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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蔡素宜(Su-Yi Tsai) | |
dc.contributor.author | Ying-Ting Lee | en |
dc.contributor.author | 李穎婷 | zh_TW |
dc.date.accessioned | 2021-06-17T02:34:37Z | - |
dc.date.available | 2020-08-24 | |
dc.date.copyright | 2020-08-24 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-17 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68772 | - |
dc.description.abstract | 人類的心臟由數十億自發節律性跳動的心肌細胞組成,而心肌間盤便是維持相鄰心肌細胞持續而穩定的機械力收縮及電生理訊號傳遞的構造。先前研究發現XIRP1高度表現在心肌間盤,其中XIN重複序列更直接與肌動蛋白結合。動物研究發現Xirp1剔除的小鼠會發展出心臟肥大、電生理異常的情形、心肌間盤破碎以及排列不規則的心肌結構,且隨著小鼠年齡越大越發展出嚴重的心肌纖維化。此外,XIRP1的點突變也在一群突發性不明原因夜間猝死症(SUNDs)的病人基因中被發現。然而,XIRP1在人類心臟中所扮演的角色仍不清楚,於是我利用CRISPR/Cas9的技術將XIRP1從人類胚胎幹細胞中剔除,去探討XIRP1在心肌細胞發育過程所扮演的角色。初步結果觀察到將XIRP1剔除後仍能成功分化成心肌細胞,且肌結構排列整齊,然而在XIRP1-/-表現型分析發現,剔除XIRP1對心肌細胞造成的影響有:1)細胞面積增大,2)多核心肌細胞數量上升,3)心肌病變相關的基因(NPPA,MTH7/MYH6 比例)顯著上升,4)鈣離子瞬變的頻率下降及振幅上升,進出細胞的鈣離子異常流動而呈現不正常的波型圖。另外,在心肌細胞的間隙連接(gap junction)和黏著型連接(adherens junction)部分,分別做間隙連接蛋白43(CX43)和N-鈣粘蛋白(CDH2)螢光免疫染色,發現剔除XIRP1後,CX43在表現量及分布方面並未觀察到差異,但有趣的是,在黏著型連接則可發現較長的N-鈣粘蛋白斑塊,而這可能造成了心肌細胞間粘著異常,進而導致心肌肥大。總體而言,藉由本次的研究,可以推論XIRP1參與在細胞間黏著型連接藉此調控機械力的傳送,進而調控心肌細胞間鈣離子的傳遞及穩定與同步性的收縮,在心肌功能正常運作上扮演重要的角色,除此之外,從我實驗的結果顯示,當XIRP1受影響時似乎活化了心肌肥大相關的信號通路表現,但更詳細心肌疾病相關的分子機制仍然需做進一步探討,也希望XIRP1的研究結果在未來有機會作為疾病模型並應用於臨床。 | zh_TW |
dc.description.abstract | The human heart is composed of billions of spontaneously beating cardiomyocytes. Mechanical stability and synchronized contraction between adjacent cardiomyocytes are orchestrated by connection structure, termed an intercalated disc. Xin repeat-containing protein 1(XIRP1) is highly expressed in the intercalated disc. Previous studies have shown that XIRP1 interacts with adherent junctions-related proteins, and the unique conserved 16-amino acid residue repeating units are known as Xin repeats, which directly bind to actin filaments. Moreover, Xirp1 knockout mice developed cardiac hypertrophy and abnormal cardiac electrophysiology. Phenotypical analyses of these knockout mice showed damaged intercalated disc and disarranged cardiac sarcomeric structure, and cardiac fibrosis. Furthermore, a point mutation in XIRP1 was found in patients with sudden unexplained nocturnal death syndrome (SUNDs). However, the role of XIRP1 in human heart remains unclear. Therefore, I carried out the CRISPR/Cas 9 technique to ablate XIRP1 gene in human embryonic stem cells (hESCs) to determine the function of XIRP1 in cardiac cell development. Based on the preliminary data, I found that XIRP1 knockout (XIRP1-/-) hESCs can still be differentiated into cardiomyocytes (CMs) with well-organized sarcomere structures. However, phenotypical analyses of the XIRP1-/- CMs, these CMs displayed 1) a larger cell size, 2) an increasing number of binucleated CMs, 3) enhanced expression of cardiomyopathy related genes (NPPA and the ratio of MTH7/MYH6), and 4) abnormal calcium images with higher amplitude and lower frequency. Interestingly, the immunostaining of the gap junction and adherens junction markers, CX43 and CDH2, revealed that CX43 did not show any difference between wild- type (WT) and XIRP1-/- CMs. However, the average length of the CDH2 plaques was longer in XIRP1-/- CMs, which might cause the altered cell-cell adhesion and contribute to hypertrophic cardiomyopathy. Taken together, XIRP1 may play a role in adherens junction and maintaining normal calcium handling. The disturbing of XIRP1 might also activate the HCM signaling pathway and lead to HCM-like phenotypes. However, the detailed molecular mechanisms of how XIRP1 involves in cardiomyopathy remain further investigation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T02:34:37Z (GMT). No. of bitstreams: 1 U0001-1708202009324400.pdf: 5056569 bytes, checksum: f8d09abedea2a282e15dd6fb4efc86c0 (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 誌謝 i 中文摘要 ii Abstract iii Contents v Introduction 1 Material and Methods 5 CRISPR/Cas9 plasmid construction and transfection 5 Human embryonic stem cells culture and differentiation to cardiomyocytes 5 Western blotting 6 Immunofluorescence Staining and microscopy 7 Calcium imaging 8 RNA extraction, cDNA synthesis and quantitative PCR 8 Transmission Electron Microscopy 8 Statistical Analysis 9 Results 10 CRISPR/Cas9-Mediated Knockout of XIRP1 in hESCs 10 XIRP1-/- hESCs were able to differentiate into hESC-CMs 10 Ablation of XIRP1 do not affect sarcomeric structure 11 Ablation of XIRP1 caused the larger size of cardiomyocytes, but not nucleus size 12 Proliferation rate was not affected in XIRP1 knockout cardiomyocytes 12 Altered adherens junction in XIRP1 knockout cardiomyocytes 13 Abnormal Ca2+ handling properties in XIRP1 knockout cardiomyocytes 13 Cardiomyopathy related genes were highly expressed in XIRP1-/- CMs 14 Discussion 16 Conclusion and Future work 20 Figures 22 Figure 1. Schematic depiction of relative positions of XIRP1 and its protein variants. 22 Figure 2. CRISPR/Cas9-Mediated Knockout of XIRP1 in hESCs 24 Figure 3. Well-organized sarcomeric structure of XIRP1-/- hESC-CMs at differentiation day30 and day50 26 Figure 4. Ablation of XIRP1 caused the larger size of cardiomyocytes (CMs) and a higher percentage of multinucleated CMs, but not nucleus size 28 Figure 5. Proliferation rate of cardiomyocytes was not affected in XIRP1-/- CMs 30 Figure 6. Expression and distribution of intercalated disc associated protein CDH2 in XIRP1 knockout cardiomyocytes 32 Figure 7. Abnormal Ca2+ handling properties in XIRP1-/- cardiomyocytes 33 Figure 8. The mRNA level expression of Ca2+ handling related genes had changes in XIRP1-/- cardiomyocytes 34 Figure 9. Expression of cardiomyopathy related genes 35 Supplementary Figures and Tables 36 Figure S1. Cardiac cytoskeleton associated proteins further confirm the well-organized sarcomere in XIRP1-/- CMs at d30. 36 Figure S2. Normal pluripotent properties of CRISPR/Cas9-edited XIRP1 knockout hESC lines. 37 Figure S3. Compensation of XIRP1-/- CMs via qPCR to examine the target genes mRNA expression level. 38 Figure S4. Cardiac fibrosis was not found in XIRP1-/-CMs at day30. 39 Figure S5. Expression and distribution of XIRP1 C terminal bind protein, filamin C (FLNC) did not be affected. 40 Figure S6. Western blotting to confirm that XIRP1 isoform C still exists in XIRP1-/- lines. 41 Figure S7. Genotyping to confirm that I failed to obtain homozygous XIRP1 isoform C knockout line in XIRP1-/- #1 using CRISPR/Cas9. 42 Table 1. sgRNA primers 43 Table 2. XIRP1 PCR primers for genotyping 43 Table 3. qPCR primers 44 References 46 | |
dc.language.iso | en | |
dc.title | 探討心肌間盤蛋白XIRP1在心肌細胞發育過程中所扮演的角色 | zh_TW |
dc.title | Investigating the role of intercalated disc protein XIRP1 in cardiac cell development | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李士傑(Shyh-Jye Lee),楊鎧鍵(Kai-Chien Yang),黃祥博(Hsiang-Po Huang) | |
dc.subject.keyword | 人類胚胎幹細胞,分化,心肌細胞,心肌間盤,心臟肥大,XIRP1基因,CRISPR/Cas 9系統, | zh_TW |
dc.subject.keyword | human embryonic stem cells,differentiation,cardiomyocyte,intercalated disc,cardiomyopathy,XIRP1 gene,CRISPR/Cas 9 system, | en |
dc.relation.page | 53 | |
dc.identifier.doi | 10.6342/NTU202003679 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-08-19 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生命科學系 | zh_TW |
顯示於系所單位: | 生命科學系 |
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