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研究生:翁任萱
研究生(外文):Ren-Shiuan Weng
論文名稱:探討基因毒性壓力調控CIRP的機制以及CIRP與癌細胞侵襲能力的潛在關聯
論文名稱(外文):CIRP: it’s regulation by genotoxic stresses and its implication in cancer cell invasion
指導教授:張嘉哲張嘉哲引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:47
中文關鍵詞:CIRPEtoposideAktp21
外文關鍵詞:CIRPEtoposideAktp21
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Cold inducible RNA binding protein (CIRP)是一個在低溫壓力下會被誘發的蛋白。在過去的研究發現除了低溫壓力之外,其他不同的藥物以及刺激之下也會導致CIRP被誘發,例如造成DNA損傷的抗癌藥Adriamycin、導致內質網壓力Thapsigargin、高滲透壓、或是缺氧的情況。因此我們可以知道CIRP是一個會受到壓力刺激而被誘發的蛋白並且CIRP的表現是細胞因應壓力的機制之一。Etoposide是一個toposiomerase II 的抑制劑,它會使DNA產生雙鍵斷裂的現象。在這篇論文當中,我們發現當細胞處理抗癌藥物Etoposide時,Etoposide會誘發CIRP的表現量。除此之外,我們還發現到Etoposide誘導CIRP蛋白質增加並會使CIRP從細胞核內移動到細胞質,這個現象是受到PI3K/Akt訊號傳遞途徑所調控。並且進一步的研究得知,在乳癌細胞株MCF-7處理Etoposide時,加入PI3K抑制劑或是Akt抑制劑使Akt失去活性時,失去活性的Akt會使CIRP mRNA表現下降以及造成CIRP蛋白質降解。然而在過去的研究中,我們知道Akt在DNA損傷藥物處理下會調控p21WAF1/CIP1的表現。令人感到有趣的是,我們觀察到在DNA損傷藥物處理下,CIRP的表現和p21WAF1/CIP1具有相同的趨勢。並且當我們構築了持續過量表現CIRP的子細胞株,發現到過量表現CIRP的子細胞株會抑制細胞的生長,同時p21WAF1/CIP1蛋白質表現量也變多。我們進一步的去研究這個現象後得知,過量表現的CIRP會造成p21WAF1/CIP1表現變多是透過調控延長p21WAF1/CIP1蛋白質的半衰期。近年來的對於利用微陣列分析在不同條件下的研究基因表現已經成為預測基因功能的工具。我們去蒐尋過去和癌症有關的微陣列分析文獻後發現,CIRP在良性腫瘤或是正常細胞中的表現量會比惡性腫瘤為高。因為這個發現我們利用乳癌細胞株以及肺癌細胞株分析細胞侵襲能力後得知,CIRP在細胞侵襲能力強的細胞株表現量較低。因此在未來我們希望釐清CIRP與細胞侵襲能力的關係,並且進一步的去探討Akt調控CIRP是否和細胞侵襲能力有關。
Cold inducible RNA binding protein (CIRP) was originally found to be a gene induced by mild cold stress in mammalian cells. Recently, some studies found that CIRP was also induced by stresses including DNA damage by Adriamycin, endoplasmic reticulum stress by thapsigargin, high osmolarity, and hypoxia. Therefore, CIRP is a stress-inducible protein and presumedly has an important role in cell responses to stresses. In this study, we found that CIRP is evidently up-regulated by the treatment of chemotherapeutic drug etoposide, a known inhibitor of toposiomerase II. In addition, we showed that phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is responsible for CIRP’s induction and the translocation of CIRP to cytoplasm by etoposide. Inactivation of Akt led to CIRP reduction by both decrease in CIRP mRNA levels and increase of proteasome-mediated degradation. In previous studies, Akt plays a critical role in the levels of p21WAF1/CIP1 induced by DNA damage drugs. Interestingly, we found that DNA-damaging stresses induce CIRP expression in the same way. Surprisingly, we demonstrated that overexpression of CIRP inhibits cell proliferation, possibly due to upregulation of p21WAF1/CIP1. Overexpressions of CIRP led to p21WAF1/CIP1 induction by the halflife of prolong p21 WAF1/CIP1. Recently, DNA microarray analysis is one of the fastest-growing new technologies in the field of genetic research. We analyzed data from different published studies attempted to profile gene expression between tumor cell and normal or benign tumor cell on the basis of DNA microarray analysis. These data showed that CIRP was downregulated in tumor cells and upregulated in normal or benign tumor cells. To this end, we used breast cancer cell line and lung cancer cell line to detect CIRP expression, and we found that CIRP was downregulated in high invasive cancer cell line. In the future, we have to find out that what the relationship between cell invasive ability and CIRP is and how Akt involves.
目次

壹、 緒論 1
一、 Cold inducible RNA binding protein 1
(一)、 Cold-inducible RNA binding protein 簡介 1
(二)、 CIRP 的結構和蛋白質功能 2
(三)、 CIRP 研究概況 3
二、 PI3K/Akt信號傳遞與腫瘤 4
(一)、 PI3K/Akt信號傳遞途徑與癌症的關係 4
(二)、 Akt的表現與乳癌細胞的關係 5
三、 Etoposide簡介 7
四、 研究動機與目的 8
貳、 實驗材料與方法 10
一、 實驗材料 10
(一)、 細胞株 10
(二)、 藥品與試劑 10
二、 實驗方法 11
(一)、 細胞培養 11
(二)、 化學藥品處理細胞株 12
(三)、 細胞總量RNA之萃取 12
(四)、 反轉錄聚合酶連鎖反應 12
(五)、 即時定量聚合酶連鎖反應 13
(六)、 CIRP基因選殖 14
(七)、 構築CIRP過量表現細胞株 15
(八)、 總蛋白之萃取及濃度分析 16
(九)、 西方墨點法 16
(十)、 細胞增生能力試驗 17
(十一)、 報導基因冷光測定 17
(十二)、 細胞侵襲能力試驗 18
(十三)、 細胞核內核外蛋白質分離 18
參、 實驗結果 20
一、 在乳癌細胞MCF-7處理Etoposide觀察CIRP表現 20
二、 Etoposide誘導CIRP與PI3K/Akt訊號途徑的關係 20
三、 Akt對CIRP 的影響 21
(一)、 抑制PI3K/Akt訊息傳遞途徑影響細胞核內CIRP的表現 21
(二)、 抑制PI3K導致CIRP mRNA的降低 21
(三)、 抑制PI3K導致CIRP蛋白質降解 22
(四)、 Akt影響CIRP的表現 22
四、 CIRP的表現與癌症細胞侵襲能力的關係 23
五、 過量表現CIRP對細胞的影響 23
(一)、 CIRP與細胞生長能力的關係 23
(二)、 CIRP對p21的影響 24
肆、 討論 25
一、 Etoposide誘導CIRP透過Akt 25
二、 過量表現CIRP延長p21蛋白質半衰期 26
三、 CIRP表現與細胞侵襲能力呈負相關 27
四、 結論 28
五、 未來展望 28
實驗數據圖 30
參考文獻 40
附錄一:SDS PAGE膠體濃度配置 43
附錄二:實驗所使用藥劑配置 44
附錄三:實驗所使用抗體稀釋比例 45
附錄四:參與96年度榮興計畫研討會(2008) 46
附錄五:參與第二屆基因體與蛋白質體學術研討會(2009) 47
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