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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 陳玉怜 | |
| dc.contributor.author | Tzu-Lin Lee | en |
| dc.contributor.author | 李紫琳 | zh_TW |
| dc.date.accessioned | 2021-06-17T01:20:18Z | - |
| dc.date.available | 2020-09-08 | |
| dc.date.copyright | 2017-09-08 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-11 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67113 | - |
| dc.description.abstract | 心血管疾病是已開發國家和發展中國家造成死亡的主要原因。心肌缺血/再灌流（ischemia/reperfusion, I / R）會導致心肌細胞損傷，包括細胞凋亡和纖維化。研究指出收集間質幹細胞(mesenchymal stem cell, MSC)的條件培養液(conditioned medium, CM)，可有效減緩I/R後所造成的傷害，脂肪間質幹細胞(adipose-derived stem cell, ADSC)是間質幹細胞其中一種。本研究主旨為探討脂肪間質幹細胞的條件培養液(ADSC-CM)對於心肌缺血再灌流(I/R)的心肌凋亡和纖維化的調節機制。藉由結紮小鼠的心臟冠狀動脈左前降支(left anterior descending coronary artery, LAD)造成心肌缺血30分鐘後再灌流，作為心肌受損的(I/R組)動物模式，將ADSC-CM注射到缺血後的瘢痕和邊界中(I/R+CM組)，並在術後3小時及3天取其心臟組織進行觀察。使用TUNEL染色計數凋亡細胞及運用免疫組織化學染色評估心肌凋亡及心臟纖維化情形。其結果顯示I/R會誘導心肌細胞凋亡及纖維化；而在ADSC-CM處理組顯著改善心肌凋亡和減少心臟纖維化情形。除此之外細胞凋亡相關蛋白PUMA在ADSC-CM處理組皆有表現量下降的情形，其中PUMA已被認為是miR221/222的目標基因，在RT-PCR的結果中可以發現，I/R組別中miR221/222表現量減少而在ADSC-CM處理組中有回升的情形。另外在miR221/222 knockout (KO) mice中，同樣觀察到ADSC-CM處理後會減緩細胞凋亡和細胞纖維化的現象。 在細胞模式方面，我們利用缺氧再灌氧環境誘導H9c2心肌細胞模擬動物缺血再灌流的傷害。缺氧再灌氧處理後H9c2心肌母細胞的確產生細胞凋亡和細胞纖維化的情形，而在ADSC-CM治療組當中與細胞凋亡及纖維化相關的蛋白表現有減少的狀況，我們認為ADSC-CM可減緩心肌細胞凋亡及纖維化的情形並保護心肌缺血後再灌流所造成的傷害且miR221/222參與在其中。 | zh_TW |
| dc.description.abstract | Cardiovascular disorders is the leading cause of death in both developed and developing countries. Myocardial ischemia/reperfusion (I/R) leads cardiomyocyte injury, including apoptosis and fibrosis. The present study was aimed at determining the effect and regulatory mechanism of conditioned media from adipose-derived stem cells (ADSC-CM) on cardiac apoptosis and fibrosis. The mouse myocardial I/R model was established to induce by ligating the left anterior descending coronary artery for 30 min and then reperfusion for 3 h or for 3 days (I/R group). ADSC-CM treatment significantly reduced I/R-induced cardiomyocyte apoptosis by TUNEL staining. Moreover, the expression of the apoptosis related proteins, p53 upregulated modulator of apoptosis (PUMA), and the fibrosis-related proteins, fibronectin and collagen III, was significantly reduced in cardiomyocytes of I/R mice with ADSC-CM treatment. PUMA and Ets-1 have been reported to be the target genes of miR221/222. I/R operation dramatically decreased miR221/222 expression, which was increased with ADSC-CM treatment by RT-PCR. We also observed that cardiac I/R operation remarkably increased cell apoptosis and cell fibrosis in miR221/222 knockout (KO) mice, which was decreased with ADSC-CM. We next established the in vitro cell model with H9c2 cells under hypoxia /reoxygenation (H/R) treatment. ADSC-CM increased cell viability of H/R-treated H9c2 cells by MTT assay. Furthermore, ADSC-CM decreased cell apoptosis by TUNEL assay. In addition, ADSC-CM treatment decreased H/R-induced PUMA and Ets-1 expression by Western blot assay. ADSC-CM decreased fibrosis-related proteins, collagen III and fibronectin expression by Western blot assay. Based on these above findings, ADSC-CM could protect myocardial I/R-induced injury against apoptosis and fibrosis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T01:20:18Z (GMT). No. of bitstreams: 1 ntu-106-R04446008-1.pdf: 3592303 bytes, checksum: d59029b04553d013eb38e354d0b24295 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 口試委員審定書…………………………………………………………………………I 誌謝……………………………………………………………………………………...II 中文摘要……………………………………………………………………………….VI 英文摘要…………………………………………………………………………........VII 壹、 緒論………………………………………………………………………………1 一、 心臟的構造和功能…………………………………………………………1 二、 心肌缺血再灌流傷害(ischemia / reperfusion injury ,I/R injury)…………. 2 三、 心肌缺血再灌流傷害與細胞凋亡(apoptosis)的關係……………………...4 四、 心肌缺血再灌流傷害與細胞纖維化的關係(fibrosis)……………………..5 五、 脂肪幹細胞(Adipose-DerivedStem Cells, ADSCs)………………………...6 六、 微核糖核酸 (MicroRNAs, miRNAs, miRs)………………………………..7 七、 微核糖核酸-221/222(miR-221/222)與細胞凋亡和細胞纖維化的關係…...7 八、 研究動機………………………………………………………...…………..9 貳、 實驗材料………………………………………………………………………...10 一、 儀器設備………………………………………………………...…………10 二、 實驗材料與試劑…………………………………………...………………11 三、 實驗用溶液配方…………………………………………………...………15 參、 實驗方法………………………………………………………..……………….17 動物實驗 (In vivo)……………………………………...………………………17 一、 建立小鼠心臟缺血再灌流的動物模式……………………………….…..17 二、 乳酸去氫酵素 (lactate dehydrogenase, LDH)之測定………………….…18 三、 ADSC條件培養液(conditionedmedia)收集……………………..…..……18 四、 組織石蠟包埋………………………………………………………..….…19 五、 蘇木精-伊紅染色(hematoxlin-eosin staining)……………………….……19 六、 西方墨點法 (Weatern blotting)…………………………………..….……19 七、 Terminal deoxynucleotidyl transferase dUTP nick end labeling assay …...21 八、 即時定量聚合酶連鎖反應法測定mRNA表現………………..…..….…22 九、 免疫組織化學染色法……………………………………………..…….…23 體外細胞實驗 (In vitro) ……………………………………...………………………24 十、 人類成體脂肪幹細胞培養…………………………………………...……24 十一、 心肌母細胞培養………………….……………………………………25 十二、 細胞活性分析法……………………………………………….………25 十三、 流式細胞技術………………………………………………….………26 十四、 數據統計分析……………………………………………….…………26 肆、 實驗結果………………………………………………………………………...27 一、 在形態方面ADSC-CM改善心臟缺血再灌流後的傷害…………….…..27 二、 心臟缺血再灌流後誘導產生氧化壓力傷害及細胞受損的情形……...…27 三、 ADSC-CM可以減緩心臟缺血再灌流後的細胞凋亡之情形……………28 四、 ADSC-CM處理下缺血再灌流後的心臟組織降低PUMA表現……..…28 五、 利用ADSC-CM處理可改善心臟缺血再灌流後細胞纖維化情形…...…28 六、 使用ADSC-CM治療能提高心肌缺血再灌流後組織miR221/222表現.29 七、 miR221/222與心臟缺血再灌流後的細胞凋亡之間的關係…………..…29 八、 miR221/222與心臟缺血再灌流後的細胞纖維化之間關係………..……30 九、 ADSC-CM可以增加H9c2細胞在缺氧再灌氧的細胞生存率……….…31 十、 ADSC-CM減緩缺氧環境再灌氧下H9c2細胞細胞凋亡情形………….31 十一、 使用ADSC-CM減緩缺氧環境再灌氧下H9c2細胞之纖維化……..32 十二、 使用ADSC-CM會增加缺氧環境再灌氧下H9c2細胞miR221/222表現……………………...……………………………………………………32 十三、 ADSC-CM中miR221/222的表現……………………………………33 伍、 討論與結論………………………………………………...……………………34 陸、 參考文獻………………………………………………………………...………37 柒、 附圖……………………………………………………………………………...43 一、 ADSC-CM對心臟缺血再灌流後形態的影響……………………………44 二、 ADSC-CM對心臟缺血再灌流動物模式中心臟細胞損傷的影響………45 三、 ADSC-CM對心臟缺血再灌流後細胞凋亡的影響………………………46 四、 ADSC-CM治療缺血再灌流後的心臟組織之PUMA表現情形……..…48 五、 ADSC-CM處理後對心臟缺血再灌流的細胞纖維化之影響……………50 六、 心臟缺血再灌流後組織miR-221/222表現之情形………………………51 七、 miR221/222與心臟缺血再灌流後的細胞凋亡之間的關係……..………54 八、 ADSC-CM處理後影響心臟缺血再灌流後細胞纖維化之情形表現……57 九、 以細胞活性分析法觀察缺氧環境再灌氧對於H9c2細胞存活率的影響58 十、 在H/R處理下及ADSC-CM對細胞凋亡的改變情形……………..……60 十一、 使用ADSC-CM於缺氧環境再灌氧下H9c2細胞之纖維化之影響..62 十二、 使用ADSC-CM在缺氧環境下H9c2細胞miR221/222 之表現...…63 十三、 ADSC-CM中miR221/222 之表現………..…………………………64 | |
| dc.language.iso | zh-TW | |
| dc.subject | 心肌缺血/再灌流 | zh_TW |
| dc.subject | 脂肪間質幹細胞條件培養液 | zh_TW |
| dc.subject | 細胞凋亡 | zh_TW |
| dc.subject | 纖維化 | zh_TW |
| dc.subject | miR221/222 | zh_TW |
| dc.subject | Ischemia/Reperfusion | en |
| dc.subject | ADSC-CM | en |
| dc.subject | apoptosis | en |
| dc.subject | fibrosis | en |
| dc.subject | miR221/222 | en |
| dc.title | 探討脂肪幹細胞的條件培養液對心肌缺血再灌流 所引發心肌凋亡及纖維化之影響 | zh_TW |
| dc.title | The effects of conditioned medium from adipose-derived stem cells on ischemia/reperfusion-induced cardiac apoptosis and fibrosis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 江美治,王懷詩,林茂欣 | |
| dc.subject.keyword | 心肌缺血/再灌流,脂肪間質幹細胞條件培養液,細胞凋亡,纖維化,miR221/222, | zh_TW |
| dc.subject.keyword | Ischemia/Reperfusion,ADSC-CM,apoptosis,fibrosis,miR221/222, | en |
| dc.relation.page | 64 | |
| dc.identifier.doi | 10.6342/NTU201702988 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-11 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 解剖學暨細胞生物學研究所 | zh_TW |
| Appears in Collections: | 解剖學暨細胞生物學科所 | |
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