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研究生:陳冠璇
研究生(外文):Kuan-Hsuan Chen
論文名稱:發展與定性NPC2及DEPDC6蛋白單株抗體
論文名稱(外文):Generation and Characterization of MonoclonalAntibodies Against NPC2 And DEPDC6 Protein
指導教授:陳宜民陳宜民引用關係
指導教授(外文):Yi-Ming Arthur Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:公共衛生研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:56
中文關鍵詞:尼曼匹克症C2型單株抗體脂肪肝
外文關鍵詞:NPC2DEPEDC6monoclonal antibodyfatty liver
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甘胺酸氮甲基轉移酶(Glycine N-methyltransferase,GNMT)是ㄧ個肝癌抑制基因,在肝癌中可以發現GNMT表現降低。利用酵母菌雙雜合系統發現GNMT分別與DEPDC6蛋白質及具攜帶膽固醇功能的尼曼匹克症C2型蛋白(NPC2)交互作用。本研究之目的是希望發展NPC2與DEPDC6蛋白抗體,並瞭解其在臨床上各種癌症組織與脂肪肝的表現情況。利用製作NPC2與DEPDC6基因重組蛋白分別注射到兔子及老鼠,使其產生多株與單株抗體,並藉由噬菌體呈現技術篩選出單株抗體之抗原決定位。在野生型小鼠,NPC2蛋白表現在副睪丸、腎臟、肺臟、睪丸、子宮、大腸及小腸;而DEPDC6蛋白則表現在脾臟、肝臟、睪丸、胰臟、副睪及大腸。在in vitro中,NPC2蛋白表現子宮頸癌上皮細胞株(Hela)、前列腺癌細胞株(LNCaP)和肝癌細胞株(SK-Hep1 、HepG2);DEPDC6蛋白則可以在乳癌細胞株(BT-20、HS578T)、骨髓瘤細胞株(NS-1)子宮頸癌上皮細胞株(Hela)、前列腺癌細胞株(LNCaP)和肝癌細胞株(SK-Hep1、HepG2、Hep3B、HuH6、HuH7、HA22T、HA59T)偵測到。另外,生物組織晶片的免疫染色顯示,DEPDC6在正常胃、子宮頸、卵巢與食道以及在乳癌、胰臟癌、腎臟癌、與大腸癌組織可被偵測到。最後,藉由餵食MCD飼料來偵測脂肪肝形成過程中,野生型與GNMT基因剔除鼠肝臟NPC2與DEPDC6的蛋白表現。在母鼠結果中顯示,雙醣基化的NPC2蛋白表現量增加,而單醣基化的NPC2蛋白顯著減少;而在餵食MCD飼料的GNMT基因剔除鼠,DEPDC6蛋白則無顯著的差異。因此,本研究所發展的NPC2與DEPDC6蛋白抗體未來將有助於更進一步探討GNMT、NPC2與DEPDC6三種蛋白質在肝臟功能異常時所扮演的角色。
Glycine N-methylatransferase gene (GNMT) is a tumor suppressor gene, its’ expression was found to be down-regulated in HCC. By using a yeast-two hybrid system, we have recently identified that GNMT could interact with DEP domain containing 6 (DEPDC6) and Neimann-Pick disease type C2 (NPC2) proteins. The goals of this study are: (1) to generate monoclonal and polyclonal NPC2 and DEPDC6 antibodies; (2) to study the expression of NPC2 and DEPDC6 in multiple mouse organs, different cell lines and human cancer tissue arrays; (3) to investigate the expression of NPC2 and DEPDC6 in MCD (Methionine-Coline dificient)-diet induced mouse fatty liver model. Polyclonal and monoclonal antibodies against NPC2 and DEPDC6 were generated by injecting recombinant proteins into animals. The epitopes of monoclonal antibodies will be determined by phage display assay. Results from western blot analysis demonstrated that NPC2 protein is mainly expressed in the epididymis, kidney, lung, testis, utures, large and small intestines of WT mice. DEPDC6 is expressed in the spleen, liver, testis, pancrea, epididymis and large intesine of WT mice. In vitro, endogenous NPC2 could be detected in Hela, LNCaP, HepG2 and SK-Hep1 cells. On the other hand, endogenous DEPDC6 were found to express in BT-20, HS578T, NS-1, Hela, LNCaP, SK-Hep1,HepG2, Hep3B, HuH6, HuH7, HA22T and HA59T cells. We further used multiple human cancer tissue arrays to detect the expression of NPC2 and DEPDC6. The data showed that DEPDC6 expressed in the normal stomach,ovary,esophagus and cervix tissues. It is worthy to note that DEPDC6 was up-regulated in breast cancer, pancreas cancer, kidney cancer, colon cancer and rectum cancer using immunohistochemistry staining. To mimic human fatty liver formation, WT and Gnmt-/- mice were fed a MCD-diet to induce hepatic steatohepatitis, and changes in expression levels of NPC2 and DEPDC6 were detected using western blot analysis. The expression of di-glycosylated form of NPC2 was enhanced and that of the mono-glycosylated form of NPC2 was significantly reduced in MCD-fed female WT and Gnmt-/- mice. In contrast, the expression of DEPDC6 was no significant changes in Gnmt-/- mice as compared to WT littermates. Therefore, our NPC2 and DEPDC6 antibodies will be useful to explore the functional roles of GNMT, NPC2, and DEPDC6 in liver disease.
中文摘要-------------------------------------------------4
Abstract-------------------------------------------------5
第一章
前言
第一節 尼曼匹克症C2型(Niemann-Pick Disease Type C2)------7
第二節 DEPDC6(DEP Domain Containing 6)-------------------9
第二章
研究目的與特定目標--------------------------------------10
第三章
材料與方法
第一節 重組蛋白純化-------------------------------------11
第二節 多株抗體製備-------------------------------------16
第三節 單株抗體製備以及噬菌體呈現技術-------------------18
第四節 酵素免疫分析法-----------------------------------24
第五節 細胞與組織均質-----------------------------------25
第六節 動物與餵食---------------------------------------27
第七節 免疫組織染色-------------------------------------28
第四章 結果---------------------------------------------30
第五章 討論---------------------------------------------36
參考文獻------------------------------------------------39
圖表
表一 NPC2與DEPDC6單株抗體之抗原決定位與免疫球蛋白-------43
表二DEPDC6單株抗體免疫組織染色結果----------------------44
表三 DEPDC6單株抗體在人類肝癌組織之免疫組織染色---------45
圖一 4個用來在大腸桿菌系統製作NPC2與DEPDC6重组蛋白質的質體
---------------------------------------------------46
圖二 NPC2與DEPDC6重組蛋白質的誘導與純化,使用Coomassie Blue
染色的結果--------------------------------------- -47
圖三 NPC2與DEPDC6多株抗體血清的製備---------------------48
圖四 以西方墨點法確認NPC2及DEPDC6單株抗體---------------49
圖五 以西方墨點法決定NPC2單株抗體之抗原決定-------------50
圖六 NPC2及DEPDC6蛋白在小鼠不同器官的表現情形-----------51
圖七 NPC2與DEPDC6之單株抗體在小鼠肝臟的表現量-----------52
圖八 NPC2與DEPDC6在不同的肝癌或其他癌症細胞株的表現量---53
圖九 DEPDC6單株抗體免疫組織染色-------------------------54
圖十 WT及Gnmt -/- 母鼠肝臟NPC2蛋白在餵食MCD 飼料後的表現量
---------------------------------------------------55
圖十一 WT及Gnmt-/-公鼠肝臟NPC2蛋白在餵食MCD 飼料後的表現量
-------------------------------------------------56
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