Please use this identifier to cite or link to this item:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35642
Title: | 第壹部份:珍菇毒蛋白之分離;第貳部分:腎臟癌及肝癌候選基因之篩選 Part I: Purification of Novel Hemolytic Proteins from Edible Mushroom Pleurotus geesteranus Part II: Identification of Candidate Genes of Renal Cell Carcinoma and Hepatocellular Carcinoma |
Authors: | Yu-Wei Chao 趙宇薇 |
Advisor: | 林榮耀 |
Keyword: | 秀珍菇毒蛋白,腎臟癌候選基因,肝癌候選基因, Hemolytic Proteins,Pleurotus geesteranus,Renal Cell Carcinoma,Hepatocellular Carcinoma, |
Publication Year : | 2005 |
Degree: | 碩士 |
Abstract: | 第壹部份:
溶血毒蛋白(hemolysin)是一種廣泛存在於生物體內的溶細胞毒素(cytolysin),研究指出,從食用菇蕈類的所純化到溶血毒素,具有在細胞膜上形成孔洞的活性(pore-forming property),例如,過去由本實驗室所純化的金針菇毒蛋白(flammutoxin)及草菇毒蛋白(vovatoxin A2, VVA2)。 在最近的研究發現,自鮑魚菇(Pleurotus ostreatus)所純化到的pleurotolysin也具有溶血的能力, pleurotolysin是由兩種蛋白共同構成, 分別被命名為Ply A 及Ply B。這些溶血毒蛋白會和紅血球的細胞膜作用,以形成孔洞的方式讓紅血球破裂,在紅血球破裂的殘骸中也發現到會有高分子量的聚合體出現(high molecular pore complex)。 本論文將對於鮑魚菇同屬的秀珍菇(Pleurotus geesteranus),進行具溶血活性的毒蛋白純化, 起初由95% 硫酸銨(Ammonium sulfate)所沉澱出的秀珍菇蛋白, 經由溶血試驗(hemolysis assay)證實具有溶血的能力, 這些蛋白再經由陰離子交換樹脂管柱(DEAE Sephacel chromatography)膠體過濾管柱(Sephadex G-75 gel filtration)純化。 我們很意外的發現, 由膠體過濾管柱所分離的兩個蛋白峰分別和紅血球反應時, 並沒有溶血的現象出現, 當把分離的兩個峰之蛋白質再次混合與紅血球反應時,溶血的現象再次出現, 根據上述的實驗結果,推測由秀珍菇所純化的溶血毒蛋白可能是由二個以上的蛋白所構成, 至於詳細的溶血機制, 則需要進一步的研究。 第貳部份: 根據衛生署的統計, 癌症連續23年高居國人十大死因榜首, 在十大癌症排行當中, 肝癌是國人癌症的第二號殺手,在近年來的研究發現,酒精、黃麴毒素及B型及C型肝炎病毒的感染,都是造成肝癌發生的主要原因。 由於腎臟癌在初期不如其他癌症有明顯的病症,當病人檢驗出有腎臟癌時, 往往伴隨有癌細胞轉移的現象,因此,腎臟癌的五年存活率往往低於其他癌症。 隨著生物技術的進步, 致癌基因的篩檢成為熱門的研究方向, 在本實驗室所建立的腎臟癌及肝癌基因資料庫, 經由定序並與NCBI的資料庫進行比對, 在腎臟癌的基因資料庫當中,共有780個基因表現有異常的現象, 至於肝癌, 則是有464個基因有異常表現的; 在這些表現量異常的基因當中, 我們共選定了12個基因, 這些基因分別是: MAP3K2, PPP2R5A, SERPINA6, SERPINB6, SERPING1, FBXO18, NET1, NFIB, SKIIP, TTR, SYAP1及 MAWBP,利用同步定量聚合酶連鎖反應 (quantitative real-time PCR)來分析他們在癌症患者檢體的mRNA 表現量。 在腎臟癌的病人檢體當中,我們發現到數個基因mRNA 表現量的變化, 與腎臟癌的分期有關連性: 參與細胞訊息傳導的MAP3K2及PPP2R5A在癌症早期和晚期(stage)的變化, 有統計上的差異, 另外, 在細胞內擔任蛋白酶抑制劑(protease inhibitor)的SERPINA6, SERPING1和 ubiquitin ligase complex 之一的FBXO18 mRNA 表現,也被發現與腎臟癌組織病理分級(histopathologic grade)有統計結果的意義; 同時,我們也利用了西方墨點轉漬法來測定MAP3K2及TTR在蛋白質層面的表現量。 然而在肝癌患者的部分,利用同步定量聚合酶連鎖反應分析MAP3K2, SERPINA6, SERPING1, SERPINB6及TTR mRNA表現量,與患者的癌症分期並沒有觀察到他們之間的關連性。 本實驗中所發現的癌症候選基因將來有機會可以成為在臨床上癌症檢測新的標的,同時對於腎臟癌和肝癌發生的機轉也有更多的了解。 Part I: Hemolysins exist widely in living organism. Many hemolysins were purified from the basidiocarps of edible mushrooms, and their pore-forming activity and cytotoxicity were demonstrated. In the past, several hemolysins were purified from edible mushrooms in this laboratory, such as flammutoxin from Flammulina velutipes and volvatoxin A2 (VVA2) from Volvariella volvace. Recently, a two-component hemolysin was purified from Pleurotus ostreatus and was named as Pleurotolysin (Ply) which consisted of non-associated A and B proteins. Many hemolysins show their hemolytic activity by interacting with red blood cells and some of them lyse red blood cells by the mechanism of pore-forming, and these pore-forming cytolysins include flammutoxin, VVA2 and pleurotolysin which interact with red blood cell membrane and aggregate into higher molecular pore complex. In the present study, novel hemolysins were identified from Pleurotus geesteranus which was a popular edible mushroom in Oriental. The proteins of crude extracts of P. geesteranus precipitated by 95% ammonium sulfate showed a hemolytic activity. The purification of proteins with hemolytic activity was carried out by DEAE Sephacel chromatography followed by Sephadex G-75 chromatography. After DEAE Sephacel column chromatography, the flow-through fractions showed a hemolytic activity, and surprisingly, this hemolytic activity was not detected in each of the protein peaks obtained by Sephadex G-75 column chromatography. However, the combination of peak 1 and peak 2 fractions from Sephadex G-75 showed a strong hemolytic activity, and 10 μg/ ml of peak 1 protein and 35 μg/ ml of peak 2 showed a 50% hemolytic activity (HA50). These results suggest that the novel hemolysins from P. geesteranus probably consisted of two non-associated proteins. Part II: The cancers have been the first cause of death in Taiwan in last two decades. Among them, hepatocellular carcinoma (HCC) is in the second most common cancer. It has been shown that alcohol abuse, aflatoxin exposure and viral infection were associated with HCC. In addition, the incidence of renal cell carcinoma (RCC) is increasing. It is characterized by lack of early warning signs and the poor survival of RCC patients with metastasis. In this laboratory, the full-length cDNA libraries from a RCC tissue and its adjacent normal kidney tissue and of a HCC tissue and its adjacent normal liver tissue were generated. The whole full-length cDNA constructs were sequenced, and compared with those of normal kidney. It was revealed that 780 genes were up- or down-regulated in the full-length cDNA libraries of RCC tissue compared with those of normal adjacent tissue while 464 genes, in HCC. The purpose of the present study was to identify possible cancer candidate genes of RCC and HCC by their expression, and their association with cancer progression. Down-regulated genes (MAP3K2, PPP2R5A, SERPINA6, SERPINB6, SERPING1, FBXO18, SYAP1, MAWBP, NFIB, NET1, and TTR) and up-regulated gene, SKIIP in a RCC tissue, were chosen for studies. The relative mRNA expression levels of these genes in 48 RCC tissues and their adjacent normal tissues were determined by quantitative real-time PCR analysis. The statistic analyses were performed to investigate the significance between the gene expression patterns and tumor clinical stages or histopathologic grades. Significant up-regulation of MAP3K2 and PPP2R5A mRNA level was observed in RCC late stage comparing to early stage. Besides, the mRNA expression of SERPINA6 and FBXO18 was down-regulated significantly in advance RCC grade while the expression level of SERPING1 was up-regulated. The protein expression levels of two candidate genes (MAP3K2, PPP2R5A) in RCC tissues were analyzed by Western blotting, and the results showed that they were down-regulated. However, the expression level of SYAP1, MAWBP, SKIIP, NFIB, NET1 and SERPINB6 mRNA was not significant with tumor stages or histopathologic grades in RCC. The candidate genes identified in the current study would not only provide new opportunities for identifying diagnostic markers of RCC or HCC but also improve our understanding of RCC and HCC biology and help identify new therapeutic targets. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35642 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生物化學暨分子生物學科研究所 |
Files in This Item:
File | Size | Format | |
---|---|---|---|
ntu-94-1.pdf Restricted Access | 3.7 MB | Adobe PDF |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.