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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊性芳 | |
| dc.contributor.author | Nai-Hui Lin | en |
| dc.contributor.author | 林迺蕙 | zh_TW |
| dc.date.accessioned | 2021-06-13T03:32:53Z | - |
| dc.date.available | 2006-12-29 | |
| dc.date.copyright | 2006-08-03 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-27 | |
| dc.identifier.citation | Ahmed, R., and Gray, D. (1996). Immunological memory and protective immunity: understanding their relation. Science 272, 54-60.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32126 | - |
| dc.description.abstract | mcl-1屬於Bcl-2家族蛋白中具有抗細胞凋亡功能的一員，在諸多受調控的細胞生死程式中扮演重要的上游角色。組織特異基因剔除老鼠研究指出mcl-1對於早期和晚期的血球發育及維持都是不可獲缺的。Mcl-1是一個受到高度調控的基因，其表現可以被許多細胞激素或是生長因子所誘導。我們實驗室先前已經證明在Ba/F3 pro-B細胞中，IL-3主要是經由順位調控子SIE（-87）和CRE-2(-70)去誘導mcl-1基因的轉錄。為了探討這些順位調控子在生理上扮演的角色，我們製造了SIE和CRE-2突變的小鼠(Ｍcl-1mSC/mSC)。研究發現相較於野生型小鼠，突變小鼠的次級淋巴組織中CD8+ T細胞約減少了一半。同時也發現mcl-1的RNA和蛋白質表現量在突變小鼠的T細胞是比較少的，但是在B細胞中並沒有減少的現象。這些結果顯示mcl-1基因轉錄的調控在T細胞和B細胞中可能是不同的。為了更近一步的研究可能的機制，我們使用電泳速度變動分析法(EMSA)來測試T細胞和B細胞核萃取蛋白質與含有mcl-1基因啟動子片段的DNA探針反應後，比較其蛋白質核酸結合的活性有無差異。我們發現在T細胞和B細胞中在mcl-1基因啟動子-97到-65片段，都可以偵測到SIE和CRE-2 complex的形成，同時SIE和CRE-2 complex中分別具有PU.1和phsphorylated CREB。另外，我們也發現在B細胞中，mcl-1啟動子上-156位置的順位調控子(AP2-like element)上會形成B細胞較多結合的蛋白質核酸複合體(B cell enriched binding complex, BEB complex)。綜合突變小鼠的表現型分析，以及相對於T細胞，在B細胞中具有較多的BEB complex結合於mcl-1啟動子上，因此我們推測在B細胞中mcl-1基因的轉錄可能主要是受到BEB complex的調控，而在T細胞中則主要是受到SIE和CRE-2順位調控子的調控。未來尚需要更多的實驗去驗證以上的結論。 | zh_TW |
| dc.description.abstract | Mcl-1, an anti-apoptotic member of Bcl-2 family proteins, functions at an apical step in many regulatory programs that control cell survival and death. Conditional depletion of mcl-1 in lymphoid organs suggested that it is essential for the development and maintenance of lymphocytes at both early and late stages. Mcl-1 is a highly regulated gene which can be induced by many cytokines and growth factors. Our lab has previously demonstrated that in Ba/F3 pro-B cells, IL-3 stimulation of Mcl-1 gene expression is mediated mainly through two upstream DNA motifs, which are located at positions -70 (the CRE-2 site) and -87 (the SIE site). To further investigate the physiological role of these regulatory cis-elements, the mice with mutant SIE and CRE-2 (mSC) sites were generated. A 50% decrease of peripheral CD8+T cells was found in Mcl-1mSC/ mSC mice. Mcl-1 protein and RNA levels were both decreased significantly in the T-cell lineages of Mcl-1mSC/ mSC mice but not in the B-cell lineage. These data suggested that T and B cells might have different transcription regulation of the mcl-1 gene. To determine possible mechanism responsible for this difference, EMSA was performed using radiolabeled probe containing mcl-1 promoter and nuclear extracts from lymph node T and B cells. We found that in both cell types both SIE and CRE-2 complex could be detected on the mcl-1 promoter region from –97 to –65. Besides, we confirmed that the SIE and CRE-2 complexes contained PU.1 and phosphorylated CREB, respectively. Interestingly, with the mcl-1 -203/+10 promoter DNA as a probe, a B cell enriched binding complex (BEB complex) was found to be formed on an AP2-like element at position -156 of the mcl-1 promoter. Taken together, the mutant mouse phenotype and the prominently enriched amount of the BEB complex in B cells compared with that in T cells suggest that in B cells, mcl-1 transcription is mainly regulated by the BEB complex, whereas in T cells, mcl-1 transcription is mainly controlled by both the SIE and the CRE-2 elements. Further experiments would be required to confirm this conclusion. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T03:32:53Z (GMT). No. of bitstreams: 1 ntu-95-R93448005-1.pdf: 1725658 bytes, checksum: b93c5366f8ca4d0f0977a5e381318319 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | Table of content………………1
中文摘要……………………………5 Abstract…………………6 Introduction…………7 Apoptosis……………7 Intrinsic cell death pathway…………………………………………………………………7 Extrinsic cell death pathway………………………9 Apoptosis in the development of immune system……………9 Myeloid cell leukemia-1…………………………………10 Structure organization of Mcl-1……………………………11 Mcl-1 has an anti-apoptotic function……………………11 Mcl-1 acts as an apical sensor in the apoptosis……13 Mcl-1 is not a redundant player in apoptosis control……13 Regulation of Mcl-1 expression……………………………14 Regulation of Mcl-1 protein stability………………………16 Mcl-1 interacting proteins……………………………………………16 IL-3 simulates Mcl-1 expression through the SIE and CRE-2 elements……………..17 Specific aims………………18 Material and methods………………19 Plasmids……………19 Genotyping……………………19 Primary culture of bone marrow cells and IL-3 induction…………………………….20 Western blots…………………20 RNA isolation and quantitative real time PCR……………21 Isolation of total T cells and total B cells from lymph nodes…………………22 Preparation of nuclear extracts………………………………………22 Electrophoretic mobility shift assay (EMSA)……………23 Flow cytometric analysis………………………………………………24 Results………………………………26 Generation of Mcl-1mSC/mSC mice………………………26 The numbers of peripheral CD8+ single positive T cells are decreased in the Mcl-1mSC/mSC mice…………………………………………27 Stimulation of Mcl-1 expression of the Mcl-1mSC/mSC mice is attenuated in the bone marrow cells……………………………………29 Mcl-1 protein is specifically decreased in the T cell linage of lymphoid organs from Mcl-1mSC/mSC mice……………………………………………29 Mcl-1 RNA was significantly decreased in lymph node T cells, but not B cells of the Mcl-1mSC/mSC mice…………………………………………………30 Mcl-1 protein levels were not significantly altered in the non-lymphoid organs of the Mcl-1mSC/mSC mice………………………………………………………31 Both T and B cells have the SIE and CRE-2 specific binding complex.…………31 PU.1 and phosphorylated CREB are involved in the SIE and CRE-2 specific binding complexes, respectively……………………………………………………32 B cell nuclear extracts have another specific binding complex recognizing sequence within the mcl-1 (-203/+10) promoter fragment………………………………33 BEB complex recognizes a sequence within the upstream half of the mcl-1 (-203/+10) promoter……………………34 DNA motif containing an AP2-like element is the binding site of the BEB complex in the B cells………………………35 BCL1 and P3X cells have the AP2 specific BEB complex………………………35 Discussion…………………………37 A 50% reduction of peripheral CD8+ T cells in Mcl-1mSC/mSC mice is mainly attributed by mutation in the CRE-2 element……………………………………………37 Impaired survival leads to reduced numbers of CD8+ T cells in the Mcl-1mSC/mSC mice…………………………………………38 CD8+ T cell is more sensitive than other T cell lineages to reduced expression of Mcl-1………………………………39 The BEB complex may play a major role in transcription regulation of the mcl-1 gene in B cells…………………………………………………40 Figure……………42 FIGURE 1. General model of signaling pathways that mediate apoptosis………42 FIGURE 2. The mammalian BCL-2 family members…………………………43 FIGURE 3. mcl-1 is an immediate-early gene activated by the interleukin 3 (IL-3) signaling pathways……………………………………………………44 FIGURE 4. Targeted mutation of the mcl-1 gene promoter………………………45 FIGURE 5. Genotyping and Mendelian ratio…………………………………46 FIGURE 6. Flow cytometric analysis of hematopoietic cell compositions of Mcl-1wt/wt and Mcl-1mSC/mSC mice…………………………………………47 FIGURE 7. Early T cell development is normal in the thymus of Mcl-1mSC/mSC mice………………………48 FIGURE 8. Myeloid cell development is slightly attenuated in the bone marrow of Mcl-1mSC/mSC mice………………………………………49 FIGURE 9. CD8+ SP T cell deficiency in the spleen of Mcl-1mSC/mSC mice………50 FIGURE 10. CD8+ T cell deficiency in the lymph node of Mcl-1mSC/mSC mice…51 FIGURE 11. IL-3 induction of Mcl-1 protein in the bone marrow cells is attenuated in Mcl-1mSC/mSC mice………………………………………………52 FIGURE 12. The IL-3 induction of Mcl-1 RNA in the bone marrow cells of Mcl-1mSC/mSC mice is less than that of Mcl-1wt/wt mice…………………………53 FIGURE 13. Mcl-1 expression is attenuated in the T-cell lineage of Mcl-1mSC/mSC mice………………………54 FIGURE 14. Mcl-1 mRNA is significantly reduced in lymph node T cells, but not B cells of Mcl-1mSC/mSC mice……………………………………………………………55 FIGURE 15. No consistent difference in Mcl-1 levels in the non-lymphoid organs of Mcl-1wt/wt and Mcl-1mSC/mSC mice………………………………………………………56 FIGURE 16. Complex formed on the SC fragment………………………………57 FIGURE 17. The SC promoter region binds proteins complexes contained PU.1 and/or phosphorylated CREB………………………………………………58 FIGURE 18. Schematic representation of mcl-1 -203/+10 WT and mutant promoter…………………………59 FIGURE 19. The mcl-1 promoter contains an abundant protein complex (BEB) in the lymph node B cells……………………………………………………60 FIGURE 20. The BEB complex does not contain PU.1 or phosphorylated CREB...61 FIGURE 21. The BEB complex is formed on the mcl-1 promoter between -203 and -87 region…………………………………62 FIGURE 22. Identification of the nuclear protein binding site of the BEB complex……………………………………63 FIGURE 23. The BEB complex specifically forms on the AP2 element (at position -156) within the mcl-1 promoter………………………………………………………64 FIGURE 24. BCL1, A20 and P3X cells have a BEB-like complex formed on the mcl-1 promoter........65 FIGURE 25. The BEB-like complex formed on the mcl-1 promoter in the BCL1 mature B cells is very similar to that formed in the CD19+ primary B cells……66 FIGURE 26. The BEB-like complex is also formed in the P3X plasma B cells……67 Reference………68 | |
| dc.language.iso | en | |
| dc.subject | B淋巴球細胞 | zh_TW |
| dc.subject | mcl-1 | zh_TW |
| dc.subject | 轉錄 | zh_TW |
| dc.subject | T淋巴球細胞 | zh_TW |
| dc.subject | mcl-1 | en |
| dc.subject | transcription | en |
| dc.subject | B lymphocytes | en |
| dc.subject | T lymphocytes | en |
| dc.title | 探討SIE和CRE-2順位調控子在T細胞和B細胞中對於Mcl-1轉錄調控所扮演的角色 | zh_TW |
| dc.title | Characterization of the role of the SIE and CRE-2 elements in the regulation of Mcl-1 transcription in T and B lymphocytes | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 嚴仲陽,李芳仁,許秉寧 | |
| dc.subject.keyword | mcl-1,轉錄,T淋巴球細胞,B淋巴球細胞, | zh_TW |
| dc.subject.keyword | mcl-1,transcription,T lymphocytes,B lymphocytes, | en |
| dc.relation.page | 76 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-28 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| 顯示於系所單位： | 分子醫學研究所 | |
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