27 was analyzed by flow cytometry.
Results: After IPL irradiation, a dose-dependent increase in the number of viable cells was demonstrated. MMP-2 activity and both protein and mRNA levels of MMP-2 were decreased. Decreased mRNA levels of MMP-14 and TIMP-2 were also noted. Conversely, the up-regulation of collagen III and TGF-β were verified. cDNA microarray showed that a group of interferon-inducible proteins were up-regulated. IFI27 was up-regulated among other genes. The number of IFI27(+) fibroblasts wassignificantly reduced in old skin. The decrease of IFI27 expression in cultured-senescent skin fibroblasts was confirmed as well. IFI27 expression in quiescent skin fibroblasts was time-dependently induced by PDGF stimulation. MTT assay demonstrated decreased cell viability in IFI27 shRNA-transfected cells. Cell cycle distribution revealed a cycle arrest at G1 phase. Knockdown of IFI27 by shRNA lead to the decreased expressions of PCNA, cyclin A, cyclin E but not cyclin D in skin fibroblasts. Cytoflow data showed a decreased S-phase cells distribution.
Conclusion: Our results indicated that the photorejuvenating effects of IPL involved both the inhibition of ECM destruction by decreasing the MMP-2 expression, and the increase of ECM construction by up-regulating the gene expressions of collagen III and TGF-β. IFI27 was up-regulated after IPL irradiation. The expression of IFI27 decreased with fibroblast senescence. Increased expression was noticed upon reentry of cell cycle. Down-regulate of cyclin A and E in IFI27 knock down fibroblasts were also noticed. The decreased expression of IFI27 in dermal fibroblasts caused a cycle arrest at G1/S phase. Taken together, these results provide new insight into the role of IFI27 as a novel proliferation and juvenile cell marker for skin fibroblasts.

目 錄
指導教授推薦書
口試委員會審定書
授權書
iii
誌謝
iv
Abbreviations
v
中文摘要
vii
Abstract
ix
目錄
xi
Chapter 1. General Introduction
1
1.1. Photodamage
1
1.2. Matrix Metalloproteinases (MMPs)
2
1.2.1. MMPs in dermatology
2
1.2.2. MMPs and photoaging
3
1.2.3. MMPs and ultraviolet radiation (UVR)
4
1.3. Treatment Modalities of Photoaging
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1.3.1. Review of treatment modalities
10
1.3.2. Review of intense pulsed light (IPL)
10
1.4. 3D Cell Culture Systm
1.5. Characterization of Cell Senescence
12
1.6. Interferon-Inducible Protein 27 (IFI 27)
13
1.7. Specific Aims
15
1.8. Experimental Schemes
17
1.9. Table and Figure
20
Chapter 2. Establishment and characterization of 3D cultured fibroblast system
22
2.1. Background and Purposes
22
2.2. Materials and Methods
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2.2.1. Skin fibroblasts cultured in 3D collagen lattice
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2.2.2. Cell recovery and viability assay
24
2.2.3. Gelatin zymography
24
2.2.4. RNA extraction, RT/ real-time PCR
25
2.2.5. Analysis of cDNA microarray
27
2.3. Results
27
2.3.1. Morphology of dermal fibroblasts cultured in monolayer and 3D collagen lattice
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2.3.2. Activation of MMP-2 and MMP-9 syntheses by skin fibroblasts cultured in monolayer and 3D.
28
2.3.3. Kinetic changes of MMP-2 activity
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2.3.4. MMPs expression profiles of skin fibroblasts cultured in 3D collagen lattices and monolayer by RT-PCR
29
2.3.5. Difference of gene expression profiles of skin fibroblasts cultured in 3D collagen lattices and monolayer by cDNA microarray analysis
29
2.4. Discussion
30
2.5. Tables and Figures
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Chapter 3. Effects of IPL on the Matrix Metabolism and Differential Gene Expression of Fibroblasts cultured in collagen lattices
40
3.1. Introduction
40
3.2. Materials and Methods
42
3.2.1. Cell culture
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3.2.2.Irradiation procedure
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3.2.3. Fibroblasts recovery and viability test
43
3.2.4. Gelatin zymography
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3.2.5. RNA extraction and quantitative RT-PCR
44
3.2.6. Western blog analysis for MMP-2
45
3.2.7. ELISA for TGF-β1
45
3.2.8. Analysis of microarray data
45
3.2.9. Statistical analyses
46
3.3. Results
46
3.3.1. IPL irradiation on human dermal fibroblasts increased viable cells
46
3.3.2. IPL irradiation suppressed the MMP-2 activity
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3.3.3. IPL down-regulated the protein and mRNA levels of MMP-2
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3.3.4. IPL irradiation suppressed the mRNA levels of MMP-14 and TIMP-2
48
3.3.5. IPL irradiation increased the mRNA level of collagen III
48
3.3.6. IPL irradiation increased the protein and mRNA levels of TGF-β1
49
3.3.7. Analysis of IPL effects on fibroblasts cultured in collagen lattices by cDNA microarray
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3.4. Discussion
50
3.5. Figures.
57
Chapter 4. Proliferation-associated expression of interferon alpha-inducible protein 27 (IFI27) is reduced in senescent human skin fibroblasts both in vivo and in vitro
69
4.1. Background
69
4.1.1. Selecting fibroblasts stands for different senescence levels
69
4.1.2. Role of IFI27 on skin aging
71
4.2. Materials and Methods
73
4.2.1. Fibroblast culture
73
4.2.2. 3-D collagen culture
73
4.2.3. IPL irradiation
73
4.2.4. RNA extraction and RT/real-time PCR
73
4.2.5. Tissue sections
74
4.2.6. Immunohistochemical studies
74
4.2.7. MTT assay
75
4.2.8. β-galatosidase (β-Gal) staining
75
4.2.9. Cell cycle study
75
4.2.10. Gene knockdown of IFI27
75
4.2.11. Cell viability
76
4.2.12 Flow cytometry
76
4.3. Results
77
4.3.1 Cell morphology of young and old skin fibroblasts
77
4.3.2. MTT assay to evaluate the proliferation potential of fibroblasts from different age groups
77
4.3.3. Contration of collagen lattices to select fibroblasts with different proliferative potential immunofluorescent
77
4.3.4. Senescence associated (SA) b-galactosidase assay
78
4.3.5. IPL irradiation enhanced the IFI27 mRNA expression in skin fibroblasts
78
4.3.6. Expression of IFI27 protein in the skin dermis from young and old donors
79
4.3.7. Decreased expression of IFI27 in senescent cultured skin fibroblasts
79
4.3.8. PDGF time-dependently increased IFI27 mRNA expression in serum-deprived quiescent skin fibroblasts
80
4.3.9. Effect of IFI27 knockdown on the cell cycle progression
80
4.3.10. Decreased expressions of PCNA, cyclin A and cyclin E in skin fibroblasts with IFI27 knockdown
81
4.4. Discussion
81
4.5. Figures
84
Chapter 5. Future Plans
98
5.1. Background
98
5.1.1. Setting up skin organ culture
98
5.1.2. The role of IFI27 in human keratinocytes
98
5.2 Materials and Methods
101
5.3. Figures
107
References
109
Appendix: List of Publications
139

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