請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82190完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃筱鈞(Hsiao-Chun Huang) | |
| dc.contributor.author | Fa-Hsuan Chiang | en |
| dc.contributor.author | 江法萱 | zh_TW |
| dc.date.accessioned | 2022-11-25T06:33:26Z | - |
| dc.date.copyright | 2021-08-20 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82190 | - |
| dc.description.abstract | "根據呂仁實驗室陳薇如博士先前的研究，發現PODXL在人類富潛能幹細胞中，可以藉由調控膽固醇細胞的代謝維持細胞自我更新的能力。鋅手指蛋白883 (Zinc finger protein 883, ZNF883) 為PODXL的下游基因。然而，ZNF883是個新穎的基因，從未在細胞中探討過它的功能。本研究揭露ZNF883富含於未分化的人類富潛能幹細胞中，但於分化細胞中會減少表現。抑制ZNF883會導致幹細胞自我更新的能力受損，且造成KLF4 和 OCT4的表現量在人類富潛能幹細胞中下降。在人類誘導性富潛能幹細胞進行逆編程的過程中，ZNF883會在後期增加表現量。另外，我建立ZNF883過度表達的系統，細胞經去氧羥四環素 (doxycycline) 處理後，ZNF883會被誘發增加表現量。ZNF883過量表達，可促進細胞自我更新。為了進一步探討ZNF883的調控路徑，我透過互補核苷酸微陣列 (cDNA microarray)及染色質免疫沉澱-測序 (ChIP-seq)，選出五個ZNF883的下游基因BAK1、 SACM1L、ARHGAP42、FNBP1L和 EIF2AK4。當抑制BAK1時，可以挽救在人類誘導性富潛能幹細胞中剔除ZNF883所造成的幹細胞自我更新能力的損傷及細胞凋亡。不論是在有無過度表現ZNF883的細胞中減少SACM1L、ARHGAP42、FNBP1L和 EIF2AK4表達，皆會造成細胞數量及鹼性磷酸酶 (alkaline phosphatase, ALP) 的活性下降。總結以上發現，ZNF883對於維持人類富潛能幹細胞中自我更新的能力是很重要的。而ZNF883如何藉由下游基因去維持幹細胞自我更新的調控機制仍尚待研究。" | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T06:33:26Z (GMT). No. of bitstreams: 1 U0001-2507202116481800.pdf: 34793094 bytes, checksum: db696847ab04e60aa5e9e3afd144abfb (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "口試委員會審定書 i 誌謝 ii 中文摘要 iii Abstract iv Table of contents v List of Figures viii List of Tables ix Chapter 1. Introduction 1 1.1 Pluripotent stem cells (PSCs) 1 1.2 Pluripotent state cells and extended pluripotent stem cells (EPSCs) 1 1.3 hESC core transcriptional regulators in self-renewal 2 1.4 Induced pluripotent stem cells (iPSCs) and somatic cells reprogramming 3 1.5 Podocalyxin (PODXL) and its downstream factor Zinc finger protein 883 (ZNF883) 4 1.6 Aim of the study 6 Chapter 2. Materials and Methods 8 2.1 Cell lines and culture conditions 8 2.1.1 Culture of primed human PSCs 8 2.1.2 Culture of human EPSCs 8 2.1.3 Culture of HEK293T cells and fibroblasts 9 2.2 Formation of Embryoid Bodies (EBs) from hESCs 9 2.3 Lentivirus production and hESC transduction 10 2.4 Generation of inducible CRISPR iPSCs to knockout ZNF883 10 2.4.1 To design sgRNAs and clone into the gRNA expression vector 10 2.4.2 To knockout ZNF883 by inducible CRISPR iPSC line 11 2.5 Somatic cell reprogramming to generate hiPSCs 12 2.6 Generation of ZNF883 inducible overexpressing hPSCs 12 2.7 ZNF883 downstream factor EIF2AK4 inhibitor treatment 13 2.8 Alamar blue assay 13 2.9 Alkaline phosphatase activity and staining assay 13 2.10 RNA extraction and quantitative reverse transcription real-time PCR (qRT-PCR) 14 2.11 DNA extraction and PCR 14 2.12 Western blot analysis 15 2.13 Immunofluorescence staining 15 2.14 Microarray analysis 15 2.15 Statistical analysis 16 Chapter 3. Results 17 3.1 ZNF883 is enriched in undifferentiated hESCs and downregulated after differentiation 17 3.2 Knockdown of ZNF883 by shRNA impairs self-renewal and pluripotency of hESCs 17 3.3 Inducible CRISPRn knockout of ZNF883 lose the function of self-renewal and pluripotency in hiPSCs 18 3.4 Overexpression of ZNF883 moderately increases the self-renewal and delayed cell differentiation in hESCs 19 3.5 Overexpression of ZNF883 in inducible cell lines promotes hESC self-renewal 20 3.6 ZNF883 is crucial for iPSC reprogramming 21 3.7 ZNF883 has DNA binding ability 22 3.8 Identification of ZNF883-related downstream genes by ChIP sequencing and cDNA microarray in hESCs 24 3.9 BAK1 knockdown rescues the self-renewal of hiPSCs under ZNF883 inducible knockout conditions 24 3.10 Using the shRNA and GCN2iB to suppress the expression levels of SACM1L, ARHGAP42, FNBP1L or EIF2AK4 in hESCs 27 3.11 shRNA of SACM1L, ARHGAP42, FNBP1L and GCN2iB block hESC renewal in ZNF883 overexpressing cells 28 Chapter 4. Discussion 30 4.1 ZNF883 plays a crucial role in self-renewal regulation of hPSCs 30 4.2 ZNF883 mediates BAK1 to induce cell apoptosis and balance cellular proliferation 31 4.3 SACM1L regulates secretory pathway to involve in cell proliferation 32 4.4 ARHGAP42 controls cellular development upon Rho-GTPase signaling pathway 33 4.5 FNBP1L promotes actin polymerization to regulate cellular reprogramming 33 4.6 EIF2AK4 maintain self-renewal of hESCs that is similar to promote growth in tumor cells 34 4.7 ZNF883 may have secondary pathway with SACM1L, ARHGAP42 or FNBP1L 35 4.8 Conclusion and future work 36 References 38 List of Figures Figure 1. ZNF883 was highly expressed in human pluripotent stem cells. 49 Figure 2. ZNF883 knockdown impaired the cell numbers and self-renewal in hESCs. 51 Figure 3. hESCs lost the pluripotent state after ZNF883 knockdown. 53 Figure 4. The strategy of ZNF883 knockout design. 55 Figure 5. Inducible knockout of ZNF883 blocked the self-renewal in hiPSCs. 57 Figure 6. ZNF883 overexpression upregulated pluripotency moderately. 58 Figure 7. Generation of inducible ZNF883-overexpressed RUES cell lines. 60 Figure 8. ZNF883 overexpression promoted the self-renewal of RUES cell lines. 61 Figure 9. High ZNF883 expression is reestablished upon iPSC reprogramming. 63 Figure 10. Identification of ZNF883 downstream target genes in hESCs. 65 Figure 11. Knockout ZNF883 by CRISPR/Cas9 inducible system and knockdown BAK1 by shBAK1. 67 Figure 12. ZNF883 inducible knockout lost the self-renewal were rescued by BAK1 knockdown. 69 Figure 13. SACM1L, ARHGAP42 and FNBP1L were knockdown by shRNA lentiviral transduction in hESC RUES2 cell lines. 71 Figure 14. To test the inhibition efficiency of GCN2iB on EIF2AK4 gene in hESC RUES2 cell lines. 73 Figure 15. ZNF883 was upregulated by doxycycline after shRNA transduction in inducible ZNF883-overexpressed RUES cells. 75 Figure 16. SACM1L, ARHGAP42 and FNBP1L downregulated the expression levels and impaired self-renewal by shRNA transduction in inducible ZNF883-overexpressed RUES cells. 77 Figure 17. The inhibition of EIF2AK4 impaired the self-renewal in ZNF883-overexpressed RUES cells. 79 Figure 18. ZNF883 is required for supporting the self-renewal in hPSCs. 80 Appendix Figure 1. ZNF883 was low expressed in differentiated cells. 81 Appendix Figure 2. The CRISPRn construct inserted into iPSC lines. 82 Appendix Figure 3. ZNF883 overexpression delayed stem cells differentiation. 83 Appendix Figure 4. Downregulation of ZNF883 affected iPSC formation efficiency. 84 Appendix Figure 5. ChIP sequence analysis of ZNF883. 85 List of Tables Table 1. shRNA target gene sequences 86 Table 2. The primers for qRT-PCR 87 Table 3. The PCR primers used for genotyping PCR or junction PCR 88 Table 4. The antibodies used for Western blot 89 Table 5. The antibodies used for immunofluorescence staining 90 Table 6. The 19 selected genes list 91 " | |
| dc.language.iso | en | |
| dc.subject | 細胞逆編程序 | zh_TW |
| dc.subject | 鋅指蛋白 | zh_TW |
| dc.subject | 人類胚胎幹細胞 | zh_TW |
| dc.subject | 人類誘導性富潛能幹細胞 | zh_TW |
| dc.subject | 幹細胞自我更新 | zh_TW |
| dc.subject | Zinc-finger protein | en |
| dc.subject | human induced pluripotent stem cell | en |
| dc.subject | self-renewal | en |
| dc.subject | reprogramming | en |
| dc.subject | human embryonic stem cell | en |
| dc.title | 探討ZNF883在人類富潛能幹細胞中調控自我更新的功能及機制 | zh_TW |
| dc.title | The functions and mechanisms of ZNF883 in regulating self-renewal human pluripotent stem cells | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 呂仁(Jean Lu) | |
| dc.contributor.oralexamcommittee | 林劭品(Hsin-Tsai Liu),(Chih-Yang Tseng) | |
| dc.subject.keyword | 鋅指蛋白,人類胚胎幹細胞,人類誘導性富潛能幹細胞,幹細胞自我更新,細胞逆編程序, | zh_TW |
| dc.subject.keyword | Zinc-finger protein,human embryonic stem cell,human induced pluripotent stem cell,self-renewal,reprogramming, | en |
| dc.relation.page | 91 | |
| dc.identifier.doi | 10.6342/NTU202101723 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2021-08-04 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2026-08-03 | - |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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