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標題: | 第二型肝細胞生長因子活化抑制者在非小型肺癌中抑制癌細胞侵襲及調降表皮生長因子受器扮演的角色 Role of hepatocyte growth factor activator inhibitor type 2 in inhibiting Non-small-cell lung carcinoma cell invasion and down-regulation of epidermal growth factor receptor |
作者: | Chih-Che Shen 沈志哲 |
指導教授: | 李明學(Ming-Shyue Lee) |
關鍵字: | 第二型肝細胞生長因子活化抑制者,非小型細胞肺癌,表皮生長因子受體,肺癌,蛋白純化, HAI-2,NSCLC,lung cancer,EGFR,purification, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 肺癌在全世界以及台灣是致死率最高的一類癌症,肺癌可以分為兩類:小細胞肺癌(small cell lung cancer)及非小細胞肺癌(non-small cell lung cancer)。非小型細胞肺癌經常在肺癌後期才容易被診斷出來,且伴隨著較差的預後。文獻指出蛋白酶失調與非小型細胞肺癌細胞移動與侵襲力增加有正關聯性。第二型肝細胞生長因子活化抑制者 (HAI-2)是一廣泛性絲胺酸蛋白酶抑制者,被認為可以有效地降低身經膠質瘤(glioma cancer)、前列腺癌(prostate cancer)以及胃癌(gastric cancer)的產生與惡化。在本篇實驗中,將探討HAI-2在前列腺癌細胞(PC-3)及非小型細胞肺癌(CL)細胞中,在細胞侵襲及移動力,以及調控表皮生長因子受體(EGFR)所扮演的角色。從結果顯示,在高侵襲性的PC-3及CL1-5細胞內,大量表達HAI-2蛋白能降低細胞移動及侵襲性。因為HAI-2是含有兩個功能區域的膜蛋白,我進一步利用大腸桿菌及人胚胎腎細胞(HEK293T)以及中國倉鼠卵巢癌細胞(CHO)表達HAI-2重組蛋白,並純化這些蛋白且分析這些蛋白對於CL1-5細胞的移動以及侵襲能力的影響。結果顯示大腸桿菌表達的重組蛋白,在高濃度作用下能抑制細胞的侵襲及移動。而由哺乳類細胞表達的重組蛋白在低濃度作用下,就能有效地抑制肺癌細胞侵襲力。除此之外,我也發現HAI-2具有兩個N-醣基化的胺基酸位置 (Asn57與Asn94),且醣基化會影響到HAI-2抑制肺癌細胞侵襲及移動的能力。同時,本篇也證實了HAI-2抑制肺癌細胞侵襲及移動能力,主要是透過HAI-2的kunitz domain 2。因為在非小型肺癌細胞惡化過程中,EGFR與HAI-2表現量有著逆向關聯性,我們更進一步證實了在CL1-5及PC-3細胞中,HAI-2的表達能降低EGFR蛋白的量,且此EGFR蛋白量的降低主要是藉由調控蛋白層次的機制而非影響基因的表達。
總結以上結果,本論文指出了HAI-2對於癌細胞侵襲及移動扮演著負向調控的角色,且HAI-2能調降在肺癌細胞中的EGFR蛋白的量。 Lung cancer is the most lethal disease in the Taiwan and world. Lung cancer has different two types: small cell lung cancer and non-small cell lung cancer (NSCLC). NSCLC is often diagnosed at an advanced stage with poor prognosis. The dysregulation of cell surface proteolysis has been strongly implicated in the progression of NSCLC, particularly in cell invasion and metastasis. Hepatocyte growth factor activator inhibitor-2 (HAI-2) is a serine protease inhibitor and has been shown to be down-regulated in advanced human glioma, gastric and prostate cancers. In this study, I examined the role of HAI-2 in the cell invasion and migration of prostate cancer PC3 and NSCLC CL cells, as well as in modulating the protein level of EGFR. My results showed that the overexpression of HAI-2 in highly invasive CL1-5 and PC3 cells suppressed the cell invasion and migration. Since HAI-2 is a membrane-anchored protein with two extracellular functional domains, I used E.coli, mammalian HEK293T and CHO cells to express recombinant HAI-2 proteins, purified these proteins and analyzed the effects of HAI-2 recombinant proteins on CL1-5 cell invasion and migration. The results showed that the recombinant HAI-2 proteins from E. coli extracts suppressed CL1-5 cell invasion and migration within a high concentration, while a low concentration of the recombinant HAI-2 proteins purified from mammalian cell expression efficiently inhibited the cell migration and invasion. Moreover, I found that HAI-2 was glycosylated with two N glycosylation sites (Asn57 and Asn94) and the recombinant N57Q/N94Q HAI-2 proteins reduced its inhibitory effects on the cell migration and invasion, suggesting that the glycosylation was important for HAI-2 function. Furthermore, I showed that the kunitz domain 2 of HAI-2 played a major role in inhibiting NSCLC cell invasion and migration. Since EGFR is overexpressed and inversely correlated with HAI-2 expression during NSCLC progression, I further explored if HAI-2 was also involved in modulating the expression of EGFR in cancer cells and found that HAI-2 overexpression in CL1-5 and PC-3 cells was able to down regulate the EGFR protein but not the gene expression. The results taken together indicated that HAI-2 is a negative regulator for cancer cell migration and invasion, as well as to down regulate the EGFR protein levels. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42332 |
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顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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