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標題: | 肝細胞癌相關基因Fetuin-A及Fetuin-B之功能性研究 Studies on the function of HCC- associated genes, Fetuin-A and Fetuin-B |
作者: | Bo-Wen Zhan 詹博文 |
指導教授: | 林榮耀 |
關鍵字: | 胎球蛋白,肝細胞癌, HCC,fetuin,ahsg, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | 由於肝細胞癌(HCC)是一種發生率高的惡性腫瘤疾病,因此找尋其相關的診斷與治療基因標的是非常重要與迫切的課題。由本實驗室蔡家櫸先生之前建構的肝細胞癌全長cDNA資料庫中發現,有一群演化同源自cystatin的基因群,包含histidine-rich glycoprotein (HRGP)、fetuin-A (a2-HS-glycoprotein)、fetuin-B及kininogen,其基因轉錄有降低表現的狀況。
Cystatin目前認為是一種生理性的溶體(lysosome)半胱氨酸蛋白脢抑制劑,屬於cystatin superfamily的蛋白皆具有數個結構相似於cystatin的多肰序列。fetuin(胎球蛋白)即屬於其中一員。fetuin是一種negative acute phase proteins,特徵為其血清濃度會在外傷、感染及發炎反應時大幅降低。此外,fetuin對細胞生理仍具有許多功能,包括抑制insulin receptor的酪氨酸磷酸脢活性、可作為巨噬細胞去活性分子的調理素(opsonization)、調節骨骼發展及抑制血液中磷灰石的形成以避免不正常的鈣化現象。 在本研究中,我們利用即時定量聚合脢鏈鎖反應及免疫組織染色的技術,確認了在許多臨床的配對採集樣本中,fetuin-A及B的mRNA及蛋白表現在HCC組織中有表現下降的現象。針對fetuin-A的功能性研究上,過度表現fetuin-A會抑制其MMP-2的mRNA表現,這表示細胞內的fetuin-A可能會抑制HCC癌細胞的侵犯能力。 然而當我們利用西方點墨法進一步分析fetuin-A蛋白在HCC組織中的表現量時,卻發現可能由於癌化區域具有較多血管及血液輸送的關係,fetuin-A蛋白在癌化組織的累積量相較於正常組織要多。因此我們給予HCC細胞純化的fetuin-A蛋白刺激後發現,fetuin-A蛋白會增強細胞生長、抑制E-cadherin的mRNA表現、及藉由活化PI3K的訊息傳遞來增強高度侵犯性癌細胞的移動性。 因此我們認為隨著HCC癌症的發展,需較大量血液供給而使癌組織區累積較多fetuin-A蛋白,且伴隨癌細胞fetuin-A接受器表現增加,皆使得後期的HCC癌細胞易因fetuin-A的刺激而變得更具侵略性。 Hepatocellular carcinoma (HCC) is one of the most frequent neoplasms worldwide. Therefore, finding new markers or associated genes is very important in its diagnosis and treatment. In previous study, using full-length cDNA library of HCC constructed by Dr. C.C. Tsai in our laboratory, we found several cystatin-derived genes, such as histidine-rich glycoprotein (HRGP), fetuin-A (a2-HS-glycoprotein), Fetuin-B, and kininogen, showed remarkable down-regulation in tumor tissues compared to their adjacent normal tissues. Cystatins are considered physiological inhibitors of lysosomal cysteine proteases. The cystatin “superfamily” encompasses proteins that contain multiple cystatin-like sequences. Fetuins are members of cystatin superfamily and are negative acute phase proteins in that their serum concentration drops after trauma, infection, and inflammation. Fetuins have been proposed to have several cellular functions including inhibition of tyrosine kinase activity of the insulin receptor, opsonization of macrophage-deactivating molecules, regulation of osteogenesis, inhibition of apatite formation and undesirable calcification. In the present study, we demonstrated that the mRNA and protein levels of fetuin-A and -B were remarkable down-regulated in HCC tissue pairs by quantitative real-time PCR and immunohistochemical staining. Over-expression of fetuin-A in SK-Hep-1 cell line caused down-regulation of MMP-2 at mRNA level without affecting cell proliferation. However, when we further analyze the fetuin protein patterns presented in HCC tissue pairs by Western blotting analysis, we surprisingly found that the level of fetuin protein in HCC tissues was higher than that of adjacent normal tissues. This phenomenon could be caused by more blood vessels in HCC tissues than normal tissues. By treating HCC cells with fetuin-A protein, it caused the increase of cell proliferation, and down-regulation of E-cadherin, whereas it activated PI3K signal pathway to enhance the cell motility of highly invasive HCC cells. It suggested that the late-stage HCC tumor tissues possessing more receptors of fetuin-A and higher amount of fetuin-A from blood vessels, and thus they are more sensitive to fetuin-A stimulation even if the fetuin-A synthesis was down-regulated. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32853 |
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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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