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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林國儀(Kuo-I Lin) | |
| dc.contributor.author | Wei-Ju Huang | en |
| dc.contributor.author | 黃暐茹 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:38:28Z | - |
| dc.date.available | 2021-07-10T21:38:28Z | - |
| dc.date.copyright | 2020-09-10 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-13 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76847 | - |
| dc.description.abstract | 僵直性脊椎炎是一種自體發炎疾病，與人類白血球抗原-B27 (HLA-B27) 基因高度關聯，局部慢性發炎的脊椎會伴隨異常骨質新生，產生韌帶贅。但是在異常骨質新生與發炎之間的研究非常有限。間葉幹細胞具有分化成成骨細胞的能力，近幾年有研究發現從僵直性脊椎炎病人骨髓取出的間葉幹細胞硬骨分化的速度較正常人快。此外，我們實驗室也從病人的韌帶贅分離並培養出間葉幹細胞，並且找出間葉幹細胞致病的機制，其透過異常折疊的人類白血球抗原-B27，促進非專一性鹼性磷酸酶 (TNAP) 活化，進而導致異常的骨分化行為。抑制非專一性鹼性磷酸酶活性能夠在僵直性脊椎炎動物模式中減緩韌帶贅的產生。然而長期抑制非專一性鹼性磷酸酶可能會導致骨頭細胞無法正常鈣沉積，造成嚴重的副作用，因此，此研究期望能進一步找出僵直性脊椎炎間葉幹細胞加速骨分化的異常傳遞訊息路徑，並尋找能緩和異常骨化且副作用較小的藥物候選，來治療僵直性脊椎炎。我們分別利用高速藥物篩選平台以及磷酸化激酶表現陣列兩種策略來進行。本文建立 384 孔僵直性脊椎炎藥物篩選平台，並且用此在 2,464 個美國食品藥物管理局核可的藥物中找到 14 個潛力藥物。此外，在病灶區取出的間葉幹細胞中，發現細胞中磷脂酰肌醇-3激酶 (PI3K)訊息傳遞路徑在骨分化過程中異常活化，抑制磷脂酰肌醇-3激酶同時會造成剪接 X-box 結合蛋白 1 (sXBP1) 及非專一性鹼性磷酸酶表現量降低，並且減少乙型轉化生長因子 (TGFβ) 的釋放，最終減緩異常加速的硬骨分化。除此之外，我們還發現抑制乙型轉化生長因子受體活性，能有效地減緩病人細胞骨分化的速度。因此本文中，我們發現磷脂酰肌醇-3激酶的活化以及乙型轉化生長因子在病人的致病機制中扮演重要的角色，是僵直性脊椎炎異常骨質新生與發炎的關鍵之一。協同藥物篩選平台與磷脂酰肌醇-3激酶訊息傳遞路徑的揭示，期待本篇研究能提供僵直性脊椎炎的藥物研發一個新方向。 | zh_TW |
| dc.description.abstract | Ankylosing spondylitis (AS) is a HLA-B27 gene strongly associated auto-inflammatory disease, characterized by outgrowth of new bone called syndesmophyte and local chronic inflammation in axil skeleton. However, the relationship between immune responses and pathogenic osteogenesis remain largely unknown. Mesenchymal stem cells (MSCs) are osteoblast progenitors controlling new bone formation. Recent studies showed that MSCs derived from AS patients have higher osteogenic differentiation and enhanced mineralization potency as compared with MSCs derived from healthy donors. It has been demonstrated that MSCs derived from the enthesis of AS patients induced ER stress pathway through misfolded and unfolded HLA-B27 molecule, and further activated tissue non-specific alkaline phosphatase (TNAP), which caused the acceleration of osteogenesis differentiation. Treatment with TNAP inhibitors arrested osteogenesis in AS-derived MSCs and syndesmophyte formation in an AS animal model. Herein, we plan to discover new potential drugs besides TNAP inhibitors, as which may cause long-term side effects. We performed high throughput drug screening and phospho-kinase array screening to search drugs that can block osteogenesis, and abnormal kinase pathways in AS-derived MSCs. A 384-well-based drug screening workflow was established in this thesis and 14 potential drug candidates from 2,464 FDA approved drugs were preliminarily identified via high throughput drug screening platform. We also demonstrated that highly active PI3K signaling in AS MSCs accelerated osteogenic differentiation. Inhibition of PI3K signaling by selective PI3K isoform inhibitors down-regulated spliced-X-box binding protein-1 (sXBP1) and TNAP expression, decreased TGFβ secretion, and effectively ameliorated the abnormal osteogenic differentiation as compared with control MSCs. In addition, blocking TGFβ receptor I/II signaling significantly slowed down osteogenic differentiation of AS MSCs. In sum, we identified that abnormal PI3K signaling and TGFβproduction may play a key role in inflammatory regulation and pathogenic osteogenesis in AS. These findings may contribute to a new direction of drug development. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:38:28Z (GMT). No. of bitstreams: 1 U0001-1308202020175600.pdf: 14843974 bytes, checksum: 96ddbbfe037e427d3485540189c980e3 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 序言 ii 中文摘要 iii Abstract iv 1. Introduction 1 1.1. Ankylosing spondylitis (AS) 1 1.2. Treatment for AS 1 1.3. AS pathogenic pathway 2 1.4. The role of HLA-B*27 3 1.5. Mesenchymal stem cells (hMSCs) 4 1.6. Pathogenic pathways of AS hMSCs 5 1.7. Cytokines involved in AS pathogenesis 5 2. Specific Aims 8 3. Materials and Methods 9 3.1. Expansion of hMSCs in hypoxia culture 9 3.2. Flow cytometry 9 3.3. Osteogenic differentiation of hMSCs in normoxia 9 3.4. ARS staining and quantification of calcium deposition 10 3.5. hMSCs cultured under the treatment with TNAP inhibitor 10 3.6. High-throughput drug screening 10 3.7. Human Phospho-Kinase Array 11 3.8. Immunoblot analysis 12 3.9. Inhibition of AKT/PI3K/ER stress/TGFβ signaling pathway 13 3.10. Cell viability of inhibitors 13 3.11. ELISA 13 3.12. Cytokine array 14 3.13. Lentiviral transduction and siRNA transfection 14 4. Results 15 4.1. Characterization of hMSCs 15 4.2. Pre-test of high-throughput screening in 96-well-plates 15 4.3. Pre-test of high-throughput screening in 384-well plates 16 4.4. Pre-test of high-throughput screening in 384-well plates used in HTS facility 16 4.5. 2,464 FDA approved drugs were screened by high-throughput drugs screening in HTS facility 17 4.6. Identification of higher phosphorylation of AKT1/2/3 and Src in AS hMSCs during osteogenic induction by Human Phospho-Kinase Array 17 4.7. Validation of elevated phosphorylation of AKT1/2/3 in AS hMSCs 17 4.8. Identification of AKT activation in AS hMSCs osteogenic differentiation 18 4.9. The effects of AKT inhibitor or PI3K p110 inhibitor on cell viability of AS hMSCs 19 4.10. Specificity of PI3K isoforms related to aberrant AS hMSCs osteogenesis pathway 19 4.11. Abnormal osteogenesis differentiation in AS MSCs rescued by PI3K p110β/ blockade 19 4.12. PI3K may be the upstream regulator of sXBP1 and TNAP 20 4.13. Several cytokines are regulated by PI3K in AS hMSCs or control hMSCs 20 4.14. Abnormal osteogenesis differentiation in AS MSCs rescued by TGFβinhibition 21 4.15. The production of IL-6 and IL-8 may be regulated by HLA-B27 molecule in AS hMSCs 21 4.16. Identification of IRE1 inhibitor efficacy in A1 AS hMSCs under osteogenic differentiation 22 5. Discussion 23 6. Figures and legends 27 References 62 | |
| dc.language.iso | en | |
| dc.subject | 高速藥物篩選 | zh_TW |
| dc.subject | 僵直性脊椎炎 | zh_TW |
| dc.subject | 自體發炎疾病 | zh_TW |
| dc.subject | 間葉幹細胞 | zh_TW |
| dc.subject | 人類白血球抗原-B27 | zh_TW |
| dc.subject | 非專一性鹼性磷酸酶 | zh_TW |
| dc.subject | 磷脂酰肌醇-3激酶 | zh_TW |
| dc.subject | 乙型轉化生長因子 | zh_TW |
| dc.subject | High throughput drug screening | en |
| dc.subject | PI3K | en |
| dc.subject | ankylosing spondylitis | en |
| dc.subject | TGFβ | en |
| dc.subject | HLA-B27 | en |
| dc.subject | mesenchymal stem cells | en |
| dc.subject | autoinflammatory disease | en |
| dc.subject | TNAP | en |
| dc.title | 探討僵直性脊椎炎病人的間葉幹細胞異常骨化之機制 | zh_TW |
| dc.title | Study of the Osteogenesis Pathways in Mesenchymal Stem Cells of Ankylosing Spondylitis Patients | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李建國(Chien-Kuo Lee),徐立中(Li-Chung Hsu) | |
| dc.subject.keyword | 高速藥物篩選,僵直性脊椎炎,自體發炎疾病,間葉幹細胞,人類白血球抗原-B27,非專一性鹼性磷酸酶,磷脂酰肌醇-3激酶,乙型轉化生長因子, | zh_TW |
| dc.subject.keyword | High throughput drug screening,ankylosing spondylitis,autoinflammatory disease,mesenchymal stem cells,HLA-B27,TNAP,PI3K,TGFβ, | en |
| dc.relation.page | 69 | |
| dc.identifier.doi | 10.6342/NTU202003318 | |
| 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 | 免疫學研究所 | zh_TW |
| 顯示於系所單位： | 免疫學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| U0001-1308202020175600.pdf 未授權公開取用 | 14.5 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。