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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳玉怜(Yuh-Lien Chen) | |
dc.contributor.author | Yu-Chun Zhang | en |
dc.contributor.author | 張郁淳 | zh_TW |
dc.date.accessioned | 2021-06-15T12:31:09Z | - |
dc.date.available | 2021-08-26 | |
dc.date.copyright | 2016-08-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-04 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50157 | - |
dc.description.abstract | 心肌梗塞(myocardial infarction, MI)是現代化國家主要的疾病之一。根據世界衛生組織2011年的統計,每年因為急性心肌梗塞死亡的人數為九百萬人,而治療方式大多使血管有再灌流的現象產生。心臟的缺血再灌流會造成心肌細胞凋亡和細胞肥大的狀況。外吐小體(exosome)是一種直徑約為30-100 nm的小囊泡,多數的動物細胞都會分泌exosome。近年來有研究指出收集培養間質幹細胞(mesenchymal stem cell, MSC)的條件培養基(conditioned medium, CM),具有減緩心肌缺血再灌流後所造成的傷害,且CM中的exosome佔有重要的角色。但詳細的機制仍尚未明瞭,因此我們在此論文研究當中探討來自於成人脂肪間質幹細胞(adipose-derived stem cell, ADSC)的條件培養基(ADSC-CM)與外吐小體(ADSC-exosome)對於心肌缺血後再灌流(ischemia/reperfusion, I/R)的保護作用與其相關的機轉。我們使用了藉由結紮心臟左前降支冠狀動脈 (left anterior descending coronary artery, LAD)後鬆綁再灌流做為動物模式(I/R組)。在術後3小時後取心臟組織進行後續觀察。結果顯示, ADSC-CM和ADSC-exosome處理組於WGA螢光染色的實驗中有減少細胞肥大(hypertrophy)的現象。另外在TUNEL assay的結果也可看到有降低細胞凋亡的效果。除此之外細胞肥大相關蛋白ETS1和ANP及細胞凋亡相關蛋白p-p53、PUMA在ADSC-CM和ADSC-exosome處理組皆有表現量下降的情形。其中PUMA和ETS1已被認為是miR221/222的目標基因,在RT-PCR的結果中可見,I/R組別中miR221/222減少而在ADSC-CM和ADSC-exosome處理組中有回升的情形。另外在miR221/222 knockout (KO) mice中,我們同樣觀察到I/R組會有細胞凋亡和細胞肥大的狀況,同樣的,在加入ADSC-CM和ADSC-exosome後有減緩的現象。在細胞模式的部份,我們利用H2O2處理H9c2心肌母細胞來模擬缺血再灌流的氧化壓力傷害。H2O2處理後H9c2心肌母細胞有產生細胞凋亡和細胞肥大的情形,而在ADSC-CM和ADSC-exosome治療組當中有減少的狀況,在RT-PCR部份miR221/222在H2O2處理後同樣有表現量變少的情形且ADSC-CM和ADSC-exosome會有促使其回升的效果,除此之外,ADSC-CM和ADSC-exosome也會降低H2O2所誘導的PUMA和ETS1表現。綜合上述實驗結果。我們認為ADSC-CM和ADSC-exosome能有效減緩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 are the major health problem and the leading cause of death and disability in both industrialized and developing nations. Myocardial ischemia/reperfusion (I/R) leads to cardiomyocyte injury, including apoptosis and hypertrophy. Here we investigated the effects of the derivatives, conditioned media (ADSC-CM) and exosomes (ADSC-exosome), from adipose-derived stem cells on cardiac I/R mice and the underlying mechanisms. ADSC-CM and ADSC-exosome significantly attenuated I/R-induced cardiomyocyte apoptosis and hypertrophy by TUNEL and WGA staining, respectively. Moreover, the expression of the apoptosis related proteins, p-p53 and PUMA, and the hypertrophy related proteins, Ets-1 and ANP, was significantly reduced in cadiomyocytes of I/R mice with ADSC-CM or with ADSC-exosome treatment. Both 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 or with ADSC-exosome treatment by RT-PCR. We also observed that cardiac I/R operation remarkably increased cell apoptosis and cell hypertrophy in miR221/222 knockout (KO) mice, which was decreased with ADSC-CM or with ADSC-exosome. Furthermore, either ADSC-CM or ADSC-exosome protected H9c2 cardiomyocytes from H2O2-induced damages by decreasing the number of apoptosis and hypertrophy in vitro. H2O2-treated H9c2 cells significantly reduced miR221/222 expression whereas ADSC-CM or ADSC-Exo treatment reversed it. In addition, ADSC-CM or ADSC-exosome treatment decreased H2O2-induced PUMA and Ets-1 expression. Based on these findings, ADSC-CM and ADSC-exosome could serve as an effective therapy for the prevention of I/R-induced cardiac injury and the miR-221/222 are involved in this protection. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T12:31:09Z (GMT). No. of bitstreams: 1 ntu-105-R03446010-1.pdf: 4987723 bytes, checksum: 56a732c58b024b16e9ec01d13b1d0e5c (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 中文摘要 7
英文摘要 9 壹、緒論 一、心臟的構造和功能 11 二、心肌缺血再灌流傷害(myocardial ischemia / reperfusion injury , MI/R) 12 三、脂肪幹細胞(Adipose-Derived Stem Cells, ADSCs) 15 四、外吐小體(exosome) 18 五、微核糖核酸(MicroRNAs, miRNAs, miRs) 20 六、微核糖核酸-221/222(miR-221/222)與細胞凋亡和細胞肥大的關係 21 七、心肌缺血再灌流傷害與細胞凋亡的關係 22 八、心肌缺血再灌流傷害與心臟肥大的關係 23 九、研究動機 25 貳、實驗材料 26 一、儀器設備 26 二、實驗材料與試劑 27 三、實驗用溶液配方 31 參、實驗方法 34 動物實驗 (In vivo) 34 一、小鼠心臟缺血再灌流動物模式建立 34 二、組織石蠟包埋 35 三、蘇木精-伊紅染色 35 四、組織冷凍包埋 35 五、活性氧螢光染色 36 六、西方墨點法 36 七、麥胚素染色法 38 八、Terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) 38 九、即時定量聚合酶連鎖反應法測定mRNA表現 39 十、免疫組織化學染色法 41 體外細胞實驗 (In vitro) 42 十一、人類成體脂肪幹細胞培養 42 十二、心肌母細胞培養 43 十三、細胞活性分析法 43 十四、活性氧螢光染色 44 十五、流式細胞技術 44 十六、數據統計分析 44 肆、實驗結果 45 動物實驗 (In vivo) 45 一、ADSC-CM或ADSC-exosome改善心臟缺血再灌流後形態上的傷害 45 二、ADSC-CM或ADSC-exosome會減少心臟缺血再灌流後心臟組織之 表現 ROS情形 45 三、ADSC-CM或ADSC-exosome減少心臟缺血再灌流後的心肌細胞肥 大情形 46 四、使用ADSC-CM或ADSC-exosome治療後改善心臟缺血再灌流後 細胞凋亡 46 五、使用ADSC-CM或ADSC-exosome治療後提高心臟缺血再灌流後 組織表現miR221/222 47 六、使用ADSC-CM或ADSC-exosome治療後減少心臟缺血再灌流後 組織表現ETS1 47 七、使用ADSC-CM或ADSC-exosome治療後降低心臟缺血再灌流後 組織表現PUMA 48 八、使用ADSC-CM或ADSC-exosome治療後降低心臟缺血再灌流後 組織表現phospho-p53 49 體外細胞實驗 (In vitro) 50 九、使用ADSC-CM或ADSC-exosome會減少以H2O2培養後H9c2心 肌母細胞之ROS的表現 50 十、使用ADSC-CM或ADSC-exosome會減少以H2O2養後H9c2心肌 母細胞之細胞肥大 50 十一、使用ADSC-CM或ADSC-exosome會減少以H2O2培養後H9c2 心肌母細胞之細胞凋亡 51 十二、使用ADSC-CM或ADSC-exosome治療後提高H2O2培養後 H9c2心肌母細胞表現 miR221/222 52 十三、使用ADSC-CM或ADSC-exosome治療後減少ETS1、PUMA、 phospho-p53及提高Bcl2的表現量 52 伍、討論與結論 54 陸、參考文獻 60 柒、附圖 73 圖一、觀察心臟缺血再灌流及處理ADSC-CM或ADSC- exosome後, 心臟缺血再灌流後形態上的變化 72 圖二、心臟缺血再灌流後及使用ADSC-CM或ADSC- exosome處理後 組織上ROS的表現之情形 76 圖三A.B、心臟缺血再灌流及使用ADSC-CM或ADSC- exosome處理 後,觀察組織細胞肥大之情形 78 圖三C、心臟缺血再灌流後及使用ADSC-CM或ADSC- exosome治療 後,表現組織ANP之情形 80 圖四A,B、心臟缺血再灌流後及使用ADSC-CM或ADSC- exosome處 理後心臟缺血再灌流組織細胞凋亡之情形 82 圖四C、心臟缺血再灌流後及使用ADSC-CM或ADSC-exosome處理 後以西方墨點法觀察組織Bcl-2表現 84 圖五、心臟缺血再灌流後及使用ADSC-CM或ADSC- exosome處理後 組織miR-221/222表現之情形 85 圖六、心臟缺血再灌流後及使用ADSC-CM或ADSC- exosome處理後 組織ETS-1之表現情形 87 圖七、心臟缺血再灌流後及使用ADSC-CM或ADSC- exosome處理 後,心臟組織PUMA表現 89 圖八、心臟缺血再灌流後及使用ADSC-CM或ADSC- exosome處理後 組織中細胞凋亡相關蛋白phospho- p53之表現情形 90 圖九、以細胞活性分析法觀察H2O2對於H9c2心肌母細胞存活率的影 響 91 圖十、以H2O2培養H9c2心肌母細胞及ADSC-CM或ADSC-exosome 處理後ROS的表現情形 92 圖十一、以H2O2培養H9c2心肌母細胞及ADSC-CM或ADSC- exosome處理後觀察細胞肥大的表現情形 94 圖十二、以H2O2培養H9c2心肌母細胞及ADSC-CM或ADSC- exosome處理後,細胞凋亡的表現情形 95 圖十三、以H2O2培養H9c2心肌母細胞及ADSC-CM或ADSC- exosome處理後細胞miR 221/222表現之情形 97 圖十四、以H2O2培養H9c2心肌母細胞及ADSC-CM或ADSC- exosome處理後,與細胞肥大、細胞凋亡相關蛋白的表現情形 99 | |
dc.language.iso | zh-TW | |
dc.title | 探討脂肪幹細胞的條件培養液與外吐小體對於心肌缺血再灌流引發心肌損傷的影響 | zh_TW |
dc.title | To Study the Effect of Conditioned Medium and Exosomes from Adipose-Derived Stem Cells on Ischemia/Reperfusion-Iuduced Cardiac Injury | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 王懷詩(Huai-Shi Wang),江美治(Mei-Zhi Jiang),李繼源(Chi-Yuan Li),蔡佳醍(Jia-Ti Cai) | |
dc.subject.keyword | 心肌缺血後再灌流,成人脂肪間質幹細胞,外吐小體,微核糖核酸-221/222, | zh_TW |
dc.subject.keyword | myocardial ischemia/reperfusion,adipose-derived stem cell,exosome,miR-221/222, | en |
dc.relation.page | 99 | |
dc.identifier.doi | 10.6342/NTU201601862 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-04 | |
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 |
顯示於系所單位: | 解剖學暨細胞生物學科所 |
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