TGF-β-stimulated clone-22 (TSC-22)是由TGF-β所誘導表現的基因，首先在老鼠造骨細胞MCT3T3E1中發現透過TGF-β的刺激會誘發TSC-22基因的表現。人類的TSC-22基因位於染色體13q14位置。文獻指出TSC-22能夠調控胚胎的發生，以及在部分癌細胞中，發現TSC-22基因的表現具有促進細胞凋亡(apoptosis)的趨勢，另外在人類唾液腺癌細胞中抑制TSC-22的表現能加速癌細胞的生長，因此推測TSC-22可能具有抑癌基因的潛力。本研究欲探討TSC-22基因在肺癌細胞中扮演何種角色。首先，由細胞計數和MTT assay結果得知，當我們在H358細胞株以shRNA干擾方式抑制TSC-22時會增加肺癌細胞的生長與細胞形成群落能力，而在CL1-5細胞株過度表現TSC-22基因則會降低肺癌細胞的生長及細胞形成群落能力。另外當抑制TSC-22表現時，會使得細胞轉型能力增強，相反的，過度表現TSC-22會抑制癌細胞的轉型能力。接著我們利用流式細胞儀分析TSC-22對細胞週期的影響，發現抑制TSC-22的表現會造成細胞週期之S期及G2/M期百分比上升。相反地，當過度表現TSC-22會造成細胞的細胞凋亡比例增加，並且在G1期、S期及G2/M期表現比例降低。此外利用短暫轉染計數將H226細胞株過度表現TSC-22亦會造成細胞凋亡比例增加的現象。進而以西方墨點法偵測細胞週期調控蛋白的表現，結果顯示將H358細胞株抑制TSC-22會明顯抑制p16的表現量，另一方面過度表現TSC-22造成CL1-5細胞株CDK1及CDK4表現量下降，並且p16的表現量有增加的趨勢，推測TSC-22的表現與p16基因之調控具有顯著相關性，並且由以上結果可以推論TSC-22在肺癌細胞株中可能扮演抑癌基因的角色。另一方面，當H358細胞抑制TSC-22後主要經由活化p38-MAPK路徑，而將CL1-5過度表現TSC-22會造成pAkt的表現量大幅降低，這些現象與TSC-22的抑癌功能之間的關係仍需再進一步釐清。接著我們繼續探討TSC-22對細胞轉移的影響，在細胞移行試驗及細胞侵潤試驗中，我們發現在CL1-0細胞株中抑制TSC-22的表現能促進細胞的移行能力，然而過度表現TSC-22會減弱CL1-5細胞株的侵潤能力。最後我們也利用動物模式觀察到抑制TSC-22時會促進腫瘤的生長能力。綜合以上結果，TSC-22基因在部份肺癌細胞株中可能扮演抑制癌細胞生長、群落形成及轉型能力的角色，但其詳細的作用機轉及影響路徑仍有待進一步探討。

TGF-β-stimulated clone-22(TSC-22) was originally reported as a TGF-β-inducible gene in mouse osteoblastic cells, MCT3T3E1. In 1992, TSC-22 was initially isolated from mouse osteoblastic cells as an immediate early response gene of TGF-β. The human TSC-22 gene was mapped to chromosome 13q14. Recently, several roles of TSC-22 have been proposed, including the regulation of embryogenesis, and the pro-apoptotic factor in several cancer cells. It has been reported that downregulation of TSC-22 enhances cell growth in the salivary gland cell line. Accumulated data suggest that TSC-22 is a potential tumor suppressor gene. In this research, we will investigate the role of TSC-22 gene in lung cancer cells. First of all, to address the function of endogenous TSC-22 in lung cancer cells, H358 and CL1-5 are known to express higher and lower levels of TSC-22, respectively. We established H358 and CL1-5 that stably expressed shRNA for TSC-22 and wt-TSC-22, respectively. Cell counting, MTT assay and colony formation assay data showed that suppression of TSC-22 in H358 increased cell growth and cell colony formation. On the other hand, overexpression of TSC-22 in CL1-5 would decrease cell growth and form smaller and looser colonies. According to anchorage-independent growth assay, reduced TSC-22 expression has better effect in cell transformation and enhanced TSC-22 expression has worse effect in cell transformation. We used flow cytometry to analyze cell cycle distribution in H358- and CL1-5-derived cell lines. Cells with suppressed TSC-22 gene would increase the cell population in S and G2/M phases. However, the percentage of sub-G1 phase was increased and the cell population in G1, S and G2//M phases was decreased in wild-type TSC-22 gene transfected cells. Moreover, the induction of sub-G1 population was also observed in the transient transfection of wild-type TSC-22 into H226 lung cancer cells. And then, we continually observed the expressions of cell cycle regulated proteins. The expression level of p16 was obviously decreased in shRNA TSC-22 transfected cells. On the contrary, overexpression of TSC-22 in CL1-5 could decrease the expression levels of CDK1 and CDK4. Besides, the protein level of p16 was elevated in wild-type TSC-22 gene transfected cells. According to our present results indicated that TSC-22 might acts as a tumor suppressor function in lung cancer. Next, we examined the expression of MAPKs and Akt by Western blot. We observed that the activities of MAPKs and Akt were also affected in TSC-22 regulated cell lines. The relationship between above phenomena and functions of TSC-22 need further study. In the experiments of metastasis, we want to determine if TSC-22 also affected the migratory and invasive capability of lung cancer cell lines. The increased migratory ability was observed in TSC-22 knockdown CL1-0 cells. But the decreased invasive ability was observed in TSC-22 overexpressed CL1-5 cells. The molecular mechanism involved in TSC-22 mediated cell migratory and invasive ability required further study. Finally, to assess whether TSC-22 plays a causal role in tumor growth, H358/vector control and H358/RNAi-4 cells were injected in subcutaneous into the right flank of SCID mice. Compared with vector control, suppression of TSC-22 may promote ability of tumor growth. In conclusion, our results suggest that TSC-22 may play a role in reducing cell proliferation, cell colony formation, cell transformation and metastasis in some lung cancer cell lines studied here. Nevertheless, the detailed mechanisms and pathways involved in tumor suppressor activity of TSC-22 are required for further investigation.

目 錄
壹、 中文摘要 1
貳、 英文摘要 3
參、 前言
一、肺癌流行病學及危險因子 5
二、肺癌的分類及成因 6
三、鑑定參與肺癌形成的基因 7
四、癌細胞的轉移 8
五、人類TSC-22基因 10
六、細胞週期調控蛋白 11
七、細胞凋亡反應與細胞凋亡之路徑 14
八、MAPKs與Akt的簡介 15
肆、 研究動機 17
伍、 實驗材料
1.酵素 18
2.商業套組 18
3.試藥 18
4.抗體 19
5.人類肺癌細胞株來源 20
6.載體來源 20
7.實驗動物 20
8.其他 21
9.儀器 21
陸、 實驗方法
1.質體DNA的萃取 (plasmid extract) 23
2.shRNA TSC-22干擾載體(vector)的構築 24
3.細胞RNA的萃取 26
4.利用去氧核醣核酸酶除去total RNA裡的染色體DNA 27
5.反轉錄反應 (reverse transcription; RT) 28
6.定量PCR (real-time PCR) 28
7.肺癌細胞培養 (cell culture) 28
8.轉染作用 (transfection) 30
9.Stable clones的篩選 (selection)步驟 31
10.西方墨點法 (Western blot) 31
11.3-(4,5-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide分析法 (MTT assay) 34
12.細胞計數生長試驗 (cell counting) 34
13.群落形成試驗 (colony formation) 35
14.軟培養基非附著性細胞生長試驗 (anchorage-independent growth assay) 35
15.細胞轉移試驗 (modified Boyden chamber) 36
16.流式細胞分析 (flow cytometry) 37
17.動物模式 38
18.統計分析 38
柒、 實驗結果
一、 利用real time PCR定量TSC-22基因在肺癌病人正常及腫瘤組
織之相對於18S rRNA的差異表現量 40
二、 觀察人類肺癌細胞株及人類正常肺臟纖維母細胞株內生性
TSC-22之表現 40
三、構築shRNA TSC-22干擾載體及具有表現TSC-22的載體 41
四、利用轉染技術改變人類肺癌細胞株內生性TSC-22之表現 42
五、觀察TSC-22的表現對於人類肺癌細胞株生長能力的影響 43
六、觀察TSC-22對細胞群落形成與細胞型態的影響 45
七、觀察TSC-22是否藉由影響細胞轉型(transformation)來改變肺癌細胞的生長能力 46
八、觀察TSC-22對於人類肺癌細胞株的細胞週期及細胞凋亡的影響 47
九、觀察TSC-22對於人類肺癌細胞株之細胞週期調控蛋白的影響 49
十、觀察TSC-22的表現對於人類肺癌細胞株內生性MAPKs與Akt之
活化的影響 50
十一、觀察TSC-22對於人類肺癌細胞株之移行能力及浸犯能力的影響 51
十二、利用動物模式觀察TSC-22對於人類肺癌細胞株生長能力的影響 53
捌、 討論 54
玖、 圖表說明
圖一、肺癌病人之正常及腫瘤組織之TSC-22相對於18S rRNA的差異
表現量 64
圖二、觀察人類肺癌細胞株及人類正常肺臟細胞株內生性TSC-22之表現 65
圖三、構築shRNA TSC-22干擾載體 66
圖四、利用轉染技術改變人類肺癌細胞株內生性TSC-22之表現 67
圖五、利用細胞計數分析TSC-22的表現對於人類肺癌細胞株生長能力的影響 69
圖六、利用MTT assay分析TSC-22的表現對於人類肺癌細胞株生長
能力的影響 72
圖七、觀察TSC-22對於人類肺癌細胞株PCNA及β-catenin表現量的影響以及觀察TSC-22的表現對於人類肺癌細胞株型態的影響 74
圖八、利用colony formation assay觀察TSC-22的表現對於人類肺癌細胞株細胞群落形成的影響 77
圖九、利用anchorage-independent growth assay觀察TSC-22的表現對於人類肺癌細胞株轉型能力的影響 80
圖十、利用流式細胞儀分析抑制TSC-22對於人類肺癌細胞株H358
細胞週期與細胞凋亡的影響 82
圖十一、利用流式細胞儀分析過度表現TSC-22對於人類肺癌細胞株
CL1-5細胞週期與細胞凋亡的影響 83
圖十二、利用流式細胞儀分析短暫轉染TSC-22對H226細胞株細胞週
期與細胞凋亡的影響 84
圖十三、觀察抑制TSC-22表現對於H358細胞株之細胞週期調控蛋白
的影響 86
圖十四、觀察過度表現TSC-22對於CL1-5細胞株之細胞週期調控蛋
白的影響 88
圖十五、觀察抑制TSC-22表現對於H358細胞株內生性MAPKs與Akt
之活化的影響 90
圖十六、觀察過度表現TSC-22對於CL1-5細胞株內生性MAPKs與
Akt之活化的影響 91
圖十七、觀察轉染shRNA TSC-22對於CL1-0細胞株移行能力的影響 92
圖十八、觀察轉染HA-TSC-22對於CL1-5細胞株浸潤能力的影響 93
圖十九、利用動物模式觀察抑制TSC-22的表現對於腫瘤生長的影響 94
拾、 附圖
附圖一、2007年全國主要死因死亡率 96
附圖二、2007年主要癌症死亡率 97
附圖三、細胞週期參考圖 98
附圖四、實驗結果流程圖 99
拾壹 、附表
附表一、細胞株的種類 100
附表二、引子序列表 101
拾貳 、文獻探討 102

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