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研究生:石晏任
研究生(外文):Yen-Jen Shih
論文名稱:脂蛋白元H基因5'-側邊序列的分子分析及氧化壓力對於脂蛋白元H基因在轉錄層次調控的影響
論文名稱(外文):Molecular analysis of the 5'-flanking sequence of apoH gene and the effect of oxidative stress on the apoH gene regulation at transcriptional level
指導教授:姜 安 娜
指導教授(外文):An-Na Chiang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
中文關鍵詞:脂蛋白元H轉錄氧化壓力過氧化氫
外文關鍵詞:apolipoproteintranscriptionoxidative stressHydrogen peroxidaseBSOSNAPGSNOMEKK and PKA
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脂蛋白元H (apoH),又稱為b2-glycoprotein I,是一個分子量50 kDa的單鏈醣蛋白,它曾被發現可以和帶負電荷的分子結合,像是磷脂質和氧化的低密度脂蛋白 (oxLDL),apoH曾被認為參與在人類動脈粥狀硬化 (atherosclerosis) 的發展過程當中。我們之前的研究發現在apoH 基因的轉譯起始點上游 -98 ~ -218 bp 具有很強的啟動子 (promoter) 活性,並且氧化壓力 (oxidative stress) 對apoH的表現可能是一個影響因子。本研究的目的是想要找出apoH基因 5'-側邊區域的調控單元 (regulatory elements) 並探究在轉錄 (transcription) 的層次氧化壓力對apoH基因調控的影響。
為了研究apoH基因的調控,我們將一些apoH 基因5'-側邊區域選殖到luciferase報告者 (reporter) 質體並確認之;將這些帶有apoH 基因5'-側邊區域的報告者質體短暫轉染 (transfection) 至Huh7細胞中,而結果顯示,以對SV 40啟動子的作用來講,—98 ~ -46 bp是一個正向調控單元 (positive regulatory element),—1100 ~ -500 bp and —2026 ~ -1526 bp 是兩個負向調控單元,但 —1100 ~ -500 bp 對於apoH 卻是一個正向調控單元。當以H2O2 處理細胞時,帶有5'-側邊區域從 —2469 ~ -1 bp的報告基因活性會被低濃度 (50 ~ 100 μM ) 處理三小時的H2O2 輕微活化,但若處理16小時則產生強烈抑制。報告基因表現的活性會被50 ~ 2000 μM、處理16小時的BSO活化約50 % ,但是若相同濃度處理3小時則沒有有意義的改變。GSNO 和SNAP處理7小時均可抑制報告基因表現的活性,但在低濃度、3小時的處理則GSNO 和SNAP的結果有些微的不同。綜合以上來說,具有細胞毒性的氧化壓力可使報告基因的活性降低,而低劑量的氧化壓力則使基因的活性增加。
將重組的apoH 5'-側邊序列-報告質體和持續表現MEKK的質體一起轉染至Huh7細胞中發現可劇烈地降低報告基因的活性,這結果顯示apoH可能可以被壓力所誘發的MAPK路徑所負向調控。共轉染 (co-transfection) PKA和報告質體可以強烈誘發-15 ~ -218 bp片段的報告基因活性;在以異合子分析的方法篩選了116個人類基因體樣本中apoH的啟動子區域,但沒有發現異合子的多型性。
本研究找出了apoH啟動子區域的調控單元並顯示apoH可以在轉錄的層次被氧化壓力所調控,這些結果提供了調控apoH基因表現之分子機制的一個新的視野。

Apolipoprotein H (apoH), also known as b2-glycoprotein I, is a 50 kDa single chain glycoprotein. It has been found that apoH can bind to negatively charged moleculars such as phospholipids and oxidized low density lipoprotein (ox-LDL). ApoH has been implicated in the development of human atherosclerosis. Our previous studies showed that -98 ~ -218 bp from the upstream of translation start site of apoH gene displayed strong promoter activity and oxidative stress may be factor for apoH expression. The aim of this study was to determine the regulatory elements of apoH 5'-flanking region and the effect of oxidative stress on apoH gene regulation at transcriptional level.
To study regulation of the apoH gene, several 5'-flanking regions of apoH gene-luciferase fusion plasmids have been cloned and characterized. Transient transfection into Huh7 cells with the reporter plasmis containing apoH 5'-flanking region revealed a positive regulatory site in region spanning from —98 ~ -46 and two negative regulatory sites from —1100 ~ -500 and —2026 ~ -1526 for SV 40 promoter. But —1100 ~ -500 bp fragment acted as a positive regulatory element for apoH promoter . When cells were treated by H2O2, the luciferase activities were inhibited at high concentration for short term treatment. The activity of the reporter gene with 5'-flanking region from —2469 ~ -1 was weakly induced by H2O2 at low concentration (50 ~ 100 μM ) for 3h treatment but was strongly inhibited for 16h treatment. Reporter activities were all induced approximately 50 % by BSO treatment for 16h at 50 ~ 2000 μM but no significant change was found for 3h treatment at the same concentration. Both GSNO and SNAP can inhibit the reporter activity for 7h treatment but there is a slight difference between GSNO and SNAP treatment at low concentration for 3 h. Briefly, cytotoxic oxidative stress revealed inhibition of the reporter activity but low dosages of oxidative stress revealed the induction of the reporter activity.
Co-transfection of the constitutive expression MEKK plasmid with reporter constructs of apoH 5'-flanking sequence into Huh7 cells dramatically decreased the reporter activity. The result suggests that apoH may by negatively regulated by the stress- induced MAPK pathway. Co-transfection of PKA with reporter constructs can strongly induce the reporter activity of -15 ~ -218 bp fragment and there may be a PKA responsive element within this fragment. After screening 116 human genomic samples of apoH promoter region by heteroduplex analysis, no heteroduplex polymorphism was found in our study.
This study determined the cis-regulatory elements of apoH promoter region and revealed that apoH can be regulated under oxidative stress stimuli at transcriptional level. These results provide new insights into the molecular mechanism controlling apoH gene expression.

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