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研究生：	陳品均 Chen, Pin-Chun
論文名稱：	探討 RPA2蛋白在人類癌細胞株中的功能性角色 Functional Studies of RPA2 in Human Cancer Cell Lines
指導教授：	張敏政 Chang, Ming-Chung
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2011
畢業學年度：	99
語文別：	中文
論文頁數：	89
中文關鍵詞：	單股DNA結合因子、細胞凋亡、細胞老化、自發性的DNA損傷
外文關鍵詞：	RPA2, cellular senescence, apoptosis
相關次數：	點閱：145 下載：0
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Replication protein A (RPA)是真核細胞中單股DNA結合因子(single-strand DNA binding-factor)的一個家族，它是由RPA1、RPA2和RPA3三個次單元體組成。RPA被認為在DNA複製、修復和重組等DNA代謝機制中扮演不可缺少的角色。其中，RPA2在細胞周期及DNA損傷的時候會被磷酸化，進而促使RPA的結構改變並影響其與DNA結合的能力。此外，近年紛紛有研究指出RPA1和RPA2在大腸癌和腎臟癌中的高表現可做為一個病人存活率較低的指標。高度磷酸化的RPA2在頭頸癌細胞當中也可以做為一個細胞對於化療藥物具抗藥性的標誌。這些研究皆推論RPA2可能成為日後治療癌症的標的或指標。然而，RPA2在癌細胞當中扮演的角色是什麼目前尚未了解。在我們的研究發現，RPA2蛋白表現量在較惡性的人類肺癌、乳癌和大腸癌細胞株中都有較多的現象。因此，我們利用帶有RPA2短髮夾型 RNA (short-hairpin RNA; shRNA)的慢病毒 (Lentivirus)感染人類癌細胞株，試圖抑制細胞內其基因的表現，進而釐清RPA2在癌細胞中所扮演的角色。實驗結果顯示，在A549人類肺癌細胞株中抑制RPA2表現會促使細胞生長、增殖和形成聚落能力有降低的趨勢。我們也藉由Hoechst 33342/PI雙染偵測細胞凋亡實驗發現，抑制RPA2表現會引發細胞凋亡。特別地，抑制RPA2表現會使細胞變成肥大的型態，同時也誘發了SA-β-gal活性及肌動蛋白壓力性纖維累積等細胞老化的指標產生。然而，在分子機制上分析顯示，抑制RPA2表現誘發細胞老化的現象僅發生於表現正常p53蛋白的A549和MCF7細胞株，並非缺失p53基因的H1299細胞株。此外，我們也發現不管在A549、MCF7或H1299細胞株中抑制RPA2表現都會產生自發性的DNA損傷。綜合以上結果，我們推測在人類癌細胞中，RPA2缺失會讓細胞產生自發性DNA損傷，使細胞凋亡及細胞老化等DNA損傷的反應發生，最後導致癌細胞生長、增殖及形成聚落的能力降低。

Replication protein A (RPA), the main eukaryotic ssDNA binding protein, is a protein of heterotrimer composed of three tightly associated subunits of RPA1, RPA2, and RPA3. RPA has been found to be an indispensable player in DNA metabolic pathways such as DNA replication, repair and recombination. In addition, RPA2 has been shown to undergo cell cycle-dependent and damage-induced phosphorylation; it may influence RPA conformation and DNA-binding characteristics. Moreover, recent studies have shown that RPA1 and RPA2 emerged as predictors of poor survival rate in patients with colon cancer or bladder cancer. Hyperphosphorylation of RPA2 is also a marker for increased cellular resistance to chemotherapy in head and neck squamous cell carcinoma. These studies suggested that RPA2 may be a therapeutic target for cancer treatment. However, the role of RPA2 in cancer cells remains unknown. Here, we found that RPA2 protein expression was upregulated in malignant human lung, breast and colon cancer cell lines. To elucidate the role of RPA2 in tumor, we infected human cancer cells with lentiviruses that produced specific short-hairpin RNA to silence their RPA2 gene expression. The result showed that RPA2 knockdown in the A549 cells reduced cell growth, cell proliferation and colony formation. We also found that RPA2 depletion induced apoptosis by Hoechst 33342/PI double stain apoptosis detection assay. Notably, RPA2 depletion markedly caused enlarged cell morphology and induced the expression of senescence markers, such as senescence-associated β-galatosidase activity and actin stress fibers accumulation. Mechanistic dissection showed that cellular senescence induced by RPA2 knockdown was associated with p53 activation in A549 and MCF7 cells(wt-p53), but not in H1299 cells(p53-null). Furthermore, we also found that RPA2 depletion caused spontaneous DNA damage in A549, MCF7 and H1299 cells. Collectively, our findings suggest that RPA2 depletion may induce spontaneous DNA damage and then cause DNA damage responses, such as apoptosis and senescence, in human cancer cell lines.

中文摘要	I
Abstract	II
誌謝	III
目錄	V
圖表目錄	VIII
縮寫表	IX
第1章  緒論	1
前言  	2
1-1  RPA的生理功能	2
1-2  RPA三個次單元體的簡介	3
1-3  RPA2的磷酸化	6
1-4  RPA2與癌症之關聯	8
1-5  腫瘤的生成	9
1-6  細胞老化	10
1-7  細胞凋亡	11
1-8  研究動機	13
第2章  材料與方法	14
2-1  實驗菌株與培養基配方	15
2-1-1  實驗菌株	15
2-1-2  培養基配方	15
2-2  細胞培養方法	16
2-2-1  實驗細胞株	16
2-2-2  細胞解凍	16
2-2-3  細胞繼代培養	17
2-2-4  細胞計數	17
2-2-5  細胞保存	18
2-3  質體製備	19
2-3-1  聚合酶連鎖反應	19
2-3-2  構築PCR片段於質體中	20
2-3-3  E. coli 形質轉型	21
2-3-4  小量質體製備	22
2-4  蛋白質分析	23
2-4-1  蛋白質萃取	23
2-4-2  蛋白質定量	23
2-4-3  SDS-PAGE 蛋白質電泳	24
2-4-4  西方墨點法	25
2-5  在A549人類肺癌細胞建立RPA2表現抑制的穩定細胞	27
2-5-1  RPA2 shRNA 之來源	27
2-5-2  細胞轉染	27
2-5-3  利用抗生素篩選穩定細胞株	28
2-6  RPA2表現抑制的穩定細胞株對於影響腫瘤生長能力的分析	29
2-6-1  利用Cell Counting Kit-8檢測穩定細胞株生長速率	29
2-6-2  利用BrdU Cell Proliferation Assay檢測穩定細胞株增殖能力	29
2-6-3  利用Colony Formation Assay觀察穩定細胞株形成細胞群落的能力	30
2-7  RPA2表現抑制的穩定細胞株對於影響細胞凋亡和老化的分析	31
2-7-1  利用Hochest 33342/PI雙染色法檢測細胞凋亡程度	31
2-7-2  檢測穩定細胞株內senescence-associated β-galactosidase的活性	31
2-7-3  利用Rhodamine phalloidin螢光染色觀察肌動蛋白應力性纖維的分布	32
2-8  p53 siRNA的暫時轉染	33
2-9  分析RPA2表現抑制的穩定細胞株產生的自發性DNA損傷	33
2-10 統計方法	34
第3章  實驗結果	35
3-1  在不同人類癌細胞株中RPA2的表現量與腫瘤發展有關	36
3-2  在A549人類肺癌細胞建立RPA2表現抑制的穩定細胞	36
3-3  在A549細胞株中抑制RPA2表現會降低細胞增殖及細胞生長速率	37
3-3-1  利用Cell Counting Kit-8檢測穩定細胞株生長速率	38
3-3-2  利用BrdU Cell Proliferation Assay檢測穩定細胞株增殖能力	38
3-3-3  利用Colony Formation Assay觀察穩定細胞株形成細胞群落的能力	39
3-4  在A549細胞株中抑制RPA2表現會促使細胞凋亡及細胞老化發生	39
3-4-1  利用Hochest 33342/PI雙染色法檢測細胞凋亡程度	40
3-4-2  分析穩定細胞株內抗細胞凋亡及細胞凋亡相關基因的蛋白質表現	40
3-4-3  利用相位差顯微鏡觀察穩定細胞株的細胞型態	41
3-4-4  檢測穩定細胞株內senescence-associated β-galactosidase的活性	42
3-4-5  利用Rhodamine phalloidin螢光染色觀察肌動蛋白應力性纖維的分布	42
3-4-6  分析穩定細胞株內與細胞老化相關基因的蛋白質表現	43
3-5  p53參與在抑制RPA2蛋白表現所引發的細胞老化過程	44
3-5-1  分析p53 siRNA暫時轉染對於RPA2表現抑制穩定細胞株的影響	44
3-5-2  在H1299細胞株中抑制RPA2蛋白表現對於細胞老化的影響	45
3-5-3  在MCF7細胞株中抑制RPA2蛋白表現對於細胞老化的影響	45
3-6  抑制RAP2蛋白表現促使人類癌細胞株產生自發性DNA損傷	46
第4章  實驗討論	49
4-1  RPA2在癌症之中扮演之角色	50
4-2  RPA2抑制癌細胞生長的機制	51
4-3  在RPA2抑制癌細胞生長之中p53蛋白的角色	53
4-4  自發性DNA損傷的產生	54
4-5  RPA1蛋白的角色	55
參考文獻	57
實驗圖表	64
附錄	83
自述	89


                                    

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