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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 呂紹俊 | |
| dc.contributor.author | Tsung-Yu Wu | en |
| dc.contributor.author | 吳宗諭 | zh_TW |
| dc.date.accessioned | 2021-06-13T05:48:32Z | - |
| dc.date.available | 2006-08-04 | |
| dc.date.copyright | 2006-08-04 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-10 | |
| dc.identifier.citation | 藍雅馨 (2005) 轉錄因子Oct-2參與脂多醣刺激RAW264.7 細胞resistin 基因表現之研究。 臺灣大學醫學院生物化學暨分子生物研究所碩士論文
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33900 | - |
| dc.description.abstract | Inducible nitrogen synthase (iNOS) 是一個能被誘發產生一氧化氮的酵素，在免疫反應中，一氧化氮有幫助殺死外來微生物以及調控免疫反應等作用。在LPS誘導iNOS表現的過程中，需要其啟動子上-90 ~ +150區域中NF-κB及Octamer這兩個轉錄因子的結合區域。之前的研究主要對於NF-κB的參與有較深入探討，但對結合Octamer結合區的轉錄因子探討則較少。但根據 (Xie, 1997) 的研究，當這兩個區域其中任一被突變後，LPS只能誘發啟動子這兩個結合區未被突變時2~3%的活性，顯示結合到Octamer的轉錄因子在LPS誘發iNOS表現是不可或缺的，因此本論文的研究是探討在iNOS啟動子上與Octamer結合的轉錄因子。
與Octamer結合的轉錄因子有Oct-1及Oct-2；Oct-1是普遍存在於細胞中的，而Oct-2則被認為主要在B細胞及神經細胞中存在，但也有一些研究認為Oct-2可能在macrophage也有表現。我們以LPS誘發Raw264.7細胞發炎反應，發現Oct-2會隨著LPS的劑量以及作用時間增長而增加其表現量，但是Oct-1的表現量則略有下降，另外iNOS的mRNA以及蛋白質也都會被LPS作用而增加表現。從Balb/c小鼠的腹腔巨噬細胞分析也發現到Oct-2的表現，而且其表現量也會受LPS刺激而增加。將iNOS的啟動子接到會表現Luciferase的表現載體上，並且分別與Oct-1和Oct-2表現載體一起轉染到細胞中，LPS促進iNOS啟動子活化的作用都明顯的提高。另外利用293T細胞，以上述具有啟動子的載體加上NF-κB的次單元體，分別與Oct-1和Oct-2一起轉染，可以看到Oct-2的效果會比Oct-1來的強，顯示Oct-2可能參與iNOS的基因轉錄，而且在轉染p65時的活化效果就已達到p50和p65一起轉染時的程度。為了進一步探討Oct-1或是Oct-2在LPS促進iNOS啟動子的活性所扮演的角色，使用RNAi的方式分別降低Oct-2及Oct-1的表現，在LPS刺激之後觀察iNOS的表現。結果發現，當Oct-2被降低表現之後，LPS促進iNOS mRNA的表現明顯的減少了許多，降低Oct-1表現時iNOS的表現沒有受到影響，此結果與啟動子活性分析結果不同的原因，可能是因為Oct-2才是在細胞核中能與染色質體結合的轉錄因子。為了證實Oct-2能結合到iNOS啟動子DNA上，在DNA affinity precipitation assay以及Chip (chromatin immunoprecipitation) assay顯示在LPS刺激之後，可以偵測到較多的Oct-2以及NF-κB次單元體結合到iNOS啟動子上，顯示Oct-2確實能結合到iNOS啟動子DNA上。所以綜合以上結果，在LPS誘導iNOS表現時，是需要Oct-2，而不是Oct-1，參與在其啟動子活化過程中。 | zh_TW |
| dc.description.abstract | iNOS is responsible for nitric oxide (NO) production under various condition, and engaged in inflammation and killing extrinsic microorganism. Previous studies have demonstrated that iNOS promoter -90bp ~ +150bp region containing a NF-κB response and an Octamer cis-elements is required for LPS-induced iNOS expression. When either one of the two cis-elements is mutated, LPS-induced promoter activity reduced to only 2 to 3% as much activity as in wild type. Most studies have focused on NF-κB mediated signaling, but less discussed in the importance of Octamer cis-element. The main purpose of this thesis is to investigate which Octamer binding protein is involved in activation of iNOS promoter.
There are two Octamer binding transcription factors, Oct-1 and Oct-2. Oct-1 is ubiquitously expressed in most cells, while Oct-2 express primarily in B-cell and neuron cell. Several studies suggest that Oct-2 is expressed in macrophage. When RAW264.7 cells were treated with LPS, mRNA levels of Oct-2 and iNOS increased in a dose- and time-dependent manner, while Oct-1 mRNA decrease slightly. We also show that both mRNA and protein of Oct-2 increased in peritoneal macrophage from Balb/c mice. Cotransfection of iNOS (-90bp ~ +150bp)-Luc with Oct-1 or Oct-2 expression plasmid into RAW264.7 cells showing that both Oct-1 and Oct-2 were able to activate iNOS promoter. However, knockdown of Oct-2 by RNAi in RAW264.7 cells prevented LPS-induced iNOS mRNA expression, whereas knockdown of Oct-1 in RAW264.7 cells had no effect on LPS-induced iNOS mRNA expression. The RNAi results was different from promoter assay. It is possible that Oct-2 can bind to chromatin structure, but not Oct-1. DAPA and ChIP assay showing that LPS-treatment increases binding of Oct-2 and NF-κB subunits to iNOS promoter. Taken together, our results suggest that Oct-2, but not Oct-1, is involved in iNOS promoter activation in response to LPS treatment. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T05:48:32Z (GMT). No. of bitstreams: 1 ntu-95-R93442011-1.pdf: 1972796 bytes, checksum: 4c8aa74f1fe62e5cb38c5823d079f945 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 中文摘要 1
英文摘要 3 縮寫對照表 5 第一章 緒論 8 第一節 文獻回顧 9 第二節 研究動機與實驗目的 18 第二章 材料與方法 20 第一節 實驗材料 21 第二節 細胞培養 23 第三節 小鼠之iNOS啟動子報導基因質體建構 23 第四節 小鼠之iNOS promoter活性的分析 28 第五節 mRNA表現分析 30 第六節 以西方墨點法(Western blot)分析蛋白質表現 33 第七節 細胞外DNA與轉錄因子結合情形 35 第八節 細胞內DNA與轉錄因子結合的觀察 41 第九節 RNA干擾(RNA interference) 46 第三章 實驗結果 48 第一節 LPS對於iNOS的啟動子活性、mRNA、以及蛋白質表現的影響 51 第二節 在有Oct-1、Oct-2、p50、或p65轉染時，以LPS誘導iNOS啟動子-90 ~ +150區域活化的情形 51 第三節 以RNAi抑制Oct-1以及Oct-2表現後對LPS誘導iNOS表現的影響 53 第四節 iNOS啟動子-90bp ~ -47bp區域上的轉錄因子結合情形 53 第五節 以ChIP assay( chromatin immunoprecipitation assay )進行LPS誘導時，Oct-1、Oct-2、p50、以及p65在細胞中與iNOS啟動子結合的 差異分析 55 第四章 討論 56 第一節 LPS誘導Oct-2以及iNOS基因表現的時間關係 57 第二節 直接干擾Oct-2的表現對iNOS基因的表現影響 57 第三節 iNOS啟動子與Oct-2結合上的關係 58 第四節 轉錄因子之間的交互作用關係 61 第五節 總結 62 第五章 圖表 64 第六章 參考文獻 89 圖表目錄 Table 1. Primers within the iNOS promoter cloning, and Chromatin immunoprecipitation-PCR assays 65 Table 2. Specific RT-PCR primers 66 Table 3. Oligonucleotides for Electromobility shift assay 67 Table 4. Oligonuleotides for DNA affinity precipitation assay 68 Table 5. The specific oligonucleotides cloning into pLL3.7 expression small hairpin RNA for RNA interference 69 Table 6. Specific RT-PCR primers for Oct-2 isoform variable region 70 Figure 1. The sequence of selected iNOS promoter region 71 Figure 2. Time- and dose-response of Oct-1 and Oct-2 mRNA expressions to LPS treatment in RAW264.7 cells 72 Figure 3. The mRNA and protein express in peritoneal macrophage under LPS inducing 73 Figure 4. The predominant form of Oct-2 express in RAW264.7 cells 74 Figure 5. The expression of iNOS mRNA is induced by LPS treatment in a time and dose dependent manner 76 Figure 6. The iNOS protein expression in RAW 264.7 is induced 77 by LPS Figure 7. LPS-induced iNOS promoter activity 78 Figure 8. Effects of Oct-1 or Oct-2 expression plasmid on the activation of iNOS promoter with or without LPS treatment 79 Figure 9. The effects of p50 and/or p65, in combination with Oct-1 or Oct-2 on the activity of iNOS promoter in 293T cells 80 Figure 10. pLL 3.7, the shRNA expression plasmid 81 Figure 11. Knock-down Oct-2, but not Oct-1, influence LPS-induced iNOS mRNA expression 82 Figure 12. EMSA using iNOS promoter nucleotide -90bp ~ -47bp as probe 83 Figure 13. EMSA binding patterns of probe from iNOS promoter -90bp ~-47bp and nuclear extract with 100 ng/ml LPS treatment for 4 hours were competed by several transcription factors consensus binding oligonucleotide 84 Figure 14. Detection of Oct-2, p65, p50 in nuclear extract and their binding to iNOS promoter assayed by DNA Affinity Precipitation Assay 85 Figure 15. Sonication condition and Chromatin immunoprecipitation (ChIP) assay result 86 Figure 16. LPS treatment increased Oct-2, p65, and p50 binding to iNOS promoter in RAW264.7 87 Figure 17. OCAB mRNA in RAW 264.7 and Peritoneal macrophage 88 | |
| dc.language.iso | zh-TW | |
| dc.subject | 巨噬細胞 | zh_TW |
| dc.subject | Oct-2 | zh_TW |
| dc.subject | iNOS | zh_TW |
| dc.subject | 轉錄因子 | zh_TW |
| dc.subject | LPS | en |
| dc.subject | iNOS | en |
| dc.subject | Oct-2 | en |
| dc.subject | macrophage | en |
| dc.title | 轉錄因子Oct-2參與在脂多醣誘發巨噬細胞中iNOS啟動子活化的研究 | zh_TW |
| dc.title | Oct-2 transcription factor mediates in LPS-induced increase of iNOS promoter activity in macrophage | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 姜安娜,張淑芬,游偉絢,羅?升 | |
| dc.subject.keyword | 巨噬細胞,轉錄因子,iNOS,Oct-2, | zh_TW |
| dc.subject.keyword | iNOS,LPS,Oct-2,macrophage, | en |
| dc.relation.page | 97 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-11 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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