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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 劉扶東(Fu-Tong Liu) | |
dc.contributor.author | Meng-Ping Lu | en |
dc.contributor.author | 呂孟萍 | zh_TW |
dc.date.accessioned | 2021-05-16T16:22:52Z | - |
dc.date.available | 2018-09-24 | |
dc.date.available | 2021-05-16T16:22:52Z | - |
dc.date.copyright | 2013-09-24 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-07-19 | |
dc.identifier.citation | References
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6195 | - |
dc.description.abstract | 半乳糖凝集素-3和-7是動物凝集素成員之一,共同具有可辨認beta型半乳糖修飾醣分子的醣類辨識區域(carbohydrate recognition domain, CRD)。許多研究指出半乳糖凝集素-3可參與細胞之間的交互作用、幫助格狀結構的形成、影響免疫細胞凋亡和分化過程、發炎反應以及腫瘤的發展,半乳糖凝集素-7的研究則著重於維持皮膚生理平衡的角色。先前我們實驗室發現,半乳糖凝集素-3可結合至一個蛋白質-Alix (ALG-2 linked protein-X),Alix本身可以影響細胞凋亡、細胞膜內吞運輸和細胞骨架的重組,近期實驗結果更指出,半乳糖凝集素-3可以幫助Alix維持上皮細胞生長因子受體(epithelial growth factor receptor, EGFR)在細胞膜上的動態平衡和下游訊息傳遞,進而調控老鼠皮膚角質細胞的移動。
我的研究主題是探討半乳糖凝集素-3和-7於人類角質細胞移動的角色,和討論可能影響的細胞機制,內容包括半乳糖凝集素-3是否要透過影響Alix進而調控人類角質細胞的移動,以及半乳糖凝集素-7是否參與了組合蛋白(integrin)調控細胞移動的訊息傳遞。由實驗結果得知,半乳糖凝集素-3基因敲落和Alix基因敲落的人類角質細胞株-HaCaT細胞其傷口癒合能力均下降,並已證實是細胞移動性下降而非細胞增生或死亡因素所造成。在討論細胞移動中半乳糖凝集素-3和Alix相互關係時,發現部分半乳糖凝集素-3和Alix會聚集在移動細胞的前端,但不管是角質細胞在正常培養狀態抑或是脂多醣、alpha腫瘤壞死因子或gamma干擾素刺激下,都無法測到半乳糖凝集素-3和Alix的結合。藉由比較半乳糖凝集素-3和Alix共同敲落HaCaT細胞,其傷口癒合能力較單一基因敲落HaCaT細胞下降來得多,由此可推論在半乳糖凝集素-3和Alix協調人類角質細胞移動的方式也許不大相同。另外,我們也發現半乳糖凝集素-7基因敲落HaCaT細胞其移動性和傷口癒合能力下降,證實半乳糖凝集素-7會正向調控細胞的移動。而實驗室先前利用mRNA微陣列(microarray)技術,發現HaCaT細胞中許多基因的表現會受到半乳糖凝集素-7基因敲落的影響,其中包括許多組合蛋白,以及其下游調節與細胞移動相關的訊息分子。因此,在探討半乳糖凝集素-7基因敲落HaCaT細胞中組合蛋白調控細胞移動的訊息傳遞時,發現一些角質細胞主要表現的組合蛋白alpha2、alpha3、alpha6、beta4和beta6的mRNA有高量表現。在組合蛋白下游訊息傳遞分子中,儘管焦點黏結酵素(focal adhesion kinase)的早期活化並無受到影響,而vinculin做為焦點黏結的重要組成分子之一,其表現量也未受到影響,但我們發現,Rho結合酵素ROCK-1和ROCK-2在半乳糖凝集素-7基因敲落HaCaT細胞中有較高的蛋白質表現量。 總結我們在探討人類角質細胞中半乳糖凝集素-3和-7角色的實驗結果,發現內源性半乳糖凝集素-3確實可幫助人類角質細胞的移動,但不一定需要透過Alix的幫助;另外,內源性半乳糖凝集素-7也被證實可以幫助人類角質細胞的移動,且可能是透過影響組合蛋白的訊息傳遞中特定組合蛋白的表現,或是所引導的Rho酵素的表現,進而造成一個促進細胞移動的訊息。目前仍需更多實驗去探討半乳糖凝集素-7如何調控角質細胞的移動,我們希望透過此研究,能進一步了解半乳糖凝集素對於人類傷口癒合的影響。 | zh_TW |
dc.description.abstract | Galectin-3 and galectin-7 are animal lectins that recognize beta-galactoside-containing glycoconjugates through their carbohydrate recognition domains (CRDs). Many studies have emphasized the multiple functions of galectin-3, including cell-cell interaction, lattice formation, apoptosis and differentiation of immune cells, inflammation, and tumor progression. The functions of galectin-7 are mainly emphasized in regulation of skin homeostasis. Our recent study showed galectin-3 modulates mouse keratinocytes migration by regulating epithelial growth factor receptor (EGFR) homeostasis through Alix (ALG-2 linked protein-X), which is involved in apoptosis, endocytic membrane trafficking, and cytoskeleton remodeling.
In this study, we investigated the role of galectin-3 and galectin-7 in modulating human keratinocyte migration, and further examined the underlying mechanism, including whether Alix is indispensable in galectin-3-mediated cell migration and the involvement of galectin-7 in integrin-mediated migration. Our results suggested that both galectin-3-knockdown and Alix-knockdown HaCaT cells, a human keratinocyte cell line, have impaired wound healing abilities, which are verified to be a consequence of impaired single cell motility not proliferation rate. In the investigation of relationship between galectin-3 and Alix in regulating human keratinocyte migration, we found both galectin-3 and Alix can translocate to the leading edge of migrating human keratinocytes, but the interaction between galectin-3 and Alix could not be detected under both resting condition and LPS, TNF-alpha, or IFN-gamma stimulation. By comparison of the wound healing ability, galectin-3-Alix double knockdown HaCaT cells were found to have more impaired cell motilities compared with galectin-3-knockdown and Alix-knockdown HaCaT cells. Thus, galectin-3 may mediate human keratinocyte cell migration through an Alix-independent pathway. On the other hand, the positive role of galectin-7 in human keratinocyte migration was also confirmed by impaired wound-healing abilities and single cell motilities of galectin-7-knockdown HaCaT cells. According to the microarray data, expression of many genes was found to be changed after galectin-7 knockdown, including integrins and related downstream signal molecules. Therefore, we further examined the integrin-related cell migration in galecitn-7-knockdown HaCaT cells. Among the abundantly expressed integrins in keratinocytes, the amount of alpha2, alpha3, alpha6, beta4, and beta6 subunit mRNA transcripts are higher in galectin-7-knockdown HaCaT cells. In integrin downstream signaling, the early activation of focal adhesion kinase is not affected in galectin-7-knockdown HaCaT cells, so is the protein expression of focal contact component, vinculin. However, Rho-kinases, ROCK-1 and ROCK-2, protein expression are higher in galectin-7-knockdown HaCaT cells. In summary, endogenous galectin-3 has a pro-migratory effect in human keratinocytes and Alix may not be the major cellular effector of galectin-3-mediated human keratinocyte migration. Endogenous galectin-7 also has a pro-migratory effect in human keratinocytes. In integrin-related cell migration, galectin-7 can regulate certain integrins and Rho kinases expression, which may contribute to the pro-migratory function of galectin-7 in human keratinocytes. Additional studies are required to elucidate how endogenous galecitn-7 regulates keratinocyte migration, which may also hlp us understand its role in human wound repair. | en |
dc.description.provenance | Made available in DSpace on 2021-05-16T16:22:52Z (GMT). No. of bitstreams: 1 ntu-102-R00449006-1.pdf: 3595749 bytes, checksum: eea4500a2f5a50008db381b3f2e9c0ed (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | Content
誌謝 I 摘要 III Abstract V Introduction 1 1. Keratinocytes 1 1.1. Keratinocytes in maintaining skin barrier 1 1.2. Role of keratinocytes in the wound-healing process 1 2. Cell migration 2 2.1. A highly integrated multistep process 2 2.2. Integrin-mediated cell adhesion and migration 3 2.3. Role of integrins in regulating keratinocyte migration 4 3. Galectins 5 3.1. beta-galactoside-binding proteins – the galectin family 5 3.2. The functions of galectin-3 and galectin-7 6 3.3. The role of galectin-3 and galectin-7 in keratinocyte homeostasis 7 4. Alix (ALG-2 linked protein-X) 8 4.1. Multiple functions 8 4.2. The role of Src kinase in the regulation of Alix function 9 4.3. The role of Alix in cell adhesion and migration 9 Experimental design 10 Materials and Methods 13 1. Cell lines and reagents 13 2. Generation of galectin-3- , galectin-7, or Alix-stable knockdown HaCaT cells 13 2.1. Construction of shRNA-expressing lentiviral plasmid and preparation of lentiviruses 13 2.2. Generation of galectin-3-, galectin-7, or Alix-knockdown HaCaT by shRNA- expressing lentiviral infection 14 3. Analysis of protein expression by western blot 14 4. Scratch-based wound-healing assay 15 5. Single cell migration 16 6. Cell proliferation assay 16 7. Cell apoptosis assay 17 8. Immunoprecipitation 17 9. Immunofluorescence assay 18 10. Generation of transient galectin-3 and Alix double knockdown HaCaT cells by siRNA transfection 19 11. Quantitative PCR 19 12. Statistic analysis 20 Results 21 Part I. The involvement of Alix in galectin-3-mediated human keratinocyte migration 21 1. The effect of galectin-3 and Alix on human keratinocyte migration 21 1.1. Expression of galectin-3 and Alix in HaCaT cells 21 1.2. Preparation of stable galectin-3-knockdown and Alix-knockdown HaCaT cells by shRNA-expressing lentivirus infection 21 1.3. The effects of galectin-3 and Alix on skin re-epithelialization in vitro 22 2. The relationship between galectin-3 and Alix in HaCaT cells 24 2.1. Cellular distribution of galectin-3 and Alix in HaCaT cells under normal culture condition and during scratch stimulation. 24 2.2. Interaction between galectin-3 and Alix in HaCaT cells under normal culture conditions and under LPS, TNF-alpha, or IFN-gamma stimulation. 24 2.3. Preparation of transient galectin-3 and Alix double knockdown HaCaT cells. 26 2.4. The relationship between galecitn-3 and Alix in regulation of human keratinocyte migration. 27 Part II. The role of galectin-7 in integrin-mediated human keratinocyte migration 28 1. The effect of galectin-7 on human keratinocyte migration 28 1.1. Expression of galectin-7 in HaCaT cells 28 1.2. Preparation of stable galecitn-7-knockdown HaCaT cells by shRNA-expressing lentivirus infection 28 1.3. The effect of galectin-7 on skin re-epithelialization in vitro 30 2. The role of galectin-7 in the regulation of integrins and related signaling 30 2.1. Expression of integrins in galectin-7-knockdown HaCaT cells 30 2.2. Expression and localization of focal contact proteins and kinases in galectin-7-knockdown HaCaT cells 31 2.3. Verification of integrin downstream- RhoA-ROCK signal in galecton-7- knockdown HaCaT cells 33 Discussion and Future work 34 References 41 Figure content Figure 1. Knockdown of galectin-3 or Alix expression in HaCaT cells. 52 Figure 2. Galectin-3-knockdown and Alix-knockdown HaCaT cells showed impaired wound healing ability. 53 Figure 3. Gaelctin-3 and Alix modulate cell motility, not cell proliferation or viability, of HaCaT cells. 54 Figure 4. The cellular distribution of galectin-3 and Alix under normal and scratch conditions. 55 Figure 5. Alix does not interact with galectin-3 in HaCaT cells after LPS, TNF-alpha, or IFN gamma-stimulation. 56 Figure 6. Knockdown of galectin-3 expression in Alix-knockdown HaCaT cells. 57 Figure 7. Galectin-3 and Alix double knockdown HaCaT cells showed more impaired wound-healing ability. 58 Figure 8. Knockdown of galectin-7 expression in HaCaT cells. 59 Figure 9. Galectin-7-knockdown HaCaT cells showed impaired wound-healing ability. 60 Figure 10. Gaelctin-7 modulates cell motility, not cell viability, of HaCaT cells. 61 Figure 11. mRNA expression of integrins in galectin-7-knockdown HaCaT cells. 62 Figure 12. Expression of vinculin and phosphorylation of FAK (pY397) in galectin-7-knockdown HaCaT cells. 63 Figure 13. Focal contacts in galectin-7-knockdown HaCaT cells. 64 Figure 14. Expression of ROCK-1 and ROCK-2 in galectin-7- knockdown HaCaT cells. 65 Appendix content Appendix-1 66 Appendix-2 69 | |
dc.language.iso | en | |
dc.title | 半乳糖凝集素-3和-7於人類角質細胞移動中所扮演的角色 | zh_TW |
dc.title | The Role of Galectin-3 and -7 in Human Keratinocyte Migration | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 嚴仲陽(Jeffrey Jong-Young Yen),賈景山(Jean-San Chia),林國儀(Kuo-I Lin) | |
dc.subject.keyword | 半乳糖凝集素-3,半乳糖凝集素-7,人類角質細胞,細胞移動,Alix,組合蛋白(integrin), | zh_TW |
dc.subject.keyword | Galectin-3,galectin-7,human keratinocyte,cell migration,Alix,integrin, | en |
dc.relation.page | 69 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2013-07-19 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 免疫學研究所 | zh_TW |
顯示於系所單位: | 免疫學研究所 |
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