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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林俊彬(Chun-Pin Lin) | |
| dc.contributor.author | Hsin-Wu Mi | en |
| dc.contributor.author | 秘心吾 | zh_TW |
| dc.date.accessioned | 2021-06-13T06:25:09Z | - |
| dc.date.available | 2012-10-05 | |
| dc.date.copyright | 2011-10-05 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-07-25 | |
| dc.identifier.citation | Abdallah BM, Haack-Sorensen M, Burns JS, Elsnab B, Jakob F, Hokland P, Kassem M. 2005. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive proliferation. Biochem Biophys Res Commun 326: 527-538.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34728 | - |
| dc.description.abstract | 我們成功的表現並制造「第四型骨成形蛋白」（BMP4）與「強化的綠色螢光蛋白」（EGFP）的融合蛋白（BMP4-EGFP）在生產細胞的分泌管道中。融合蛋白（BMP4-EGFP）的螢光只能在融合蛋白的輸送制造中斷後獲得，方法是在一個低溫（20°C）的環境中培養細胞，而且此融合蛋白的單分子動態, 可利用單分子顯微鏡予以追蹤檢視。經西方墨點分析法確認，在低溫培養細胞，有助於BMP4-EGFP融合蛋白的完整形成。在本研究中，我們首次能夠為附著於人類牙周纖維母細胞細胞膜上的螢光 BMP4 分子造影。進一步我們觀察到EGFP的一個步驟光漂白,以及量子點（quantum dots）的「閃爍」行為顯示, 有些螢光BMP4亮點確實代表單一分子的活動事跡。對單一 BMP4 分子的追蹤更顯示，BMP 的接受器（BMP receptors） 在受到 BMP4 的激發後，會發生二聚化現象；或者一個 BMP4 分子和一個已成二聚體的 BMP接受器的複合體首先形成，接著便是第二個 BMP4 分子的附著。再者，實驗發現 BMP4-EGFP 融合蛋白可以強化人類牙周纖維母細胞的成骨分化，但須透過涉及 BMP 的接受器的信號轉導。此一單分子的造影技術，或可成為一項有力的工具，以利未來 BMP4 基因療法和以人類牙周纖維母細胞為目標細胞的再生醫學的發展。
由於人類牙周韌帶細胞內可能含有先驅細胞或幹細胞群, 所以被認為對臨床牙周再生是一個有利的細胞來源。 而「第四型骨成形蛋白」(BMP4)和「人類端粒酶」(hTERT)，在幹細胞屬性的調節過程中，都扮演很重要的角色。在本研究中，我們首次發現，共同BMP4 and hTERT的基因轉殖於人類牙周纖維母細胞，顯著提高並強化了人類牙周纖維母細胞的多功化能力的分化效率，並在體外細胞培養實驗成骨、成脂、以及神經生成的分化過程中，以及在活體動物實驗牙骨質或牙周韌帶類似組織的再生過程中顯現出來。 結果發現，強化了人類牙周纖維母細胞的多功化能力的原因，至少部分要歸功於有些重要幹細胞標記的表現增加，例如scleraxis，Stro-1，CD146，以及憑藉了增強的Smad 1/5/8的基本磷酸化階度，而使為活化BMP訊息所需的BMP濃度門檻降至極低，而又產生更好的分化結果。此外, 如加入Noggin去抑制BMP4的作用，實驗結果發現CD146 和 CD90表現程度顯著降低，如此更確認了BMP4加諸於類似幹細胞表型上的直接效果, 而此種表型係對人類牙周纖維母細胞的基因轉殖而獲得。再者, 此等細胞呈現了一種高度神經系統的分化能力（> 75%）。而在將這群細胞移植到NOD/SCID老鼠身上之後, 這些基因修改後的人類牙周纖維母細胞，在載體的表面，產生了類似牙骨質或牙周韌帶的結構。這些基因修改後的人類牙周纖維母細胞的多功分化能力表現，對未來牙周病治療及再生醫學，提供了幹細胞的潛在來源。 | zh_TW |
| dc.description.abstract | We expressed bone morphogenetic protein 4 (BMP4) fused with enhanced green fluorescent protein (BMP4-EGFP) in the secretory pathway of producer cells. Fluorescent EGFP was only acquired after interrupting the transport of BMP4-EGFP by culturing cells at a lower temperature (20°C) and the dynamics of BMP4-EGFP can be monitored using single molecule microscopy. Western blotting analysis confirmed that exposure to low temperature helped the integrated formation of BMP4-EGFP fusion proteins. In this study, we, for the first time, could image the fluorescently labeled BMP4 molecules localized on the plasma membrane of living periodontal ligament cells (PFs). The one-step photobleaching with EGFP and the “blinking” behavior of quantum dots suggest that the fluorescent spots represent the events of single BMP4 molecules. Single-molecule tracking showed that the BMP receptors (BMPR) dimerize after BMP4 stimulation or that a complex of one BMP4 molecule and a preformed BMPR dimer develop first, followed by the binding of the second BMP4 molecule. Furthermore, BMP4-EGFP enhanced the osteogenic differentiation of PF cells via signal transduction involving BMP receptors. This single-molecule imaging technique might be a valuable tool for the future development of BMP4 gene therapy and regenerative medicine mediated by PFs. Because periodontal ligament (PDL) cells are reported to contain progenitor or stem cell populations, they are considered a beneficial cell source for clinical periodontal regeneration. Both bone morphogenetic protein 4 (BMP4) and human telomerase reverse transcriptase (hTERT) play essential roles in the modulation of stem cell properties. Here, we report for the first time that the combined ectopic expression of BMP4 and hTERT significantly enhanced the multipotent differentiation efficiency and capacity of human periodontal ligament fibroblasts (PFs) as shown by osteogenic, adipogenic, and neurogenic differentiation in vitro, and cementum/PDL-like tissue regeneration in vivo. These findings may be attributed, at least in part, to the original upregulation of important stem cell markers, such as scleraxis, Stro-1, CD146 and the extremely lowered threshold for BMP concentration to activate BMP signaling by enhanced basal phosphorylation levels of Smad 1/5/8. In addition, the significantly reduced expression levels of CD146 and CD90 with the presence of Noggin confirms the direct effect of BMP4 on the stem cell-like phenotype of genetically modified PF cells (BT-PFs). Furthermore, BT-PFs exhibited a high neural differentiation capacity (> 75%). After transplantation into NOD/SCID mice, genetically modified-PFs generated cementum/PDL-like structures on the surface of the carrier. The multipotency of these modified cells potentially provides an attractive source of stem cells for therapeutic purposes and regenerative medicine. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T06:25:09Z (GMT). No. of bitstreams: 1 ntu-100-D93422003-1.pdf: 3655950 bytes, checksum: 0a03738436436a54636fedaab709064d (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 口試委員會審定書………………………………………………… i
誌謝…………………………..…………………………………… ii 中文摘要…………………………………………………………. iii 英文摘要……………………………………………………………. v Chapter one: Single-molecule imaging of BMP4 dimerization on human periodontal ligament cells ….......…………… 1 1.1 INTRODUCTION…........................................ 2 1.2 MATERIALS AND METHODS…........................... 8 1.3 RESULTS…...……................................. 16 1.4 DISCUSSION…..................................... 23 Chapter two: Highly efficient multipotent differentiation of human periodontal ligament fibroblasts induced by combined BMP4 and hTERT gene transfer…………………. 30 2.1 INTRODUCTION…...……........................... 31 2.2 MATERIALS AND METHODS…......................... 39 2.3 RESULTS…....................................... 47 2.4 DISCUSSION….................................... 56 REFERENCES………………………………………………………. 65 FIGURES and FIGURE LEGENDS…………………………………. 91 Figure 1….......................................... 91 Figure 2…...……................................... 92 Figure 3….......................................... 94 Figure 4….......................................... 95 Figure 5….......................................... 97 Figure 6….......................................... 99 Figure 7…......................................... 100 Figure 8…......................................... 102 Figure 9…......................................... 103 Figure 10…........................................ 105 Figure 11…........................................ 107 Figure 12…........................................ 109 Figure 13…........................................ 110 Figure 14…........................................ 112 Figure 15…........................................ 114 Figure 16…........................................ 115 Figure 17…........................................ 117 Figure 18…........................................ 118 Figure 19…........................................ 119 Figure 20…........................................ 120 Figure 21…........................................ 121 Figure 22…........................................ 122 Figure 23…........................................ 123 Figure 24…........................................ 125 Figure 25…........................................ 127 TABLE 1…………………………………………………………. 129 ABBREVIATIONS…………………………………………………. 132 | |
| dc.language.iso | en | |
| dc.subject | 第四型骨成形蛋白 | zh_TW |
| dc.subject | 單分子造影技術 | zh_TW |
| dc.subject | 強化的綠色螢光蛋白 | zh_TW |
| dc.subject | 人類牙周纖維母細胞 | zh_TW |
| dc.subject | 量子點 | zh_TW |
| dc.subject | 人類端粒酶 | zh_TW |
| dc.subject | 分化效率 | zh_TW |
| dc.subject | single-molecule imaging | en |
| dc.subject | human periodontal ligament cells | en |
| dc.subject | enhanced green fluorescent protein | en |
| dc.subject | bone morphogenetic protein 4 | en |
| dc.subject | differentiation efficiency | en |
| dc.subject | human telomerase reverse transcriptase | en |
| dc.subject | quantum dots | en |
| dc.title | 第四型骨成形蛋白和人類端粒酶共同基因轉殖可誘發人類牙周纖維母細胞的高效率多功分化能力 | zh_TW |
| dc.title | Highly efficient multipotent differentiation of human periodontal ligament fibroblasts induced by combined BMP4 and hTERT gene transfer | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 周綠蘋(Lu-Ping Chow),江俊斌(Chun-Pin Chiang),陳羿貞(Yi-Jane Chen),傅鍔(Earl Fu) | |
| dc.subject.keyword | 單分子造影技術,第四型骨成形蛋白,強化的綠色螢光蛋白,人類牙周纖維母細胞,量子點,人類端粒酶,分化效率, | zh_TW |
| dc.subject.keyword | single-molecule imaging,bone morphogenetic protein 4,enhanced green fluorescent protein,human periodontal ligament cells,quantum dots,human telomerase reverse transcriptase,differentiation efficiency, | en |
| dc.relation.page | 133 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-07-25 | |
| dc.contributor.author-college | 牙醫專業學院 | zh_TW |
| dc.contributor.author-dept | 臨床牙醫學研究所 | zh_TW |
| 顯示於系所單位： | 臨床牙醫學研究所 | |
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