臺灣博碩士論文加值系統

Clusterin 是一種分泌型的醣蛋白，具有增加細胞聚集的能力，普遍存在於組織及人類體液中。在我們的研究結果得知，clusterin 表現高的 H1355 肺癌細胞株對化療藥物 Doxorubicin、Gemcitabine 及 Taxotere 的感受性呈現較不敏感的反應。而後，利用短暫轉染 clusterin siRNA 的方法，抑制了 H1355 細胞株的 clusterin 蛋白表現，則會使 H1355 細胞株對化療藥物的感受性變為較敏感的情形，可見細胞死亡率增加。接著，我們篩選出穩定大量表現 clusterin 的 H1299 和 A549 肺癌細胞株，亦發現對化療藥物 Doxorubicin 具低感受性的情形。為了得知影響肺癌細胞對化療藥物的感受性為位在細胞質中的 sCLUc 蛋白還是成熟會分泌到細胞外的 sCLUs 蛋白的作用，則利用 clusterin 表現低的 H1299 細胞株，分為控制組是不含 sCLUs 蛋白的新鮮培養液及實驗組是含有 sCLUs 蛋白的 H1355 之 conditioned medium，處理化療藥物，觀察 H1299 細胞株對化療藥物 Doxorubicin、Gemcitabine 及 Taxotere 的感受性變化，實驗結果間接證明了是分泌至細胞外的 clusterin 蛋白 (sCLUs) 具有保護細胞的作用，使得肺癌細胞對化療藥物感受性變差，而我們有純化出 sCLUs 蛋白，之後可外加至細胞培養液中，直接證明就是 sCLUs 蛋白的作用，保護細胞對化療藥物感受性低。另外，我們也利用穩定大量表現 clusterin 的 H1299 和 A549 細胞株，探討 clusterin 對肺癌細胞移行的影響。從實驗結果得知，clusterin 會抑制肺癌細胞的爬行能力，特別是在 H1299 細胞株較有明顯的移行能力受抑制的情形。
我們認為 clusterin 在肺癌治療及轉移上扮演著重要的角色，或許將來可當作在肺癌臨床上的指標蛋白之一，而對於 clusterin 之作用機制尚未清楚，是值得往後再深入探討的方向。

Clusterin is a secretory glycoprotein that can be found in all human fluids and enhances protection as well as aggregation of a variety of cells in vitro. To investigate the role of clusterin in lung cancer, the expression of clusterin was examined with various lung cancer cell lines followed by drug sensitivity assay. We found that H1355 cells which express a large amount of clusterin were less sensitized to doxorubicin, gemcitabine and taxotere. Inhibition of H1355 cells clusterin expression by transiently transfected clusterin siRNA resulted in enhanced chemosensitivity to doxorubicin, gemcitabine and taxotere in vitro. The clusterin stably overexpression clones of H1299 and A549 cells were selected by G418 antibiotics and treated with chemotherapeutic drugs for cytotoxicity assay. Our data showed that the clusterin stably overexpression cells reduced sensitive to doxorubicin. To study whether secreted clusterin protein has a protection role in cancer cells, conditioned medium that contained clusterin was collected from H1355 cells and combined with chemotherapeutic drugs to treated H1299 cells. The results indicated that H1299 cells in conditioned medium were less sensitive to doxorubicin, gemcitabine and taxotere than in fresh medium. Therefore, indirect evidences showed that secreted clusterin is protective and decreased the cytotoxicity of chemotherapeutic drugs for lung cancer cells. Moreover, we also purified secreted clusterin protein to confirm the protective function of secreted clusterin directly. In addition, we investigated the effect of clusterin in cell migration and found that the clusterin stably overexpression clones migrated slowly. The data indicated that high amount of clusterin expression may inhibit lung cancer cells migration.
Our data suggested that clusterin plays an important role in the drug susceptibility and metastasis of lung cancer cells. Clusterin could be a target protein for lung caner therapy. However, the regulation mechanism of clusterin in lung cancer is not clear that deserve further investigation.

目錄

頁次
壹、 中文摘要 1
貳、 英文摘要 2
參、 縮寫表 4
肆、 緒論
一、Clusterin
1.起源背景 5
2.基因及蛋白構型 5
3.相關功能、腫瘤生成及治療之角色 6
二、肺癌 8
三、化療藥物
1. Doxorubicin 9
2. Gemcitabine 9
3. Taxotere 10
伍、 研究動機 11
陸、 實驗材料與設備
一、實驗材料
1.藥品及試劑 12
2.抗體 12
3. Kit 12
4.其他 12
二、實驗設備 13
柒、 實驗方法
一、微量質體 DNA 抽取 14
二、洋菜凝膠 (garose gel) 之電泳分析
(1) 2﹪agarose gel 的製備方法 14
(2)電泳操作 15
三、中量質體 DNA 抽取 15
四、細胞培養 (Cell culture)
(1)解凍細胞株 16
(2)細胞株培養 16
(3)細胞繼代培養 (passage) 及種細胞 17
(4)回凍細胞 17
五、細胞轉染作用 (Transfection)
(1) TransFastTM Transfection Reagent 18
(2) LipofectamineTM 2000 Transfection Reagent 18
(3) TurboFectTM in vitrro Transfection Reagent 19
六、抑制細胞中 clusterin 蛋白表現之短暫轉染 (Transient transfection of clusterin siRNA) 19
七、Stable clone 的挑選 (seletion) 及培養
(1)經由 G418 篩選 stable clone 19
(2) Stable clone 的挑選 20
(3) Stable clone 的培養 20
八、西方點墨法 (Western blotting)
(1)製備 cell lysate 20
(2)製備 conditioned medium 21
(3)蛋白濃度定量分析 21
(4) SDS-聚丙烯醯胺板膠之製備與操作 21
(5)抗體作用及偵測 22
九、細胞毒性試驗 (MTS 及 MTT assay)
(1) MTS assay 23
(2) MTT assay 23
十、細胞傷口癒合試驗 (Wound healing assay) 24
十一、細胞移行試驗 (Boyden chamber cell migration assay) 24
十二、純化分泌性 Clusterin (sCLUs) 蛋白 (Purification of Secreted Clusterin Protein) 24
捌、 實驗結果
一、H1299、A549、H1355、H460、CaLu-1、BEAS-2B 細胞株及培養液中，clusterin 蛋白的表現量之差異 26
二、與 clusterin 表現量較低的 H1299 及 A549 細胞株相比，
clusterin 表現量較高的 H1355 細胞株對化療藥物Doxorubucin、Gemcitabine 和 Taxotere 的感受性較不敏感 27
三、短暫抑制 H1355 細胞株的 clusterin 蛋白表現會增加對化療藥物的感受性，變為較敏感的情形 28
四、利用 anti-clusterin 抗體確定 H1299 及 A549 細胞株穩定大量表現 clusterin 29
五、穩定大量表現 clusterin 的細胞株生長速度比親代細胞慢 29
六、穩定大量表現 clusterin 的細胞株對 Doxorubicin 感受性較差 30
七、H1299 細胞株在含有分泌性 clusterin (sCLUs) 蛋白的 H1355
conditioned medium 下對化療藥物具較低的感受性 30
八、純化分泌性 clusterin (sCLUs) 蛋白 31
九、相較於親代細胞株，穩定大量表現 clusterin 的細胞株之移行能力降低 32
玖、 討論 34
拾、 圖表說明
圖一、H1299、H460、A549、H1355、CaLu-1、BEAS-2B 細胞株及培養液中 clusterin 表現量的情形 38
圖二、比較不同 clusterin 蛋白表現量之 H1355、A549及 H1299細胞株對化療藥物的感受性 39
圖三、H1355 細胞株及培養液中短暫抑制 clusterin 表現的情形 41
圖四、短暫抑制 clusterin 蛋白表現之 H1355 細胞株對化療藥物的感受性情形 42
圖五、比較親代細胞 (H1299、A549) 細胞株與穩定大量表現
clusterin 細胞株之 clusterin 的表現量 44
圖六、比較親代細胞株與穩定大量表現 clusterin 細胞株的生長速度 45
圖七、比較親代細胞株與穩定大量表現 clusterin 之細胞株對化療藥
物 Doxorubicin 的感受性 46
圖八、比較 H1299 細胞株在新鮮培養液和 conditioned medium 中
對化療藥物的感受性 49
圖九、利用 SDS-PAGE 染色及西方墨點法分析純化分泌性 clusterin (sCLUs) 蛋白過程中的分布及表現量 51

圖十、利用 Wound healing assay 及 Boyden chamber cell migration
assay 比較親代細胞株和穩定大量表現 clusterin 細胞株之細胞癒合和移行的情形 53
表一、H1299、A549 及 H1355 細胞株對化療藥物之 IC70 56
表二、短暫抑制 clusterin 蛋白表現的 H1355 細胞株對化療藥物之
IC50 57
表三、親代及穩定大量表現 clusterin 細胞株對化療藥物之 IC50 58
表四、H1299 細胞株在新鮮和 conditioned medium 中對化療藥物之
IC70 59
拾壹 附表及附圖 60
拾貳、 參考文獻 70

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