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研究生:曾昭榮
研究生(外文):Chao-Jung
論文名稱:研究clusterin在人類肺癌H1299細胞的表現及功能
論文名稱(外文):Study of the clusterin expression and functions in human lung cancer H1299 cells
指導教授:許國堂
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:醫學分子毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:72
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Clusterin為一種分泌型之異二聚體醣蛋白,它廣泛的表現在組織中以及在人類所有體液中都可發現它的存在。我們的研究結果得知可以促進clusterin表現的路徑有兩條:其一是將細胞處理蛋白質體抑制劑MG132,MG132會調控熱休克蛋白因子轉錄活化區而促進clusterin啟動子的活性;另一是將細胞處理組蛋白去乙醯酶抑制劑TSA,TSA會使得染色質裡的組蛋白高度乙醯化而讓許多基因及其蛋白增加表現量,其中包括了clusterin。而在H1299人類肺癌細胞中,以上兩條路徑都有反應;但在Huh-7人類肝癌細胞,只經由組蛋白乙醯化路徑而促進clusterin的表現,同樣地,在Huh-7人類肝癌細胞中,Lg-HDAg也是經由組蛋白高度乙醯化的路徑而增加clusterin的表現。
我們將clusterin穏定地表現在H1299細胞株,處理化療藥物後,利用MTS assay去偵測細胞的存活率,結果顯示穏定表現clusterin的細胞株會對Doxorubicin產生抗藥性;而對Gemcitabine有非常明顯的敏藥性,但處理Taxotere或Vincristine,clusterin的表現量不影響細胞對藥的感受性。另外,比較野生型及穏定表現clusterin的H1299細胞株,利用Gelatin zymography去觀察MMP-2及MMP-9表現的情形,結果發現穏定表現clusterin細胞株的MMP-2表現量明顯降低,另外我們再觀察內生性clusterin表現量就較高的Huh-7及H1355,除了Huh-7的MMP-9有微量表現,其餘都偵測不到MMP-2及MMP-9的表現。由以上結果得知:clusterin對於MMP-2的表現具有其關聯性,但兩者確切的相關性究竟為何,則有待後續實驗證明之。


Clusterin is a secretory heterodimeric glycoprotein, which expressing in all tissues and can be found in all human fluids. To analyze the mechanism of clusterin induction, we generated the luciferase reporter gene constructs with 5’-regulatory sequence of clusterin gene. Our data showed that clusterin expression could be mediated by two pathways. The first pathway is through activation of heat-shock factor (HSF). Another pathway is epigenetic regulated by histone hyperacetylation of chromatin. In H1299 human lung cancer cell, both pathways are associated with clusterin induction; but in human liver cancer cell (Huh-7), only the histone acetylation pathway is exist. We also showed that expression of Lg-HDAg upregulating clusterin expression via histone acetylation in Huh-7 cells.
To study the function of clusterin in lung cancer cells, we generated the clusterin stably overexpression clone from H1299 cells and exposed to chemotherapeutic drugs for cytotoxicity assay. The results suggested that clusterin stably expressing cells increased resistance to doxorubicin but sensitive to gemcitabine treatments. There are no effect on treatment of the clusterin stably expressing cells with taxotere and vincristine when compare to wild type H1299 cells. We also investigated the expression of matrix metalloproteinase (MMP)-2 and -9 activities with clusterin stably expressing cells by gelatin zymography method, the results showed apparent reduction of MMP-2 in clusterin stably expressing cells. The data indicates that clusterin may act as a regulator for MMP-2 expression but the role of clusterin with MMPs gene needed to be further investigated in the future.


壹、 中文摘要 1
貳、 英文摘要 2
參、 前言
一、Clusterin 發現起源 3
二、Clusterin 之異構型態 3
三、Clusterin的生物功能及腫瘤生成角色 5
肆、 研究動機 7
伍、 實驗材料與實驗設備
一、實驗材料 8
二、實驗設備 9
陸、 實驗方法
一、Clusterin promoter質體之構築 10
二、Clusterin open reading frame質體之構築 17
三、細胞培養 18
四、細胞轉染作用 19
五、Luciferase assay system 21
六、Stable clone的挑選及培養 22
七、西方墨漬法 23
八、細胞毒性試驗 25
九、Gelatin zymography 26
柒、 實驗結果
一、在Huh-7細胞中,Lg-HDAg不活化clusterin promoter的活性 27
二、利用Proteasome inhibitor MG132確定所構築出的clusterin promoter具有活性,而活性表現於H1299細胞,但不表現於Huh-7細胞 28
三、Histone deacetylase inhibitor trichostatin A (TSA)誘導clusterin的表現是經由chromatin histone acetylation路徑 29
四、MG132促進clusterin表現不經由histone acetylation路徑 29
五、Lg-HDAg促使histone acetylation進而誘導clusterin表現 30
六、A549、H1355、H1299、Huh-7及HepG2 細胞株及培養液中,clusterin 的表現量各有差異 30
七、利用anti-FLAG及anti-clusterin抗體確定H1299細胞株穏定表現clusterin 31
八、穏定表現clusterin之 H1299細胞生長速度比wild type H1299細胞株慢 32
九、穏定表現clusterin的H1299細胞株對Doxorubicin具抵抗性,對Gemcitabine具敏藥性,而細胞表現clusterin的多寡,不影響細胞對Taxotere及Vincristine的敏藥性 32
十、Clusterin降低matrix metalloproteinases (MMPs)-2或MMP-9的表現量 33
捌、 討論 34
玖、 圖解說明
圖一、利用luciferase reporter assay偵測Huh-7及H1299細胞中,HDV Lg-Ag是否活化Clusterin promoter活性 38
圖二、利用luciferase reporter assay偵測Huh-7及H1299細胞中,處理MG132是否活化Clusterin promoter活性 40
圖三、偵測MG132是否影響Huh-7或H1299細胞株clusterin的表現 41

圖四、TSA對Huh-7及H1299細胞株的clusterin表現之調控作用
42
圖五、偵測MG132促進clusterin表逹確實不經由組蛋白乙醯化路徑 43
圖六、利用西方墨點法比較A549、H1355、H1299、Huh-7及HepG2 細胞株及培養液中,clusterin 所表現的情形 44
圖七、比較wild type H1299細胞株與穏定表現clusterin之 H1299細胞株,所表現clusterin的情形 45
圖八、比較wild type H1299細胞株與穏定表現clusterin之 H1299細胞株生長數速度 46
圖九、比較wild type H1299細胞株與穏定表現clusterin之H1299細胞株對化療藥物的感受性 47
圖十、比較在不同天數,wild type H1299細胞株與穏定表現clusterin之H1299細胞株對化療藥物的感受性 50
圖十一、比較clusterin與matrix metalloproteinases (MMPs)的相關性 52
拾、 附圖
附圖一、Clusterin異構型 54
附圖二、利用西方墨點法確定Huh-7細胞株有L-HDAg及S-HDAg穏定表逹。利用北方墨點法和統計圖證實clusterin具有差異性表現。 55
附圖三、Clusterin promoter質體之構築 57
附圖四、利用西方墨點法確定clusterin、acetyl-histone H3、L-HDAg蛋白的表現 61
附圖五、Clusterin open reading frame質體之構築 63
拾壹、 附表
附表一、Clusterin的各種名稱 65
附表二、引子序列表 66
附表三、SDS-page配製法 67
拾貳、 參考文獻 68


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