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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林泰元(Thai-Yen Ling) | |
| dc.contributor.author | Cheng-Wei Wang | en |
| dc.contributor.author | 汪承威 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:39:03Z | - |
| dc.date.available | 2021-07-10T21:39:03Z | - |
| dc.date.copyright | 2020-09-10 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76869 | - |
| dc.description.abstract | 我們實驗室先前已經分離出一種會表現柯薩奇病毒/腺病毒受體(CAR)的老鼠肺臟幹細胞，並命名為mPSCsCAR+，而這種肺臟幹細胞被純化出來後會分化成類第一型肺泡上皮細胞。根據之前的單細胞定序分析結果，我們發現肺臟發育中後期的某種肺臟幹細胞的細胞標誌，也會表現在mPSCsCAR+，也因此我們推論mPSCsCAR+ 可能是一種肺臟發育中後期的幹細胞。事實上，正常肺臟發育於一個低氧的環境中，也就是說肺臟幹細胞是在低氧的條件下生長以及分化成第一型和第二型肺泡上皮細胞，然而我們先前是將mPSCsCAR+培養於一般的氧氣濃度之下，這種培養環境相較於胚胎發育的環境來說是一種高氧的狀態。在這種培養環境下，許多纖維化相關的基因表現在我們的幹細胞分化成第一型肺泡上皮細胞的過程中大幅上升，而這些基因通常不會大量表現於正常的第一型肺泡上皮細胞。也因此我們改變細胞培養條件，將mPSCsCAR+放在只含1 %氧氣濃度的培養箱中，試著去模擬肺臟幹細胞真實的生長以及分化條件。結果顯示，低氧會刺激mPSCsCAR+開始分化。有趣的是，在正常氧氣濃度下用DMOG這個藥物來增加低氧誘導因子-1α(HIF-1α)的蛋白含量，也會使mPSCsCAR+分化，並增加第一型肺泡上皮細胞的細胞標誌表現，伴隨著肺臟幹細胞其細胞標誌的下降。另一方面，我們也發現如果細胞的整個分化過程都處在低氧的環境，會使纖維化相關的基因表現下降，而且和TGF-β受體抑制劑並用的話能加成性的抑制α-平滑肌肌動蛋白這類纖維化標誌的表現。除此之外，當我們選擇加入DMOG來穩定低氧誘導因子而非藉由長時間的低氧，就發現mPSCsCAR+能夠分化成更典型的第一型肺泡上皮細胞，而纖維化相關的基因表現則大幅地被抑制，暗示著這類藥物用於治療肺部纖維化或不正常肺部發育的潛能。總而言之，藉由mPSCsCAR+細胞分化模型，我們更了解低氧這個外在條件以及低氧誘導因子在肺泡細胞分化過程所扮演的腳色，同時低氧誘導因子的活化也使我們的細胞模型更為完善。 | zh_TW |
| dc.description.abstract | In our lab, we have identified a rare population of CAR positive mouse pulmonary stem/progenitor cells named mPSCsCAR+, which could differentiate into alveolar type I (AT1)-like cells. According to the result of single-cell RNA sequencing, mPSCsCAR+ expressed Id2 and Foxp1 in high and Sox9 in middle; therefore, we suggest that mPSCsCAR+ might be similar to distal lung progenitor. It has been reported that Id2+/Sox9+ distal lung progenitors give rise to alveolar type I and type 2 (AT1/AT2) epithelial cells at the late stage of embryonic development, which contains less oxygen than atmosphere air. Precisely, it indicated that the distal lung progenitors differentiated into alveolar epithelial cells at the hypoxic condition. Originally, growth and differentiation of mPSCsCAR+ were in normoxia. However, fibrosis-related genes were upregulated during the differentiation process. To improve the protocol of AT1 cells formation, we adjusted the culture condition from normal oxygen level (20%), which is relatively hyperoxic for fetal lung development, to low oxygen tension (1%). In hypoxia, differentiation of mPSCsCAR+ surrounded by stroma cells was initiated. Interestingly, HIF-1α stabilizer DMOG also induce mPSCsCAR+ flattening in normoxia. In addition, T1α expression in mPSCsCAR+ microenvironment was upregulated and Sox9 expression was inhibited in dose dependent manner by DMOG. On the other hand, when cells were cultured in hypoxia for the entire process of differentiation, myofibroblasts markers α-SMA and calponin were decreased accompanied by increased expression of T1α. Furthermore, hypoxia combined with TGF-β type 1 receptor inhibitor: A83-01 had synergic effect on downregulation of α-SMA protein level. In addition, treatment of DMOG in normoxia led to obviously blockade of α-SMA, calponin expression, significant elevation of tight junction protein ZO-1 and T1α. Downregulating the products of TGF-β signaling activation including α-SMA revealed the potential role of HIF-1α stabilizer in treating lung diseases associated with abnormal lung development. In conclusion, hypoxia and HIF-1α stabilizer could optimize the AT1 cells formation from mPSCsCAR+. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:39:03Z (GMT). No. of bitstreams: 1 U0001-1208202017124300.pdf: 21028121 bytes, checksum: 14157e492aea1d8abe8171266988db9d (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | Contents 口試委員審定書 I 誌謝 II 中文摘要 III Abstract IV Abbreviation list VI Chapter 1 Introduction 1 1.1 Stem/progenitor cells in lung development 2 1.2 Alveolar type 1 (AT1) cells 3 1.3 CAR+ mouse pulmonary stem/progenitor cells (mPSCsCAR+) 3 1.4 The role of TGF-ß signaling in lung development 4 1.5 The role of TGF-ß signaling in epithelial to mesenchymal transition (EMT) and lung fibrosis 5 1.6 TGF-β signaling is involved in the differentiation process of mPSCsCAR+ 7 1.7 Hypoxia 8 1.8 Oxygen and HIF-1 activity 9 1.9 The role of hypoxia and HIF-1α in normal and abnormal lung development 10 1.10 The effect of hypoxic culture condition on the generation of distal lung cells 12 1.11 The known impact of oxygen tension on mPSCsCAR+ 13 1.12 Aim of the study 13 Chapter 2 Materials Methods 15 2.1 Primary culture of mPSCsCAR+ 16 2.2 Isolation of mPSCsCAR+ 17 2.3 Culture of mPSCsCAR+-derived AT1-like cells 18 2.4 Chemicals 18 2.5 Real-time qPCR 18 2.6 Immunofluorescence staining 19 2.7 Cell apoptosis assay 20 2.8 Western blotting and antibody list 20 2.9 Statistical analysis 21 Chapter 3 Results 22 3.1 Hypoxia induced differentiation of mPSCsCAR+ in the mPSCsCAR+ microenvironment 23 3.2 TGF-β signaling was involved in hypoxia-induced mPSCsCAR+ differentiation 23 3.3 Exposure to hypoxia for 5 days didn’t induce EMT in mPSCsCAR+ 24 3.4 HIF-1α stabilizer: DMOG promoted mPSCsCAR+ differentiation in dose-dependent manner 25 3.5 Duration of exposure to hypoxia resulted in differentially enhanced AT1, epithelial cell markers and inhibited expression of myofibroblast markers in mPSCsCAR+-derived AT1-like cells 26 3.6 Time-course protein expression of mPSCsCAR+ differentiation under normoxia and hypoxia 27 3.7 Combination of hypoxia and TGF-ß receptor inhibitor/Dexamethasone showed synergistic inhibition of myofibroblast marker 28 3.8 Stabilization of HIF-1α by DMOG optimized AT1 cells formation 28 3.9 No synergic effect of tight junction protein expression was observed when cells were treated with DMOG and A83-01 29 Chapter 4 Figures 30 4.1 The morphology and gene expression of mPSCsCAR+ were altered in hypoxia 34 4.2 TGF-β signaling was associated with hypoxia-induced cell flattening 38 4.3 Expression of EMT-related genes in mPSCsCAR+ in hypoxia 40 4.4 Impact of DMOG on mPSCsCAR+ microenvironment 45 4.5 Effect of hypoxia on the early stage of mPSCsCAR+ differentiation 48 4.6 Cells cultured in hypoxia for the entire differentiation process showed less mesenchymal phenotype 55 4.7 Effect of hypoxia on mPSCsCAR+ was observed in different differentiation stage 58 4.8 Combined effect of Dexamethasone/A83-01 and hypoxia on α-SMA expression was investigated 60 4.9 DMOG significantly improved the formation of mPSCsCAR+-derived AT1 cells 68 4.10 Combination of DMOG and A83-01 could not enhance the expression of ZO-1 at cell-cell border 70 Chapter 5 Tables 71 Table 1. List of primers used for real-time qPCR 72 Table 2. Summary of different conditions for optimizing AT1 cells formation 73 Table 3. Summary of mPSCsCAR+ phenotype in different conditions before sorting 73 Chapter 6 Discussion 74 6.1 Possible mechanism of hypoxia-stimulated lung stem cells differentiation 75 6.2 Beneficial effect of hypoxia and HIF-1α accumulation on preventing abnormal mPSCsCAR+ differentiation in normoxia 76 6.3 Possible mechanism of HIF-1α stabilizer DMOG-induced inhibition of TGF-β -induced myofibroblast marker expression 77 6.4 Possible reason for the different inhibition effect of hypoxia and DMOG on myofibroblast marker 77 Chapter 7 References 79 | |
| dc.language.iso | en | |
| dc.subject | 第一型肺泡上皮細胞 | zh_TW |
| dc.subject | 肺臟幹細胞 | zh_TW |
| dc.subject | 纖維化 | zh_TW |
| dc.subject | 低氧 | zh_TW |
| dc.subject | 低氧誘導因子 | zh_TW |
| dc.subject | hypoxia-inducible factor | en |
| dc.subject | lung stem cells | en |
| dc.subject | AT1 cells | en |
| dc.subject | fibrosis | en |
| dc.subject | hypoxia | en |
| dc.title | 氧氣分壓調控第一型肺泡細胞型態發生之探討 | zh_TW |
| dc.title | The study of alveolar type 1 (AT1) cells morphogenesis regulated by oxygen tension | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳惠文(Huei-Wen Chen),曹伯年(Po-Nien Tsao),董奕鍾(Yi-Chung Tung),郭青齡(Chin-Lin Guo) | |
| dc.subject.keyword | 肺臟幹細胞,第一型肺泡上皮細胞,纖維化,低氧,低氧誘導因子, | zh_TW |
| dc.subject.keyword | lung stem cells,AT1 cells,fibrosis,hypoxia,hypoxia-inducible factor, | en |
| dc.relation.page | 85 | |
| dc.identifier.doi | 10.6342/NTU202003131 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-14 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 藥理學研究所 | zh_TW |
| 顯示於系所單位： | 藥理學科所 | |
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