DC 欄位 | 值 | 語言 |
dc.contributor | 楊雅倩 | zh-TW |
dc.contributor | 臺灣大學:醫學檢驗暨生物技術學研究所 | zh-TW |
dc.contributor.author | 賴彥君 | zh-TW |
dc.contributor.author | Lai, Yen-Chun | en |
dc.creator | 賴彥君 | zh-TW |
dc.creator | Lai, Yen-Chun | en |
dc.date | 2009 | en |
dc.date.accessioned | 2010-05-11T07:56:19Z | - |
dc.date.accessioned | 2018-07-06T08:01:51Z | - |
dc.date.available | 2010-05-11T07:56:19Z | - |
dc.date.available | 2018-07-06T08:01:51Z | - |
dc.date.issued | 2009 | - |
dc.identifier.other | U0001-1108200913212500 | en |
dc.identifier.uri | http://ntur.lib.ntu.edu.tw//handle/246246/182963 | - |
dc.description.abstract | 大腸直腸癌高居國人癌症致死率第三位,已知致癌路徑主要有三個:造成基因不穩定的失異合性、微衛星不穩定,以及表觀基因修飾的調控。本實驗室先前分析大腸直腸癌檢體在第四號染色體上微衛星標記的缺失情形,進而發現PCDH10(protocadherin 10) variant 1 mRNA 在大腸腫瘤檢體表現量顯著較正常黏膜組織低,並因受到DNA 高度甲基化而降低基因表現,此初步結果支持PCDH10 可能為大腸直腸癌相關之抑癌基因,因此選擇PCDH10 基因為研究目標,了解PCDH10在大腸直腸癌致癌路徑中是否扮演抑癌基因的角色。PCDH10 有兩種mRNAvariants,本論文經由建構PCDH10 表現載體於大腸直腸癌細胞株HCT116 進行蛋白質表現,以研究此兩種基因產物之可能功能。結果顯示暫時性大量表現PCDH10variant 1 會抑制細胞生長及侵犯能力,而大量表現PCDH10 variant 2 僅影響細胞生長能力。由於無法獲得會表現PCDH10 蛋白的混合穩定株(mixed stable clones),我們利用兩株PCDH10 variant 1 單一穩定株(single stable clones)及一株PCDH10 variant 2 單一穩定株進行功能研究。結果顯示在PCDH10 variant 1 蛋白表現量高時具有抑制細胞生長及群落形成能力,而PCDH10 variant 2 的表現則不影響細胞的這些特性。此外,為確認PCDH10 在細胞中表現位置,建構表現PCDH10 和綠色螢光蛋白融合的載體,也以免疫螢光染色檢PCDH10 兩種蛋白在細胞中的位點。以共軛焦顯微鏡觀察發現PCDH10 variant 1 和variant 2 均表現在細胞膜上,並且兩者有共同存在(co-localization)的現象。由以上結果我們推測PCDH10 於細胞膜上並進行蛋白功能,其中,PCDH10 variant 1 在大腸直腸癌細胞株HCT116 可能具有抑癌基因功能。 | zh-TW |
dc.description.abstract | Colorectal cancer (CRC) is one of the most common malignancies and is the third leading cause of cancer death in Taiwan. Three major pathways, loss of heterozygosity, microsatellite instability and epigenetic regulation are demonstrated in CRC tumorigenesis. To identify putative tumor suppressor genes associated with CRC, deletion mapping on chromosome 4 was performed by loss of heterozygosity analysis with colorectal cancer paired tissues. Previous data figured out that expression of PCDH10 variant 1 is significantly down-regulated in tumor tissues than their paired normal mucosa. On the other hand, the expression of PCDH10 is regulated by epigenetic modification. These data support that PCDH10 might be a putative tumor suppressor gene associated with in colorectal cancer. It has been reported that PCDH10 gene is transcribed into 2 kinds of mRNA variants. In the present study, PCDH10 mammalian expression vectors were constructed and over-expressed in colorectal cancer cell line, HCT116, and then the cells were analyzed by functional assays. The results showed transient over-expression of PCDH10 variant 1 could suppress cell proliferation and invasion, while over-expression of PCDH10 variant 2 only inhibited cell proliferation. Furthermore, we got two of PCDH10 variant 1 single stable clones and one of PCDH10 variant 2. PCDH10 variant 1 single stable clones could repress cell growth and colony formation in protein expression level-dependent manner. There is no same effect in PCDH10 variant 2 stable expression clone. Moreover, through construction of PCDH10-GFP fusion protein vector and immunofluorecent stain of theCDH10 variant 1 single stable clone, we identified the localization of PCDH10 protein in cells. Both PCDH10 variant 1 and variant 2 were expressed on cell membrane and seemed to be co-localizated. In conclusion, PCDH10 variant 1 can suppress cell proliferation and invasion in HCT116, suggesting that PCDH10 variant 1 might be a putative tumor suppressor gene associated with colorectal tumorigenesis. | en |
dc.description.tableofcontents | Content謝...............................................................................................................................i要...............................................................................................................................iibstract .........................................................................................................................iiibbreviation ..................................................................................................................vigure Content ............................................................................................................viiiHAPTER 1. INTRODUCTION .............................................................................1.2. Colorectal cancer ...................................................................................... 1.1.1. The colon .......................................................................................... 1.1.2. Risk factors ....................................................................................... 2.1.3. Staging .............................................................................................. 3.1.4. Pathways involved in colorectal tumorigenesis................................ 6.1.5. LOH study on chromosome 4........................................................... 9.2. Protocadherin 10 ..................................................................................... 11.2.1. Protocadherin family....................................................................... 11.2.2. Structure of PCDH10...................................................................... 12.2.3. Functional study of PCDH10 ......................................................... 12.2.4. PCDH10 as a novel tumor suppressor gene in various cancers...... 13.2.5. Previous study at our lab................................................................. 14.3. Objectives in the thesis ........................................................................... 16HAPTER 2. MATERIALS AND METHODS .....................................................17.1. Cell lines ................................................................................................. 17.2. Vector construction ................................................................................. 17.2.1. PCDH10 mammalian expression vector ......................................... 17.2.2. PCDH10-GFP mammalian expression vector ................................ 19.2.3. Gel purification ............................................................................... 20.2.4. Preparation of competent cells........................................................ 21.2.5. Transformation................................................................................ 21.2.6. Mini-preparation of plasmid DNA ................................................. 22.3. Cell transfection...................................................................................... 22ii.4. Immunofluorescent stain......................................................................... 23.5. Cell proliferation assay ........................................................................... 23.6. Colony formation assay .......................................................................... 24.7. Matrigel invasion assay .......................................................................... 24HAPTER 3. RESULTS.........................................................................................25.1. Construction of PCDH10 mammalian expression vectors ..................... 25.2. PCDH10 expression in HEK293T.......................................................... 26.3. HCT116 as a model cell line................................................................... 26.4. PCDH10 expression in HCT116............................................................. 27.5. Inhibition of cell invasion ability by transient over-expression ofCDH10 variant 1 .................................................................................. 27.6. Suppression of cell proliferation by transient over-expression ofCDH10.................................................................................................. 27.7. Stable expression clones of PCDH10 in HCT116 .................................. 28.8. Suppression of cell proliferation and colony formation ability in oneCDH10 variant 1 stable clone .............................................................. 28.9. PCDH10 variant 1 and variant 2 expressed on cell membrane .............. 29.10. PCDH10 variant 1 and variant 2 are co-localized with homophilicnteraction ............................................................................................... 30HAPTER4. DISCUSSION........................................................................................32EFERENCE...............................................................................................................38PPENDIX..................................................................................................................59 | en |
dc.format | application/pdf | en |
dc.format.extent | 1520345 bytes | - |
dc.format.mimetype | application/pdf | - |
dc.language | en | en |
dc.language.iso | en_US | - |
dc.subject | 大腸直腸癌 | zh-TW |
dc.subject | 抑癌基因 | zh-TW |
dc.subject | PCDH10 | zh-TW |
dc.subject | colorectal cancer | en |
dc.subject | tumor suppressor gene | en |
dc.subject.classification | [SDGs]SDG3 | - |
dc.title | PCDH10 基因於大腸直腸癌之抑癌功能的鑑定 | zh-TW |
dc.title | Identification of a putative tumor suppressor gene,CDH10, in colorectal cancer | en |
dc.identifier.uri.fulltext | http://ntur.lib.ntu.edu.tw/bitstream/246246/182963/1/ntu-98-R96424015-1.pdf | - |
item.languageiso639-1 | en_US | - |
item.grantfulltext | open | - |
item.fulltext | with fulltext | - |
顯示於: | 醫學檢驗暨生物技術學系
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