臺灣博碩士論文加值系統

染色體端粒酶的再活化使正常細胞具有高度的細胞增殖速率，進而走向不朽化並提高正常細胞轉型成惡性腫瘤的機率。我們過去研究曾發現肺癌病人組織中端粒酶的活性與惡化程度有關。因此推測端粒酶活性是肺癌的一種預後因子，特別是端粒酶活性低的病人其預後表現較佳。所以端粒酶的活性抑制可能是發展新型治療癌症策略的主要目標之一。近年來中草藥在抗癌方面的能力陸續被證實，尤其是在抑制染色體端粒酶活性。靈芝一直以來被視為傳統中藥的翹礎，有效成分為靈芝多醣體，靈芝高三帖類和靈芝免疫調節蛋白。在細胞和動物實驗已證實靈芝多醣體和靈芝高三帖類具有抗腫瘤等功能，但靈芝免疫調節蛋白尚未被證實。綜合以上觀點本研究想探討松杉靈芝免疫調節蛋白是否具有抗癌能力，並且著重於抑制端粒酶活性作用機制。本實驗室主要以遺傳工程方式重組松杉靈芝免疫調節蛋白質reFIP-gts，也已將此基因選殖並純化其蛋白質。本實驗證實重組靈芝免疫調節蛋白(reFIP-gts)會造成A549，CaLu-1C和H1355肺癌細胞生長抑制。然而不會影響人類正常細胞株的生長。此外我們也證實reFIP-gts會造成肺癌細胞細胞週期停滯在G1期並走向未成熟的衰老(premature senescence)反應並明顯增加lysosome胞器增大，此為衰老細胞重要特徵。以染色體端粒酶重覆增幅步驟(Telomeric Repeat Amplification Protocol，TRAP assay)分析，結果發現隨著reFIP-gts濃度增加其A549肺癌細胞株端粒酶活性依次減少。且以半定量(RT-PCR)和定量(REAL-Time PCR)分析結果發現端粒酶反轉錄酶hTERT mRNA表現量亦隨著reFIP-gts濃度增加而減少，並以Reporter gene assay活性分析結果發現隨著reFIP-gts濃度增加而減少hTERT啟動子活性。利用含有一系列缺失短小突變的hTERT啟動子reporter gene發現reFIP-gts作用在-196 to -177的c-Myc 結合區域(E-box site)。利用Western blot 和in vitro及in vivo DNA-Protein binding assay，我們發現reFIP-gts並不會影響c-Myc蛋白的表現量但會明顯降低c-Myc蛋白與E-box結合能力。透過免疫螢光染色法及共軛焦顯微鏡觀察發現reFIP-gts會累積於內質網胞器內並誘發ER stress造成ER stress marker GADD153 mRNA和BiP/ GRP78表現量增加。利用Fluo-3 AM和細胞內鈣離子螯合劑BAPTA-AM證實reFIP-gts會誘發細胞內鈣離子的生成並以鈣離子作為第二訊息來抑制端粒酶活性。此外，以半定量RT-PCR和TRAP分析結果發現reFIP-gts會減少H1299肺癌細胞株hTERT mRNA表現量卻無法抑制其端粒酶活性，而以Western blot和蛋白合成抑制劑Cycloheximide分析證實H1299細胞端粒酶的半衰期較A549細胞長，顯示H1299細胞端粒酶較為穩定。另外，在A549細胞中以核輸出蛋白抑制劑Leptomycin B及蛋白分解酶抑制劑MG132發現reFIP-gts會透過CRM1-dependent 核輸出機制將hTERT 蛋白從核內轉移到細胞質且被蛋白分解酶分解，同時也發現reFIP-gts會抑制Akt激酶的活性，而Akt激酶的活性下降影響hTERT蛋白無法與NF-κB結合導致hTERT蛋白無法進入細胞核。因此推論reFIP-gts也會利用轉譯後修飾來抑制A549端粒酶活性。最後由動物實驗證實reFIP-gts具有抑制腫瘤生長的能力。綜合上述研究發現在肺癌細胞中reFIP-gts抑制端粒酶活性的過程中影響hTERT 蛋白的座落位置(轉譯後修飾的調控)比抑制hTERT基因的表現(轉錄的調控)更顯得重要。本實驗研究結果證實松杉靈芝菌絲體中純化的免疫調節蛋白具有抗癌的功效。此免疫調節蛋白可應用於抑制肺癌細胞的端粒酶活性。所以將來有發展出治療或搭配其他抗癌藥物一起處理抵抗腫瘤並減少腫瘤細胞的再復發。

ABSTRACT
Telomerase expression is the hallmark of tumor cells in which this ribonucleoprotein complex activation may therefore be a critical step in cellular immortalization and oncogenesis. Fungal immunomodulatory proteins, FIP-gts, were found in Ganoderma tsugae. This protein has been implicated in activating human peripheral blood mononuclear cells. However, the effect of FIP-gts on cancer cells has not clearly been described. In the present study, we expressed and purified the recombinant fungal immunomodulatory protein (reFIP-gts) in E. coli, and demonstrated its role in tumor suppressor activity. Treatment of A549 cells with reFIP-gts significantly inhibited their growth but did not affect MRC-5, which is human normal lung fibroblast. We further demonstrated that reFIP-gts suppressed telomerase activity in a concentration-dependent manner. Repression of telomerase activity can be accounted by a decrease in expression of the telomerase catalytic subunit (hTERT) as revealed by decreasing its mRNA level, suggesting that the reFIP-gts may directly or indirectly regulate trnascirption of telomerase subunit. These results were also confirmed by luciferase reporter assay, in which transient transfection of A549 cells with a construct containing the luciferase reporter gene driven by a functional hTERT promoter. Further deletion analyses of hTERT promoter revealed a 19-bp region (-196 to -177) that clearly showed transrepression activiy of FIP-gts . Since this region contains an E-box, we performed electrophoretic mobility shift assays and Chromatin immunoprecipitation assay to demonstrate that the binding activity of c-Myc transcriptional factor to the E-box sequence of the hTERT promoter was inhibited in response to reFIP-gts treatment. `

Here, we proved that reFIP-gts entry into the cell and localization in endoplasmic reticulum can result in ER stress, thereby increasing ER stress markers (CHOP/GADD153) and intracellular calcium release in A549 cells. The use of calcium chelator restores reFIP-gts–mediated reduction in telomerase activity. These results strongly suggest that ER stress induces intracellular calcium release and results in inhibition of telomerase activity. Although reFIP-gts decreased hTERT mRNA level in both A549 and H1299 cells, only the telomerase activity in A549 cells was inhibited. Surprisingly, we found that reFIP-gts induces translocation of hTERT from the nucleus into the cytosol in A549 cells but not in H1299 cells. Using leptomycin B, nuclear export inhibitor, we showed that hTERT is not transported. Using MG132, a proteasome inhibitor, reFIP-gts also prevents hTERT translocation from proteasome degradation. Based on these findings, the posttranslational modifications of hTERT protein might play more important role than down-regulation of hTERT transcriptional activity in mediating reFIP-gts-induced suppression of telomerase activity in lung cancer cells. We also demonstrated that reFIP-gts-treated lung cancer cells were arrested at G1 phase by flow cytometry and its morphological phenotype was consistent with cellular senescence. The premature senescent nature of these cells was supported by positive staining for senescence-associated β-galactosidase activity , increased lysosomal content, and up regulation of cyclin-dependent kinase 2 inhibitor p27, but lacking of telomere shortening in A549 lung cancer cells. Finally, reFIP-gts grew more slowly and formed significantly fewer A549 cell colonies in soft agar. Importantly, in mouse model, A549 cells treated with reFIP-gts grew significantly slower than the cells treated with PBS alone. This study evidences that reFIP-gts strongly inhibits tumorigenesis of lung cancer, and thus it may turn into an attractive agent for chemoprevention and antineoplastic therapy.

目錄
謝誌 2
目錄 4
縮寫表 7
論文題目 9
中文摘要 10
英文摘要 12
壹、前言 14
一、染色體穩定與端粒序列 14
二、端粒酶結構與其活性表現 15
三、染色體端粒酶與細胞腫瘤之間關係 17
四、發展抑制端粒酶活性策略 18
五、細胞衰老與抑制腫瘤生成的關係 21
六、靈芝簡介 22
七、松杉靈芝免疫調節蛋白 24
八、松杉靈芝中之免疫調節能力 24
九、靈芝免疫調節蛋白的抗癌研究 26
十、研究動機 27
十一、實驗設計 28

貳、研究材料與方法 33
研究材料 33
一、reFIP-gts與純化 35
二、MTS Assay 37
三、細胞週期分析-流式細胞儀 37
四、細胞衰老分析 38
五、染色體端粒酶重覆增幅步驟 39
六、反轉錄酶鏈聚合酶連鎖反應(RT-PCR) 40
七、Transient transfection和Reporter gene assay 41
八、EMSA (Electrophoretic mobility shift assay) 42
九、Chromatin immunoprecipitation assay (ChIP). 44
十、免疫沉澱法 45
十一、西方點墨法(Western Blot) 45
十二、細胞免疫染色法 47
十三、以Fluo-3AM測定細胞內鈣離子 49
十四、染色體端粒長度分析 49
十五、動物腫瘤實驗 50



参、實驗結果
一、大腸桿菌中reFIP-gts的表現與純化 51
二、reFIP-gts對肺癌細胞株與正常細胞株的存活率 51
三、reFIP-gts對肺癌細胞株細胞週期的影響 52
四、reFIP-gts對肺癌細胞株誘發未成熟的細胞衰老反應
premature cellular senescence 52
五、reFIP-gts處理肺癌細胞A549後有效抑制染色體端粒酶活性 54
六、reFIP-gts處理肺癌細胞後有效抑制端粒酶反轉錄催化單元
mRNA 55
七、reFIP-gts抑制端粒酶反轉錄催化單元的轉錄活性 55
八、分析端粒酶反轉錄催化單元上與reFIP-gts的response
element 56
九、reFIP-gts不會影響c-Myc蛋白的表現 56
十、reFIP-gts抑制c-Myc結合到端粒反轉錄酶啟動子的能力 57
十一、訊息傳遞激酶抑制劑對reFIP-gts抑制A549細胞中端粒酶
活性的影響 57
十二、reFIP-gts處理A549細胞後的座落位置 58
十三、reFIP-gts誘發A549細胞內質網壓力 58

十四、reFIP-gts處理肺癌細胞後透過鈣離子的誘發而抑制染色體
端粒酶活性 59

十五、reFIP-gts抑制肺癌細胞染色體端粒酶的活性與p53的表現
無關 60

十六、reFIP-gts藉由轉譯後修飾機制改變hTERT蛋白座落位置 61
十七、reFIP-gts 改變hTERT蛋白座落位置與NF-κB位置改變
有關 63

十八、reFIP-gts抑制裸鼠腫瘤的形成 64














肆、討論 65

伍、未來展望 76

陸、圖表與附表 77

柒、參考文獻 132

捌、個人資料表 142

玖、已發表之論文抽印本 144

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