台灣翼狀贅片致病機轉研究：( I ) BPDE-Like DNA鍵結物含量和細胞色素p450 1A1與麩氨基硫轉移酶M1基因多型性的相關性( II )細胞週期蛋白D1的蛋白表現與β-連環蛋白表現位置的相關性

The study of the pterygium pathogenesis in Taiwan:(I)An Association Between BPDE-Like DNA Adduct Levels and CYP1A1 and GSTM1 Polymorphisms(II)CyclinD1 protein expressed in pterygia is associated with β-catenin protein localization

童瑞年(Jai-Nien Tung)

指導教授 ： 鄭雅文

中山醫學大學/醫學院/醫學研究所/博士(2011年)

https://doi.org/10.6834/CSMU.2011.00126

摘要

( I )中文摘要
目的：BaP 7,8-diol 9,10-epoxide（BPDE）是 benzo(a)pyrene (BaP) 的終極代謝產物，會攻擊去氧鳥糞核糖甘(deoxyguanosine )而形成一種BPDE-N2-dG 鍵結物，導致 p53 基因的突變。有文獻指出細胞色素p450 1A1 (CYP1A1)和麩氨基硫轉移酶 M1 (GSTM1) 參與多環芳香族烴(PAHs)的代謝。然而在翼狀贅片組織之 BPDE-like DNA 鍵結物的含量與 CYP1A1 及 GSTM1 的基因多型性關係並不清楚。本研究中將檢測 BPDE-like DNA adducts 與 CYP1A1 及 GSTM1 基因多型性的關係，以尋求更多分子學上的証據，來了解在翼狀贅片裡BPDE-like DNA adducts 形成翼狀贅片的原因。
方法：在本研究中，針對 103 個翼狀贅片檢體以多株抗體進行免疫組織化學染色方法來偵測 BPDE- like DNA adduct。為了分析 CYP1A1 和GSTM1 基因的多型性，所有 DNA 都是取自於翼狀贅片組織的上皮細胞，再利用限制片段長多型性聚合酶鍵反應(RFLP-PCR)及聚合酶鍵反應(PCR)來決定 CYP1A1 和 GSTM1 的基因型。
結果：在 33%(34/103)的翼狀贅片組織可偵測到 BPDE-Like DNA adduct。DNA 鍵結量含量的差異與 CYP1A1 基因多型性相關，但與 GSTM1 基因多型性無統計上的相關性。另外， CYP1A1 的 m2/m2(T/T) 及 m1/m2(C/T)基因型的患者較 CYP1A1的 m1/m1(C/C)基因型者易形成BPDE-Like DNA 鍵結物 (risk: 9.675, p=0.001, 95% CI：2.451 –38.185)。
(II)中文摘要
背景：Wnt 訊息傳遞途徑的連鎖反應(Wg/Wnt signaling cascade )在細胞發生癌化過程中扮演一個重要的角色。本研究室過去的研究指出在翼狀贅片組織中β-連環蛋白(β-catenin)有蛋白表現位置改變(aberrant localization)的現象。因此，本次研究欲了解翼狀贅片組織中β-連環蛋白與其下游基因的細胞週期蛋白D1(cyclin D1)的蛋白表現的相關性。
方法：利用免疫組織化學染色的方法偵測β-連環蛋白及細胞週期蛋白D1的表現。本研究採150個翼狀贅片的檢體及30個正常的結膜組織。
結果：在150個翼狀贅片檢體中β-連環蛋白及細胞週期蛋白D1之免疫染色陽性反應者分別為73（48.7%）及60（40.0%），其中β-連環蛋白表現在細胞核/細胞質者其細胞週期蛋白 D1的蛋白表現明顯高於 β-連環蛋白表現在細胞膜或不表現者(p值<0.0001)。另外，翼狀贅片多肉型其細胞週期蛋白D1的表現比萎縮型及中間型的表現來的更顯著(p值=0.006)。
結論：本研究顯示在細胞核/細胞質有β-連環蛋白表現的翼狀贅片，會增加細胞質中細胞週期蛋白(cyclin D1)的蛋白表現，進而引起翼狀贅片細胞的增生。

關鍵字

翼狀贅片 ； 細胞週期蛋白D1

並列摘要

(I)英文摘要
Purpose: BaP 7,8-diol 9,10-epoxide (BPDE), an ultimate metabolite of benzo(a)pyrene (BaP), attacks deoxyguanosine to form a BPDE-N2-dG adduct resulting in p53 mutations. Both cytochrome P4501A1 (CYP1A1) and glutathione S-transferase M1 (GSTM1) have been demonstrated to be involved in the metabolism of polycyclic aromatic hydrocarbons (PAHs). The relationship between BPDE-like DNA adduct levels and CYP1A1 and GSTM1 gene polymorphisms in pterygium is not clear. Therefore, BPDE-like DNA adducts, and CYP1A1 and GSTM1 gene polymorphisms were detected in this study to provide more molecular evidence to understand the cause of BPDE-like DNA adduct formation in pterygium.
Methods: In this study, immunohistochemical staining, using a polyclonal antibody on BPDE-like DNA adducts, was performed on 103 pterygial specimens. For the analysis of CYP1A1 and GSTM1 gene polymorphisms, DNA samples were extracted from epithelial cells and then subjected to restriction fragment length polymorphism and polymerase chain reaction for the determination of mutation and genotype of the CYP1A1 and GSTM1 genes.
Results: BPDE-like DNA adducts were detected in 33.0% (34/103) of the pterygium samples. The differences in DNA adduct levels were associated with the genetic polymorphisms of CYP1A1 but not GSTM1. Additionally, the risk of BPDE-like DNA adducts formation for patients with CYP1A1 m2/m2 and m1/m2 was 9.675-fold than that of patients with m1/m1 types (p=0.001, 95% CI 2.451 –38.185).
Conclusion: Our data provides evidence that the BPDE-like DNA adduct formation in pterygium samples was associated with CYP1A1 gene polymorphisms.
(II)英文摘要
Background: The Wnt (Wg/Wnt) signaling cascade plays an important role in tumorigenesis. Our previous report indicated that aberrant localization of β-catenin proteins was a feature of pterygia. Therefore, this study aimed to analyze the association of β-catenin protein and expression of a downstream gene, cyclin D1, in pterygial tissues.
Methods: Using immunohistochemistry, β-catenin and cyclin D1 protein expression was studied, in 150 pterygial specimens and 30 normal conjunctivas.
Results: Seventy-three (48.7%) and 60 (40.0%) pterygial specimens tested positive for β-catenin and cyclin D1 protein expression, respectively. Cyclin D1protein expression was significantly higher in β-catenin-nuclear/cytoplasmic positive groups than in β-catenin membrane positive and negative groups (p<0.0001). In addition, cyclin D1 expression was significantly higher in the fleshy group than in the atrophic and intermediate groups (p=0.006).
Conclusions: Our study demonstrated that β-catenin expressed in nuclei/cytoplasm increases cyclinD1 protein expression, which invokes pterygial cell proliferation.

並列關鍵字

BPDE-Like DNA ； CYP1A1 ； GSTM1 ； CyclinD1 ； β-catenin

參考文獻

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