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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蘇剛毅(Kang-Yi Su) | |
| dc.contributor.author | Chih-Jung Chen | en |
| dc.contributor.author | 陳祉蓉 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:40:18Z | - |
| dc.date.available | 2021-07-10T21:40:18Z | - |
| dc.date.copyright | 2020-09-10 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-10 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76913 | - |
| dc.description.abstract | 粒線體是一種動態的胞器，它會進行分裂或融合進而調控粒線體的功能以適應細胞環境的改變。然而，粒線體動力學的失衡會導致多種疾病的臨床進展，包括神經退化性疾病，代謝性疾病和癌症。近年許多研究指出，粒線體的功能會隨著年齡增加而逐漸下降，衰老的細胞常伴隨著粒線體動力學的失衡、ATP製造減少、ROS製造增加等等的粒線體功能缺失，而此與老化相關疾病的進展有相當大的關聯。然而，目前有效的抗老化粒線體藥物並不多。因此，為了找出能夠調節粒線體型態的潛力藥物，我們利用高通量螢光影像分析系統(High Content Screening system)以篩選Library of Pharmacologically Active Compounds (LOPAC)中可明顯調控粒線體型態的藥物。我們初步地利用肉眼判讀結果並以未加藥的控制組作為標準後，根據藥物改變粒線體型態的程度打分數。經由初步篩選1280個藥物後，先挑選出14個會明顯改變粒線體型態的藥物進行進一步驗證。而後，從14個藥物中挑選出以劑量依賴性的方式調節粒線體型態的3個藥物(Cortisone, TBBz, Diphenyleneiodonium chloride(DPI))進行後續的機制探討、粒線體功能測試以及探討其對老化的影響。在機制探討的方面，3個藥物皆不會影響粒線體動力學相關基因的表現量，亦不是藉由改變粒線體分裂蛋白Drp1 Ser616位點的磷酸化以及粒線體融合蛋白Opa1切割、Mfn2的表現量來調控粒線體型態。在粒線體功能測試方面，Cortisone和TBBz皆不會對糖解作用及氧化磷酸化造成影響，而DPI則是會抑制氧化磷酸化、促進糖解作用及ATP的產生，且其亦會導致氧化磷酸化相關的基因下調。此外，推測DPI可能是藉由促進粒線體分裂而減緩氧化壓力所誘導的細胞衰老。為了了解DPI是否可以成為預防老化的藥物，我們也積極測試用於長期治療的適當濃度，並且將利用小鼠胚胎成纖維細胞(MEF)作為細胞模型觀察藥物對於延緩複製性衰老的作用。 | zh_TW |
| dc.description.abstract | Mitochondria are a highly dynamic organelle that constantly undergoes fission and fusion, and this plays an important role in regulating mitochondrial functions. Imbalance of mitochondrial dynamics contributes to the clinical progression of a variety of diseases, including neurodegenerative diseases, metabolic diseases, and cancers. According to the previous studies, imbalance of mitochondrial dynamics and mitochondrial dysfunction are observed in senescent models, and this is extremely associated with the progression of age-related disorders. However, there are few effective anti-aging drugs targeting mitochondria nowadays. Therefore, we combined the LOPAC®1280 compound library with the High Content Screening system to screen potential drugs that can significantly modulate mitochondrial morphology. Mitochondrial morphology is observed and scored according to comparison with the untreated control group to select drugs. There are fourteen drugs selected for further validation after the preliminary screening of 1280 compounds. Among them, three drugs (Cortisone, TBBz, Diphenyleneiodonium chloride(DPI)) modulating mitochondrial dynamics in a dosage-dependent manner are selected for the subsequent experiments. Cortisone can induce fusion while TBBz and DPI can induce fission. There is no difference in the expression of mitochondrial dynamics-associated mRNA after treatment with these three drugs. They seem to regulate mitochondrial dynamics via neither phosphorylating DRP1 at Ser616, OPA1 cleavage, nor MFN2 expression. Cortisone and TBBz do not influence mitochondrial respiration, while DPI can inhibit oxidative phosphorylation (OXPHOS), promote glycolysis and ATP production, and it causes downregulation of OXPHOS-associated genes. In addition, we speculate that DPI may be able to inhibit oxidative stress-induced senescence through mitochondrial fission. Finally, in order to investigate whether DPI can act as an aging-preventing drug, the optimal concentration for long-term treatment is necessary to be determined first. In the future, we will assess the effect of DPI on ameliorating replicative senescence in MEFs. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:40:18Z (GMT). No. of bitstreams: 1 U0001-1008202012175600.pdf: 4988129 bytes, checksum: 07e46939b787ef83cde2009dbb0dbc73 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 論文審定書...................................................................... i 致謝........................................................................... ii 中文摘要........................................................................ iii ABSTRACT....................................................................... iv Chapter 1 Introduction........................................................ 1 1.1 Mitochondria and Mitochondrial dynamics.................................... 2 1.2 Mitochondrial diseases..................................................... 4 1.3 Cellular senescence........................................................ 5 1.4 Mitochondria and Cellular senescence....................................... 7 1.5 Mitochondrial therapy and Anti-aging therapy............................... 8 1.6 High Content Screening..................................................... 9 Chapter 2 Specific aims....................................................... 11 Chapter 3 Materials and Methods............................................... 13 3.1 Cell culture............................................................... 14 3.2 FCCP and H2O2 treatment.................................................... 15 3.3 Trypan blue viability test................................................. 16 3.4 LOPAC drug library......................................................... 17 3.5 Protein extraction and Western blotting analysis........................... 17 3.6 MitoTracker staining....................................................... 19 3.7 Fluorescent imaging........................................................ 19 3.8 MTT assay.................................................................. 20 3.9 RNA extraction and Quantitative PCR........................................ 21 3.10 Lactate assay............................................................. 22 3.11 NAD/NADH assay............................................................ 23 3.12 ATP assay................................................................. 24 3.13 SA-β-gal staining......................................................... 25 3.14 Statistical analysis...................................................... 25 Chapter 4 Results............................................................. 26 4.1 The experimental design.................................................... 27 4.2 Establishmentof mitochondrial morphology Analysis with NIH3T3 cell lines and high content screening.............................................................. 28 4.3 Result of LOPAC®1280 compound library screening............................ 29 4.4 Three potential drugs modulate mitochondrial dynamics in a dosage-dependent manner and are no cytotoxicity................................................. 30 4.5 Investigation of potential drugs regulating the mechanism of mitochondrial dynamics....................................................................... 31 4.6 The effects of the potential drugs on mitochondrial respiratory capacity... 32 4.7 The effect of DPI on oxidative stress-induced senescence................... 33 4.8 Determination of the appropriate DPI concentration for long-term treatment in MEFs........................................................................... 35 Chapter 5 Discussions......................................................... 36 5.1 Application of High Content Screening system............................... 37 5.2 Analysis of mitochondrial morphology....................................... 37 5.3 Discussion on preliminarily selected drugs................................. 38 5.4 Discussion on three potential drugs........................................ 39 Chapter 6 Conclusion.......................................................... 45 Chapter 7 Figures............................................................. 47 Figure 1. Drug screening flowchart............................................. 48 Figure 2. Dosage-dependent of FCCP for mitochondrial fission................... 52 Figure 3. Result of LOPAC®1280 compound library screening...................... 56 Figure 4. Potential drugs for the modulation of mitochondrial dynamics in a dosage-dependent manner............................................................... 58 Figure 5. Cytotoxicity test of potential compounds............................. 59 Figure 6. Mitochondrial dynamics in cells treated with potential compounds..... 61 Figure 7. Mitochondrial respiration in cells treated with potential compounds.. 63 Figure 8. The effect of DPI on H2O2-induced senescence......................... 66 Figure 9. Determination of the optimal DPI concentration for treating replicative senescence in MEFs............................................................. 69 Chapter 8 Table............................................................... 70 Table 1. Antibodies used in Western blotting................................... 71 Table 2. Primer sequences used in quantitative RT-PCR.......................... 72 Table 3. Information of selected drugs......................................... 74 Table 4. The effect of the multiple dosages of selected drugs on mitochondrial morphology..................................................................... 75 References..................................................................... 76 | |
| dc.language.iso | en | |
| dc.subject | LOPAC 1280 | zh_TW |
| dc.subject | 粒線體動力學 | zh_TW |
| dc.subject | 抗老化 | zh_TW |
| dc.subject | 高通量螢光影像分析系統 | zh_TW |
| dc.subject | Mitochondrial dynamics | en |
| dc.subject | LOPAC 1280 | en |
| dc.subject | High Content Screening system | en |
| dc.subject | Anti-senescence | en |
| dc.title | 篩選調控粒線體型態之潛力藥物及其於抗衰老之應用 | zh_TW |
| dc.title | Drugs Identification for Mitochondrial Dynamics Modulation for Anti-Senescence | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林亮音(Liang-In Lin),楊雅倩(Ya-Chien Yang),郭靜穎(Ching-Ying Kuo) | |
| dc.subject.keyword | 粒線體動力學,抗老化,高通量螢光影像分析系統,LOPAC 1280, | zh_TW |
| dc.subject.keyword | Mitochondrial dynamics,Anti-senescence,High Content Screening system,LOPAC 1280, | en |
| dc.relation.page | 87 | |
| dc.identifier.doi | 10.6342/NTU202002781 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-11 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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