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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李建國(Chien-Kuo Lee) | |
dc.contributor.author | wei-chun chou | en |
dc.contributor.author | 周威君 | zh_TW |
dc.date.accessioned | 2021-06-12T18:00:53Z | - |
dc.date.available | 2013-02-20 | |
dc.date.copyright | 2008-02-20 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-01-26 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27311 | - |
dc.description.abstract | 在老鼠的骨髓中,多元性前趨細胞(MMP, multipotential progenitor)能不能分化為B淋巴球是取決於兩種重要因素-細胞激素如IL-7和Flk2/Flt3的作用,以及轉錄因子如E2A、EBF及Pax5等的調控。我們的研究發現STAT3此轉錄因子缺乏時,會使B淋巴球的發育產生缺陷,而使成熟B細胞的數量在骨髓或週邊血液都變少。在STAT3基因剔除鼠(STAT3 knockout mice) 的骨髓中, pro-B、pre-B以及未成熟B細胞的百分比及數目均有顯著下降,然而pro-B 的前趨細胞pre-pro B的數目則有增加的現象。另外,在IL-7的刺激下,我們發現缺乏STAT3的pro-B及pre-B的增殖現象比控制組來得低,其中原因之一是因為STAT3剔除鼠對IL-7有反應的細胞數目降低,而不是因為這些細胞表面的IL-7接受器數目的減少或IL-7及其接受器訊息傳遞的下游分子STAT1或STAT5活化有缺陷。此外,缺少STAT3的 pro-B在沒有存活因子的培養條件下,細胞凋亡的百分比相對於控制組也比較高。因此,至少有兩種機制 (降低增殖以及增加細胞凋亡)使得STAT3基因剔除鼠的B細胞減少。同樣地,在IL-7的刺激下,缺少STAT3的 骨髓細胞之抗凋亡分子Mcl-1的表現下降。最後,我們利用骨髓移植實驗去確認B細胞發育的缺陷,乃是起因於骨髓細胞本身缺少STAT3所造成。
除此之外,我們也利用反轉錄定量聚合酵素鏈鎖反應的方式去觀察其B細胞發育所需之基因如: EBF、E2A、Pax5、Rag1/2、TdT、VperB、λ5、Igα、Igβ等的表現,然而在STAT3剔除鼠的pro-B及pre-B細胞其表現量和正常老鼠並無顯著差異。有趣的是,我們發現缺乏STAT3的pro-B細胞,不管是germline或重組過的遠端VHJ558的基因表現有下降的情形。另一方面,利用體外培養供給Flt3L的條件下,使Lin-c-kit+IL-7Rα+前趨細胞分化成B220+ IL-7Rα+之B細胞的實驗結果顯示,對IL-7有反應的前趨細胞數目降低之原因,可能是更早期藉由Flt3L生長的前趨細胞增殖能力下降所造成。 總而言之,我們是第一個證實了STAT3在早期B細胞的發育中扮演正向調控的角色,而其中原因可能是影響B淋巴球的早期發育過程及Flt3L或IL-7 參與其前趨細胞之存活與增殖。 | zh_TW |
dc.description.abstract | B lymphopoiesis involves the expression of multiple factors, including cytokine receptors and transcription factors. Among the cytokine receptors, Flt3 and IL-7R are known to be critical for early B-cell development. Likewise, transcription factors including E2A, EBF, and Pax5 are critical for specification and commitment of the development of B lymphocytes from multipotential progenitor cells in the bone marrow (BM). Here, we show that the absence of STAT3 impaired B-cell development. Mice selectively lacking STAT3 in BM progenitor cells displayed reduced numbers of mature B cells, both in the BM and in the periphery. The reduction in the B-cell compartment included reduced percentages and numbers of pro-B, pre-B, and immature B cells in the absence of STAT3, whereas the number of pre-pro-B cells was increased. We found that pro-B and pre-B-cell populations lacking STAT3 were hyporesponsive to IL-7 because of a decreased number of IL-7-responsive cells rather than decreased expression of IL-7Rα or reduced activation of STAT1 and STAT5 downstream of IL-7/IL-7Rα signaling. Moreover, STAT3-deficient mice displayed enhanced apoptosis in the pro-B population when deprived of survival factors, suggesting that at least two mechanisms (impaired proliferation and enhanced apoptosis) are involved in the mutant phenotype. Likewise, the expression of anti-apoptotic molecule Mcl-1 in response to IL-7 was slightly reduced in STAT3KO mice. Last, BM transplantation confirmed that impaired B lymphopoiesis in the absence of STAT3 was caused by a cell autonomous defect.
Moreover, we found that the expression of B cell specific regulators such as EBF, E2A, Pax5, Rag1/2, TdT, VpreB, λ5, Igα, and Igβ was comparable in control and STAT3KO cells. Interestingly, the dramatic reduced of distal VHJ558 germline and rearranged transcriptions were shown in the absence of STAT3. In addition, we also found that the reduced IL-7-responsive progenitors might due to the insufficient proliferative ability of progenitors in response to Flt3L by the in vitro culture of Lin-c-kit+IL-7Rα+ progenitors that were differentiated into B220+ IL-7Rα+ cells. In sum, these studies defined a specific role for STAT3 in early B-cell development, probably acting at or before the pre-pro-B transition by contributing to the survival and proliferation of Flt3L or IL-7-responsive progenitors. | en |
dc.description.provenance | Made available in DSpace on 2021-06-12T18:00:53Z (GMT). No. of bitstreams: 1 ntu-97-D91449001-1.pdf: 2562033 bytes, checksum: c9fb2ad00381e1d30d47b06be4dcc429 (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | Acknowledgement i
List of figures vi Abbreviations vii 中文摘要 x Abstract xii Chapter I Introduction 1 1.1 Phenotypic Delineation of B cell Developmental Stages in bone marrow 1 1.2 Cytokines involved in B cell development 3 1.3 Transcription Factors involved in B cell Commitment 6 1.4 Regulation of immunoglobulin heavy chain V-to-DJ recombination —a critical event in early B-cell development 12 1.5 JAK-STATs signaling pathway 15 1.6 Physiological role of STAT3 17 1.7 Rationale and significance 18 Chapter II Materials and Methods 20 2.1 Mice 20 2.2 Cell line 20 2.3 Genotyping for targeted alleles 21 2.4 Single cell PCR 22 2.5 Flow cytometric analysis 23 2.6 Purification of B cells 23 2.7 Purification of LK (Lin-c-kit+) cells 24 2.8 Colony formation assay 24 2.9 In vitro proliferation assay 25 2.10 In vitro apoptotic assay 25 2.11 BM transplantation 26 2.12 Intracellular staining of STAT3 and phospho-STAT3 26 2.13 Isolation of mRNA, preparation of cDNA and QPCR 27 2.14 Semi-quantitative RT-PCR and VDJ transcripts 29 2.15 Western blot analysis 30 2.16 Purification of Flt3L-Ig Protein 31 2.17 Functional assay of Flt3L-Ig Protein 31 2.18 Statistical analysis 32 Chapter III Results 33 3.1 Defected B cell development in the absence of STAT3 33 3.2 Reduced response to IL-7 in STAT3-deficient BM cells 35 3.3 Enhanced apoptosis of pro-B cells in the absence of STAT3 37 3.4 Cell-autonomous requirement of STAT3 for normal B cell development 39 3.5 Higher expression and activation of STAT3 in pro-B cells 40 3.6 Comparable B-specific gene expression profiles in control and STAT3KO mice 41 3.7 Reduced VH7183 and VH J558 germline and rearranged transcription in STAT3KO mice 43 3.8 Purification and characterization of Flt3L-Ig 44 3.9 Flt3L-induced IL-7Rα expression in control and STAT3KO mice 46 Chapter IV Discussions 49 4.1 STAT3 is required for early B cell development through cell-autonomous mechanism 49 4.2 STAT3 might be required in IL-7-mediated B cell development 53 4.3 Normal B cell development but impaired differentiation in CD19CreSTAT3KO mice 58 4.4 Potential role of Flt3/Flt3L signaling in B cell development 59 4.5 Potential role of Notch signaling in B cell development 60 References 62 Figures and Tables 88 Appendix 129 | |
dc.language.iso | en | |
dc.title | STAT3正向調控早期B淋巴球之發育與生成 | zh_TW |
dc.title | STAT3 Positively Regulates an Early Step in B Cell Development | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 賴明宗(Ming-Zong Lai),許秉寧(Ping-Ning Hsu),廖楓(Liao, F.),嚴仲陽(Yen J.J.Y.) | |
dc.subject.keyword | B 淋巴球,發育, | zh_TW |
dc.subject.keyword | STAT3,B cell development, | en |
dc.relation.page | 129 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2008-01-28 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 免疫學研究所 | zh_TW |
顯示於系所單位: | 免疫學研究所 |
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