臺灣博碩士論文加值系統

器官在結構變換的過程中如何妥善佈署幹細胞，對於生理恆定及損傷後的再生皆扮演著關鍵的角色。毛囊生長週期包含三個時期，分別是生長期(anagen)、衰退期(catagen)、及休止期(telogen)，各時期的毛囊結構與細胞組成皆不盡相同。在此，我們利用毛囊當作模式來探討器官結構變換時，幹細胞的佈署與器官再生之間的關聯性。在臨床上利用放射線或化學治療癌症的病人都不可避免地伴隨著許多副作用，落髮是其中的一種，且目前並無有效的治療方法，造成病患除了生理的不適外也承受了心理上極大壓力。臨床上，生長期毛囊受損的反應及受損後毛囊再生的機轉還有待更深入瞭解。為了讓基礎研究的結果能解決臨床上的問題，我們使用臨床上廣泛用來治療癌症的游離輻射與化學治療藥cyclophosphamide誘導生長期毛囊的損傷，並探討以下問題: (1)游離輻射與cyclophosphamide造成的細胞效應，(2)再生過程中不同細胞族群的動態，(3)毛囊萎縮與再生過程中的訊息調控機制。我們發現游離輻射和cyclophosphamide主要在基質細胞引起DNA雙股斷裂並造成細胞凋亡，其受損的程度隨劑量增加而遞增。另高劑量的游離輻射與cyclophosphamide皆造成與臨床上相似的落髮現象。結合BrdU標定與細胞系追蹤，我們發現根據受損程度的不同，生長期毛囊可透過異位前驅細胞的調動與去分化這兩種不同機制，使毛囊再生進而防止繼續萎縮。在低劑量游離輻射後，毛囊球基底層的K5+細胞會被快速活化，並在12到72小時內補充毛球內因細胞凋亡而失去的細胞。我們稱此為早期再生反應。在早期再生反應中，突部毛囊幹細胞則維持靜止狀態並無參與此再生過程。當高劑量輻射造成所有的基質細胞與毛球基底層K5+細胞都走向細胞凋亡時，這時存活下來的外根鞘細胞即透過去分化獲得與正常休止期毛囊中的次級毛胚幹細胞相似的幹細胞特性，並在72到120小時內進行毛囊再生。我們稱此為晚期再生反應。突部毛囊幹細胞則在輻射後第五天活化並分化成上部的外根鞘細胞。免疫染色與次世代定序分析結果顯示，游離輻射傷害會誘導p53表現，並且短暫抑制Wnt signaling。72小時之後，當p53停止表現，Wnt signaling即再度被活化。若在毛囊啟動自發性再生反應之前給予WNT 抑制劑或是剔除Wnt下游基因表現，毛囊就無法再生。此結果顯示毛囊再生反應的啟動與否需要Wnt signaling來調控。於是我們嘗試在高劑量游離輻射與cyclophosphamide傷害後立即以皮下植入方式給予Wnt3a重組蛋白，此處理雖無法降低p53表現，但透過Wnt3a持續活化Wnt signaling，進而刺激異位前驅細胞的活化，可以防止輻射及化療造成的落髮。我們的研究結果顯示生長期的毛囊透過廣泛佈署多能性細胞以因應損傷發生時，毛囊能迅速再生。透過活化異位前驅細胞可做為一個新的策略來改善臨床上因放射線或化療造成的落髮。

How organs deploy their stem cell (SC) pools during metamorphic remodeling is not only vital for homeostasis but also for regenerative potential after injury. Hair follicles (HFs) serve as an exceptional model to study this question because they undergo life-time cyclic growth with structural transformation between anagen (active growth), catagen (regression), and telogen (relative rest). Clinically, radiotherapy and chemotherapy are often inevitably accompanied by various side effects, such as hair loss, which causes great psychological distress in patients. Currently, there is no effective treatment for radiotherapy and chemotherapy-induced hair loss. Up to date, how the growing HFs respond and regenerate after radiotherapy and chemotherapy are not well understood. To investigate this, ionizing radiation (IR) and genotoxic cyclophosphamide (CYP) were employed to damage anagen HFs in mice. We aim to explore the following topics: (1) the cellular effects of IR and CYP injuries on anagen HFs, (2) the cell dynamics of distinct cell populations during HF regeneration from such injuries, (3) the molecular mechanisms of dystrophy and regeneration in anagen HFs. We found that both IR and CYP induced a dose-dependent DNA damage and apoptosis mainly in the proliferative matrix cells. Hair loss was observed after high-dose of IR and CYP injuries. Combining BrdU incorporation and lineage tracing, we found that, depending on the severity of IR injury, anagen HFs activated two distinct regenerative attempts to regenerate hair bulbs and to avoid regression through mobilization and dedifferentiation of two different populations of ectopic progenitor cells. After low-dose of IR, the K5+ basal hair bulb cells, rather than bulge stem cells (BgSCs), were quickly activated to replenish the germinative and matrix cells lost by apoptosis and regenerated concentric layers of various differentiations between 12 and 72 hrs. We named it “the early regenerative attempt”. After high-dose of IR, when all basal hair bulb, germinative, and matrix cells were depleted by apoptosis, the surviving radioresistant outer root sheath cells rapidly dedifferentiated toward the SC-like state and fueled HF regeneration between 72 and 120 hrs. We named it “the late regenerative attempt”. These dedifferentiated ectopic progenitor cells were functionally similar to the secondary hair germ stem cells (ShgSCs) in telogen HFs. BgSC activation was detected at day 5 when the hair bulb has been regenerated by the dedifferentiated ectopic progenitor cells. By immunostaining and RNA sequencing, we found that Wnt/β-catenin signaling was transiently suppressed in a p53-dependent manner, and Wnt/β-catenin signaling reactivation was a prerequisite for regenerative attempts from IR injury. Inhibiting Wnt/β-catenin signaling reactivation abolished the regenerative attempts. Finally, we demonstrated that both IR and CYP-induced alopecia could be largely prevented by enhancing the ectopic SC mobilization through augmenting Wnt/β-catenin signaling. These results demonstrate that the wide deployment of multipotent cells in anagen HFs confers flexible regenerative strategies upon them to enable prompt regeneration from genotoxic injury. Enhancing ectopic progenitor mobilization can be a potential strategy to prevent hair loss from radiotherapy and chemotherapy.

誌謝……………………………………………………………………………………i
中文摘要………………………………………………………………………………ii
Abstract……………………………………………………………………………….iv
Index of Figures………………………………………………………………………x
Index of Tables……………………………………………………………………….xii
Index of Supplementary Information……………………………………………...xiii
CHAPTER 1. INTRODUCTION…………………………………………………….1
1.1. The strategy of quick regeneration from injury to an organ is required…………1
1.2. Epithelial-mesenchymal interaction and the hair cycle……………………………1
1.3. Hair follicle structures…………………………………………………………………3
1.4. Cell dynamics during structural transformation between distinct phases of the hair cycle………………………………………………………………………………...4
1.5. Cellular response to ionizing radiation………………………………………………7
1.6. IR-induced cellular damage in hair follicles………………………………………...8
1.7. Hair cycle disruption: the response of an organ to IR injury……………………..9
1.8. IR injury and regeneration: learn from other organs and their mechanisms…..11
1.9. Anagen repair/regenerative activity presents after chemotherapeutic damage..13
1.10. Reserve stem cells in other organs. Are they also present in anagen HFs?.....14
CHAPTER 2. MATERIALS AND METHODS……………………..……………..17
2.1. Animals………………………………………………………………………………….17
2.1.1. C57BL/6 mice……………………………………………………………………..17
2.1.2. K5CreER mice……………………………………………………………………….17
2.1.3. Lgr5EGFP-IRES-CreERT2 mice………………………………………………………..17
2.1.4. K19CreER mice…………………………………………………………….............18
2.1.5. Rosa26tdTomato mice………………………………………………………............18
2.1.6. Ctnnbl flox/flox mice………………………………………………………………..18
2.1.7. p53 null mice……………………………………………………………………..18
2.1.8. p53flox/flox mice……………………………………….........................................18
2.1.9. K5CreER; R26tdTomato, Lgr5EGFP-IRES-CreERT2; R26tdTomato, and K19CreER; R26tdTomato mice………………………………………………………………….19
2.1.10. K5CreER; Ctnnblflox/flox and K5CreER; p53flox/flox mice……………………........19
2.2. Genomic DNA extraction and genotyping………………………………………….19
2.3. IR and chemotherapy exposure of mice…………………………………………….20
2.4. Mapping proliferative cells by BrdU incorporation………………………………21
2.5. Lineage tracing experiment…………………………………………………………..21
2.6. Inhibition of Wnt/β-catenin signaling……………………………………………….22
2.7. Histology, immunostaining and TUNEL staining………………………………….22
2.8. In situ hybridization……………………………………………………………………25
2.9. Image acquisition and quantification……………………………………………….25
2.10. Quantification of γ-H2AX foci………………………………………………………26
2.11. RNA-sequencing analysis……………………………………………………………27
2.12. Quantitative RT-PCR………………………………………………………………...27
2.13. FACS……………………………………………………………………………………28
2.14. Protein administration experiment…………………………………………………28
2.15. Statistics……………………………………………………………………………….29
CHAPTER 3. RESULTS…………………………………………………………….30
3.1. Dose-dependent HF dystrophy after IR and CYP injuries………………………..30
3.2. Cellular effects of anagen HFs after IR and CYP injuries……………………….31
3.3. K5+ basal hair bulb keratinocytes preferentially proliferate during the early regenerative attempt…………………………………………………………………..33
3.4. K5+ basal hair bulb cells replenish the germinative population and contribute to all concentric layers of recovered HFs…………………………………………….35
3.5. K5+ ORS cells remodel into the epithelial strand with dedifferentiation into stem cell-like progenitors that contribute to the late regenerative attempt………….37
3.6. Lower tip cells and BgSCs undergo stepwise activation for the late regenerative attempt………………………………………………………………………………….38
3.7. IR injury disrupts WNT/β-catenin signaling that is required for the late regenerative attempt…………………………………………………………………..40
3.8. Dual roles of p53 in IR-induced HF dystrophy and the suppression of WNT/β-catenin signaling…………………………………………………………….44
3.9. Local delivery of Wnt ligands prevents IR- and CYP-induced alopecia by enhancing WNT/β-catenin signaling-driven compensatory proliferation of ectopic progenitor cells………………………………………………………………46
CHAPTER 4. DISCUSSION………………………………………………………...48
4.1. Compensatory proliferation of multipotent basal hair bulb cells during the early regenerative attempt…………………………………………………………………..48
4.2. Remodeling and dedifferentiation of ORS cells to orchestrate the late regenerative attempt…………………………………………………………………..50
4.3. Activation of WNT/β-catenin signaling facilitates compensatory proliferation of ectopic progenitor cells and rescues IR- and chemotherapy-induced hair loss………………………………………………………………………………………53
CHAPTER 5. REFERENCES……………………………………………………....55
CHAPTER 6. FIGURES AND FIGURE LEGENDS……………………………...67
CHAPTER 7. TABLES……………………………………………………………..100
CHAPTER 8. SUPPLEMENTARY INFORMATION…………………………...106

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