| Abstract: | 翳狀贅肉(pterygium)是一種慢性疾病，它的特徵是結膜組織伴著新生血管長入角膜中。翳狀贅肉長久以來被認為是一種退化性疾病。有些研究發現翳狀贅肉之p53基因會發生突變以及蛋白表現異常，而被認為可能是一種腫瘤。但是p53基因發生異常之成因至今未明，因此本研究假設眼角膜長期受到紫外線照射或許會造成反應性氧化物 (reactive oxygen species, ROS)，而引起DNA氧化性傷害所致。本研究首先分析台灣翳狀贅肉組織中p53基因之突變率，以及其突變形式是否和氧化性傷害有關。在收集的51個翳狀贅肉組織，以DNA定序法來定序p53基因，結果發現8 (15.7%) 個翳狀贅肉發生p53基因突變，其中有6個為取代 (substitution) 突變，2個是缺失 (deletion) 突變。在所有8個基因突變中，有3個 (37.5%) 突變是屬於和氧化性壓力有關的突變形式，包括2個G:C → C:G和1個G:C → T:A；另外有1個G:C → A:T的突變位在dipyrimidines 位置，這種突變代表紫外線造成之氧化性傷害所致。
8-hydroxy-deoxyguanosine (8-OHdG)是一種氧化性DNA傷害的生物指標。在收集52個翳狀贅肉組織和6個正常結膜組織，以免疫組織化學法(immunohistochemistry) 分析8-OHdG的表現。發現23.1% (12 of 52)的翳狀贅肉組織有8-OHdG的存在，而8-OHdG染色都只表現於上皮細胞細胞核中。在正常結膜組織及翳狀贅肉上皮下的結締組織中，則染不出8-OHdG的存在。因此在眼睛翳狀贅肉中，確有氧化性DNA的傷害。
ROS會引起氧化性DNA傷害，也會經由NF-kB訊號路徑造成環氧蛋白(Cycloxygenase-2, Cox-2)之表現。因此擬以Cox-2表現來間接驗證眼睛翳狀贅肉中有ROS的形成。本實驗共收集90個翳狀贅肉組織和10個正常結膜組織，以免疫組織化學法分析Cox-2的表現。發現有高達83.3% (75 of 90)的翳狀贅肉組織中有Cox-2的表現，而Cox-2只表現於上皮細胞細胞質中。在正常結膜組織及翳狀贅肉上皮下的結締組織中，則沒有Cox-2的存在。因此眼睛翳狀贅肉中確實存有Cox-2，而間接提供了翳狀贅肉組織中存有氧化性壓力的可能性。
hOGG1是一種DNA修補基因，主要參與8-OHdG的修復。本研究擬了解紫外線引發翳狀贅肉組織的氧化性傷害與hOGG1之相關性。以52個翳狀贅肉組織和6個正常結膜組織，用免疫組織化學染色方法分析8-OHdG 和hOGG1的表現。結果發現23.1% (12 of 52)的翳狀贅肉組織有8-OHdG的存在，13.5% (7 of 52) 的翳狀贅肉組織有hOGG1的存在。而hOGG1的表現和8-OHdG的表現有統計上的正相關 (p=0.04)。 而正常結膜組織及翳狀贅肉上皮下的結締組織中，則染不到hOGG1蛋白。顯示經紫外線照射在翳狀贅肉會引起DNA氧化性傷害產生8-OH-dG，進而誘發hOGG1之表現。
為了解其他參與DNA修補之基因多型性與翳狀贅肉生成是否有關？以病例控制組研究法 (case-control study)，用restriction fragment length polymorphism (RFLP)方法分析進行XRCC1、XPA和XPD等三種DNA修補基因之基因多型性與翳狀贅肉發生之相關性。結果顯示XRCC1 codon 399的基因多型性，在翳狀贅肉組和正常組達到統計意義。即有一個或二個Gln基因型者發生翳狀贅肉的危險性，是Arg/Arg基因型的3.06倍 (OR=3.06, 95%信賴區間為1.78-5.26)。至於XPA A23G和XPD codon 751的基因多型性在翳狀贅肉組和控制組的分佈差異就沒有統計意義。
Glutathione S-transferase M1 (GSTM1)是一個抗氧化之保護蛋白，同樣以病例控制組研究法來探討GSTM1無效型 (null type) 與翳狀贅肉之相關性。結果顯示在病例和控制組兩組間之GSTM1的無效型和有效型的分佈，並沒有達到統計上的差異，但若以年紀區分，GSTM1無效型在年輕患者與年輕的控制組間，就達到統計上的意義(p = 0.007)，而這樣的差別在老年組中並沒有意義。因此GSTM1的無效型和早發型翳狀贅肉之間有相關性。因此抗氧化保護蛋白在翳狀贅肉形成上的確扮演一些角色，而抗氧化保護蛋白的缺失，容易導致早發型翳狀贅肉的發生。
總之，翳狀贅肉組織中的確實存有氧化壓力所造成之氧化性DNA傷害，這些氧化性傷害可能與該組織之p53基因發生突變有些相關。以下綜合本論文之結果，提示氧化壓力在翳狀贅肉組織引起p53基因突變之可能路徑。
Pterygium is an ocular surface lesion characterized by the encroachment of a fleshy triangle of conjunctival tissue into the cornea. The disease has long been considered as degeneration; however, after abnormal expression of p53 protein being found in the epithelium, more and more researchers feel that pterygium is a tumor.
Though UV irradiation plays the most important role in the pathogenesis of pterygium, the molecular mechanism responsible for the p53 gene mutation in pterygium is unknown. We propose UV causes oxidative stress in cornea and conjunctiva via the production of reactive oxygen species, and oxidative stress plays an importance role in p53 gene mutation in pterygium.
Firstly, we evaluate the prevalence of p53 mutation in pterygium, and whether the mutational spectra are related to oxidative stress. In this study, p53 abnormalities in pterygium were evaluated by DNA sequencing and immunohistochemistry (IHC). IHC staining was performed on 127 pterygial specimens, and DNA sequencing was performed on 51 pterygial specimens. Among the 127 pterygial samples for IHC, there were 29 specimens (22.8%) positive for p53 expression. P53 staining was limited to the nuclei of the epithelial layer. Among the 51 pterygial samples for DNA sequencing, DNA samples were extracted from epithelial cells and subjected to DNA sequencing for examination of mutations in exons 4, 5, 6, 7, and 8 of the p53 gene. Mutations within the p53 gene were detected in 8 pterygial samples (15.7%) with only one mutation found in each sample. All the mutations observed were point mutations, with 6 being substitutions and 2 deletions. Three of the 8 (37.5%) point mutations belong to reported ROS related mutational spectra, including 2 G:C → C:G and 1 G:C → T:A. There was 1 G:C → A:T at dipyrimidine site, which is the UV molecular signature. The other 4 mutations were non-specific.
8-hydroxydeoxyguanosine (8-OHdG) is an oxidative DNA damage and a ubiquitous marker of oxidative stress. Immunohistochemical staining using a monoclonal antibody to 8-OHdG was performed on 52 pterygial specimens and 6 normal conjunctiva. There were 12 (23.1%) pterygial specimens were positive for 8-OHdG staining. The staining was limited to the nuclei of the epithelial layer. All normal controls were negative for 8-OHdG staining. This study represent that there is oxidative stress and oxidative DNA damage in pterygium.
Besides oxidative DNA damage, ROS induces cyclooxygenase 2 (COX 2) formation via activation of NF-kB signaling pathway. We try to evaluate the presence of COX 2 in pterygium to support the hypothesis that there is oxidative stress in pterygium. Immunohistochemical staining using a monoclonal antibody to COX 2 was performed on 90 pterygial specimens and 10 normal conjunctiva. There were 75 (83.3%) specimens were positive for COX-2 staining. COX-2 staining was limited to the cytoplasm of the epithelial layer and predominantly over basal epithelial layer. All normal controls were negative for COX-2 staining. This study shows that there is COX 2 in pterygium and further supports the hypothesis that there is oxidative stress in pterygium.
The human 8-oxoguanine glycosylase (hOGG1) is the key component of DNA damage repair system responsible for the removal of 8-OHdG. We try to evaluate whether there is oxidative DNA repair system responsible for the removal of oxidative DNA damage in pterygium. Immunohistochemical staining using a monoclonal antibody to 8-OHdG and hOGG1 was performed on 52 pterygial specimens and 6 normal conjunctiva. There were 12 (23.1%) pterygial specimens were positive for 8-OHdG staining and 7 (13.5%) specimens were positive for hOGG1 staining. HOGG1 expression was significantly associated with the 8-OHdG positive staining (p=0.04). All normal controls were negative for 8-OHdG and hOGG1 staining. This study represents that UV causes oxidative DNA damage and induces the formation of 8-OHdG in pterygium. Accumulated 8-OHdG induces the expression of hOGG1.
For evaluating the association of genetic polymorphisms of DNA repair system and pterygium formation, we conducted a case control study to evaluate the association between pterygium and XRCC1 codon 399, XPA A23G, and XPD codon 751 polymorphisms by restriction fragment length polymorphism (RFLP) method. In our study, the frequency of the genotypes and alleles of XRCC1 codon 399 polymorphism in the pterygium group and control group were significantlly different (p<0.05). Individuals who carried at least 1 A (Gln) allele had a 3-fold increased risk of developing pterygium compare to those who carried the GG (Arg/Arg) wild-type genotype (OR = 3.06; 95% CI: 1.78-5.26). The frequency of the genotypes and alleles of XPA A23G, and XPD codon 751 polymorphisms in the pterygium group and control group were not different.
GSTM1 is an antioxidant defense enzyme and protects cells against oxidative stress. We conducted a case control study to evaluate the association between pterygium formation and GSTM1 null type. In our study, the percentage of GSTM1 null type in case and control group was not different. After further stratification by age of 60 years, there was a significantly higher frequency of the GSTM1 null genotype in young patients when comparing to the young individuals in control group (p = 0.007). Moreover, all pterygium patients younger than 50 years were found to be GSTM1 null genotype. The difference was not significant in old patients and controls. Hence, GSTM1 null genotype is associated with early onset pterygium. This study indicates that the antioxidant defense enzyme plays a role in pterygium development, and deficiency of antioxidant defense enzyme results in early onset pterygium.
In conclusion, there is oxidative stress and oxidative DNA damage in pterygium. The oxidative stress may be highly involved in the mutagenesis of the p53 gene in pterygium and the development of pterygium. According to our results, we propose a pathway of p53 gene mutation in pterygium. |
