FANCD2之活化與BRCT 的交互作用及其FANCD2基因多形性與乳癌發生關聯之分子遺傳學研究

中山醫學大學機構典藏 CSMUIR > 醫學院 > 生化微生物免疫研究所 > 研究計劃 > Item 310902500/3105

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/3105

Title:	FANCD2之活化與BRCT 的交互作用及其FANCD2基因多形性與乳癌發生關聯之分子遺傳學研究 Functional Activation of FANCD2 and the Risk Association of Genotypic Polymorphisms of FANCD2 in Breast Cancer
Authors:	鄭鈞文;黃俊升;俞志誠;沈志陽;余忠泰 Cheng, Chun-Wen;Huang, Chiun-Sheng;Yu, Jyh-Cherng;Shen, Chen-Yang
Contributors:	中山醫學院生物化學研究所
Keywords:	乳癌、染色體雙股錯結、酵母菌雙重雜交 Breast cancer？BInterstrand crosslink？BYeast two-hybrid assay？B BRCA1？BFANCD2
Date:	2005
Issue Date:	2010-12-07T09:24:03Z (UTC)
Abstract:	Fanconi貧血症(Fanconi anemia)是一種關於染色體異常的體隱性遺傳疾病，臨床上常會伴隨著惡性細胞轉形的發生。FANC群體複合蛋白會參與細胞因為染色體在分裂時可能發生交錯連結(interstrand crosslink)異常的修復作用。BRCA1協同DNA雙股斷裂修復基因Rad51, Rad52, Nbs1和Mre11等，參與DNA同源染色體重組修復作用。最近，在細胞遺傳學的研究證據顯示，於乳癌的致癌機轉中，FANCD2基因會受到BRCA1的活化並共同移位至細胞核中以修復雙股斷裂的染色體。然而，在乳癌細胞株HCC1937(BRCT truncated protein)卻無法活化FANCD2進行染色體斷裂的修復。因此，本研究即是以酵母菌雙重雜交試驗來探討BRCA1之BRCT區域和FANCD2 之間的交互作用。實驗中，我們首先對BRCT部份以定位突變建構此區域基因多型性的重組質體，並以核酸序列確認。另外，針對FANCD2藉由反轉錄作用，設計特定序列引子，將FANCD2基因區分為三個部分，並進行其基因片段的分子選殖。之後，將BRCT多型性基因片段各自連結於pGBT9質體(含DNA結合部位)和FANCD2的次選殖片段載入pACT2質體(含DNA活化部位)。最後，再將兩者以配對的方式分別殖入酵母菌LV40中，分析beta-galactosidase(beta-半乳糖脢)的酵素活性。現階段，我們已成功地完成了15個BRCT多形性變異基因的選殖和建構。再者，亦完整地選殖出FANCD2基因的三個次選殖部分，並完成其重組質體的建構。在後續的研究工作中，將針對BRCT多型性基因片段和FANCD2的次選殖片段分批共同殖入酵母菌中，評估分析酵母菌beta-半乳糖脢的酵素活性，以確實釐清BRCA1與FANCD2真正作用的區段。台灣地區婦女乳癌的年輕化和高發生率已是一個不容忽視的公共衛生問題，其中因為染色體交錯異常亦常常是導致乳癌發生的關鍵原因之一。本實驗著眼於因為染色體異常的修復作用所可能造成細胞癌化的病理成因探討，其了解BRCT和FANCD2的作用機轉，除了能夠更進一步探討台灣近年來乳癌發生率高漲的可能致癌原因外，其後續對乳癌致癌診斷和治療的預後評估其貢獻將相當可觀。 Fanconi anemia (FA) is an autosomal recessive disorder with diverse clinical symptoms and markedly predisposition to cell malignancy. FANCD2 can colocalize with BRCA1 in nuclear foci, associating with chromosomal damage repair. Underlying the rationale that FANCD2 can repair endogenous DNA interstrand crosslinks damage caused by estrogen exposure (the most important risk factor of breast cancer) and interact with BRCA1, showed that initiation of FANCD2 protein against hypersensitivity to such DNA break lesions.We thus, aimed at exploring the possible interaction between BRCA1 BRCT and FANCD2 proteins using yeast two-hybrid system. In the current study, we succeeded in constructing and sequencing 15 various polymorphic sites in BRCT region using site directed mutagenesis technique. On the other hand, the partial cDNA fragments of FANCD2 were also separately subcloned into 3 portions. The BRCT clones, containing different polymorphic sites of C-terminus domains were further fused into vector pGBT9, having Gal4 A binding domain (DBD). In addition, three FANCD2 subclones have been individually reconstructed into vector pACT2, who has Gal4 activating domain (AD). Followingly, the works at hand are going to separately cotransfected the polymorphic allele of BRCT and FANCD2 subclones into yeast LV40 cells. To fully understand tumorigenic contribution associated with interaction between individual BRCT and FANCD2 in chromosomal damage repair, a yeast two-hybrid assay using beta-galacosidease reporter gene will be used. We are now striving to obtain cotransfectants harboring BRCT and FANCD2 subclones and examine their beta-galacosidease activities in yeast. In summary, these data will provide a comprehensive understanding and characterizing the real interactions between the unique genetic polymorphism of BRCT acting on FANCD2 fragment against DNA interstrand crosslink damage to breast cancer development. Also, this model will be further applied to explore the independent genetic risk factors in contribution to define the etiology of Chinese breast cancer development in Taiwan.
URI:	https://ir.csmu.edu.tw:8080/handle/310902500/3105
Appears in Collections:	[生化微生物免疫研究所] 研究計劃

Files in This Item:

File	Description	Size	Format
932320B040022.pdf	研究計畫成果發表	267Kb	Adobe PDF	652	View/Open

Loading...