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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉秉慧(Biing-Hui Liu) | |
| dc.contributor.author | Miao-Ju Chien | en |
| dc.contributor.author | 簡妙如 | zh_TW |
| dc.date.accessioned | 2021-06-15T14:02:41Z | - |
| dc.date.available | 2018-09-25 | |
| dc.date.copyright | 2015-09-25 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-20 | |
| dc.identifier.citation | Bamias, G., and Boletis, J. (2008). Balkan nephropathy: evolution of our knowledge. American journal of kidney diseases : the official journal of the National Kidney Foundation 52, 606-616.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52007 | - |
| dc.description.abstract | 馬兜鈴酸 (Aristolochic acid, AA) 是馬兜鈴科植物的天然成份，主要由馬兜鈴酸 I 和馬兜鈴酸 II 所組成，目前已知馬兜鈴酸 I 為主要腎毒性及致癌性的來源，研究指出服用馬兜鈴酸可能導致包括膀胱癌在內的泌尿道腫瘤形成，國際癌症研究中心 (IARC) 也將其分類為人類致癌物 (Group1)。許多臨床調查研究發現 Bladder cancer-associated protein (BLCAP) 與膀胱及其他腫瘤的進程相關，推測其 blcap 基因可能扮演著抑癌基因的角色，但是目前對其相關機轉仍不清楚。Gremlin 是骨形成蛋白 (bone morphogenetic protein, BMPs) 家族的拮抗蛋白，在細胞生長和分化佔有一席之地，近期研究也認為 Gremlin 和許多癌症的調控有關。
本研究的主要目的在於探討馬兜鈴酸 I 如何調控 blcap 基因和 gremlin 基因。首先利用不同濃度的馬兜鈴酸處理 HEK293 (人類胚胎腎臟細胞株)、NRK-52E (大鼠近曲小管上皮細胞株) 和 HT1376 (人類膀胱癌細胞株) 24 小時後，發現 blcap mRNA 表現量減少。建構含有 blcap 啟動子片段的冷光報導基因質體，並將其轉染入 HEK293 細胞株後發現馬兜鈴酸會有效抑制 blcap 啟動子所驅動的冷光活性，因此推測馬兜鈴酸在轉錄層次上調控 blcap 基因的表現，但是進一步縮短 blcap 啟動子片段長度進行冷光活性，卻無法找出馬兜鈴酸在啟動子上確切的調控位置。在另一方面，馬兜鈴酸並不會藉由 blcap 3’UTR 而影響報導基因的冷光活性，顯示馬兜鈴酸不會在後轉錄層次影響 blcap 基因的表達。在蛋白質層次上使用免疫螢光染色法觀察得知，BLCAP 蛋白質主要表現在 HEK293、 HT1376 和 HT1197 細胞株的細胞質中，而西方點墨法的結果也顯示 BLCAP 主要分布在細胞質。 降低 HT1376 細胞株中 blcap 基因的表現量導致細胞生長速度以及細胞遷移能力增加，同時提高 gremlin mRNA 表現量。進一步建構含有不同長度 gremlin 啟動子片段的冷光報導基因質體並且進行 HEK293 轉染，結果發現降低細胞中 blcap 表現可活化 gremlin 啟動子所驅動的冷光活性，並且推測 blcap 基因可能在 gremlin 啟動子 -889 ~ -380 的位置進行調控。利用不同濃度的馬兜鈴酸處理 HEK293、NRK-52E 和 HT1376 24小時會增加 gremlin mRNA 的表現量，但是在進行 gremlin 啟動子的冷光活性分析時則發現馬兜鈴酸並不會在轉錄層次上調控 gremlin 基因。最後以低劑量馬兜鈴酸長時間處理 HEK293 細胞株達 30 天，無論在細胞型態、blcap 或 gremlin 基因的表現量與對照組相比都沒有顯著差異。 綜合以上實驗數據可知，馬兜鈴酸經由轉錄層次抑制 blcap mRNA 的表現量，馬兜鈴酸雖然增加 gremlin mRNA 表現量，但是調控機轉未明。當 blcap 基因表現量下降時，細胞會出現類似癌化的現象同時也增加 gremlin mRNA，目前推測 blcap 基因藉由轉錄層次調控 gremlin mRNA 的表現。 | zh_TW |
| dc.description.abstract | Aristolochic acid (AA), a group of natural compound widely found in aristolochiaceae family of plants, is composed of AAI and AAII. AAI is nephrotoxic and carcinogenic to human and associated with urothelial carcinoma. IARC also classifies AA as a Group 1 human carcinogen. Bladder cancer-associated protein (BLCAP) with 10 kD is related with tumor development and cancer progression. Therefore, the blcap gene is supposed to be a tumor suppressor gene, but its molecular mechanisms are still not clear. Gremlin is known for its antagonistic reaction with bone morphogenetic proteins (BMPs) and plays an important role in cell growth and differentiation. Recent studies have indicated that Gremlin is involved in the regulation of cancer progression.
The main object of this study was to investigate that how AAI regulated the expression of blcap and gremlin gene. First, we treated HEK293, NRK-52E and HT1376 cells with different concentrations of AAI for 24 hours and found that the levels of blcap mRNA decreased. A series of luciferase reporter plasmids containing various promoter regions of blcap were constructed and then transfected into HEK293 cells. AAI significantly inhibited the blcap promoter-derived luciferase activity, suggesting that AAI modulated blcap gene expression at transcriptional level. However, the promoter deletion assay did not reveal the regulatory site which was modulated by AAI. On the other hand, AAI did not affect the luciferase activity of the reporter construct containing blcap-mRNA-3’UTR. From the viewpoint of protein, both immunofluorescence staining and western blotting showed that the BLCAP protein mainly located in the cytoplasmic part in HEK293, HT1376 and HT1197 cells. Decreasing the expression of blcap in HT1376 not only promoted the growth rate and migration ability of cells, but also increased the levels of gremlin mRNA. A series of luciferase reporter plasmids containing various regions of gremlin promoter were constructed and transfected into HEK293 cells. We found that reducing the blcap expression could enhance the gremlin promoter activity, and the regulatory regions on gremlin might be located at nucleotides -889 to -380. AAI treatment also increased the expression of gremlin mRNA in HEK293, NRK-52E and HT1376 cells. However, AAI did not altered the luciferase activity driven by gremilin promoter. Finally, HEK293 cells treated with low dose of AAI for 30 days did not change the cellular morphology and the levels of blcap and gremlin mRNA comparing to the solvent-treated group. In summary, the above data suggest that AAI inhibited the blcap mRNA at the transcriptional level with unknown mechanisms. Additionally, decreasing blcap mRNA resulted in cellular carcinogenesis and also promoted the gremlin expression. The blcap may regulate the gremlin expression at the transcription level. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T14:02:41Z (GMT). No. of bitstreams: 1 ntu-104-R02447010-1.pdf: 3897326 bytes, checksum: 47efee46aa4b16e02129e85deec51c04 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 中文摘要 i
Abstract iii 第一章 緒論 (Introduction) 1 1.1 馬兜鈴酸 (Aristolochic acid) 1 1.1.1 馬兜鈴酸的基本介紹 1 1.1.2 馬兜鈴酸腎病變 1 1.1.3 馬兜鈴酸的腎毒性 2 1.1.4 馬兜鈴酸的致癌性 3 1.2 膀胱癌相關蛋白質 (Bladder cancer associated protein, BLCAP) 5 1.2.1 膀胱癌相關介紹 5 1.2.2 膀胱癌相關蛋白質 (BLCAP) 相關介紹 5 1.2.3 BLCAP 的功能 6 1.3 Gremlin 蛋白質 9 1.3.1 Gremlin 蛋白質相關介紹 9 1.3.2 Gremlin的生理功能 9 1.3.3 Gremlin 蛋白質和癌症的相關性 10 1.4 研究動機 12 第二章 材料和方法 (Materials and Methods) 13 2.1實驗材料 13 2.1.1試劑 13 2.1.2質體 15 2.1.3儀器 15 2.2實驗方法 16 2.2.1 細胞培養 16 2.2.2 細胞冷凍保存 16 2.2.3 細胞解凍 17 2.2.4 核醣核酸萃取（RNA extraction） 17 2.2.5 反轉錄作用（Reverse transcription reaction） 18 2.2.6 半定量聚合連鎖反應（Semi – quantitative PCR） 18 2.2.7 洋菜瓊膠電泳 (Agarose gel electrophoresis) 18 2.2.8 質體建構 19 2.2.9 大腸桿菌之基因轉型 (E.coli Transformation) 19 2.2.10 質體萃取（Plasmid extraction） 20 2.2.11 細胞轉染 (Transfection ) 20 2.2.12 冷光報導基因活性分析 I (Luciferase reporter assay) 20 2.2.13 冷光報導基活性分析 II (Dual-Luciferase reporter assay) 21 2.2.14 免疫螢光染色 (immuonofluorescence staning) 22 2.2.15 細胞核質蛋白分離收集 22 2.2.16 西方墨點法 (Western blot) 23 2.2.17 慢病毒感染 (Lentivirus infection) 23 2.2.18 傷口癒合實驗 (Wound healing assay) 24 2.2.19 影像分析 24 2.2.20 統計分析 (Statistic analysis) 24 第三章 實驗結果 (Result) 25 3.1 馬兜鈴酸抑制 blcap mRNA 表現量 25 3.2 馬兜鈴酸抑制 blcap 啟動子的活性 25 3.3 馬兜鈴酸不會影響 blcap mRNA 3’UTR 冷光活性 26 3.4 BLCAP 蛋白質在不同細胞株中的分布情況和表現量 27 3.5 馬兜鈴酸抑制 BLCAP 蛋白質的表現量 27 3.6 blcap基因表現量降低促進 HT1376細胞生長速度及細胞遷移能力 28 3.7 blcap基因表現量的降低會增加gremlin mRNA 表現量 28 3.8 blcap基因表現量的降低會增加 gremlin 啟動子活性 29 3.9 馬兜鈴酸增加 gremlin mRNA 表現量 29 3.10 馬兜鈴酸抑制 gremlin 啟動子的活性 30 3.11 長期處理馬兜鈴酸對 HEK293 細胞的影響 30 第四章 討論 (Discussion) 32 第五章 參考文獻 (References) 37 第六章 圖表 45 第七章 附錄 59 | |
| dc.language.iso | zh-TW | |
| dc.subject | gremlin 啟動子 | zh_TW |
| dc.subject | 馬兜鈴酸 | zh_TW |
| dc.subject | blcap | zh_TW |
| dc.subject | gremlin | zh_TW |
| dc.subject | blcap 啟動子 | zh_TW |
| dc.subject | aristolochic acid | en |
| dc.subject | blcap promoter | en |
| dc.subject | gremlin | en |
| dc.subject | blcap | en |
| dc.subject | gremlin promoter | en |
| dc.title | 馬兜鈴酸對於腎臟細胞株中 blcap 和 gremlin 基因的影響 | zh_TW |
| dc.title | The effect of aristolochic acid on blcap and gremlin gene expression in kidney cell lines | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄭鈞文(Chun-Wen Cheng),楊建洲(Jiann-Jou Yang),陳佩貞(Pei-Jen Chen) | |
| dc.subject.keyword | 馬兜鈴酸,blcap,gremlin,blcap 啟動子,gremlin 啟動子, | zh_TW |
| dc.subject.keyword | aristolochic acid,blcap,gremlin,blcap promoter,gremlin promoter, | en |
| dc.relation.page | 67 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-08-20 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 毒理學研究所 | zh_TW |
| 顯示於系所單位： | 毒理學研究所 | |
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