粒線體基因突變及氧化傷害與子宮內膜異位症相關性之研究 Mitochondrial DNA mutations and oxidative damages in endometriosis
中文摘要
根據資料顯示婦女罹患子宮內膜異位症的發生率愈來愈高，其發生率約佔婦女
人口的15﹪~50﹪。依據研究顯示，子宮內膜異位症常為女性不孕的主要原因，
大約有50﹪的不孕婦女患有此一疾病。所謂子宮內膜異位症是子宮內膜上皮細
胞及腺體生長於子宮以外的部位。迄今，對於子宮異位性內膜症的致病機轉及成 因尚未清楚。許多學者認為子宮內膜異位症與氧化壓力（oxidative stress）有關，
在異位的組織受到發炎反應影響而有較高的氧化壓力，使oxidatively modified complexes 增加，並產生氧化性傷害(oxidative damage)。此外，在免疫系統上，
經由cytokine 及 chemokine 活化巨噬細胞 macrophage 也會造成發炎反應及增加 氧化性傷害。我們的研究證明在子宮內膜異位症患者的病灶處的檢體中，檢測出 高含量的氧化性傷害物質，如8-OH-deoxyguanosine (8-OHdG)及脂質過氧化物 (lipoperoxide)及粒線體 DNA 突變(mitochondrial DNA mutation)的堆積。尤其於巧 克力囊腫(chocolate cyst)的組織中，檢測出遠高於正常值 5.5 倍的 8-OHdG 含量。
正常人週邊血球為1.66 ± 0.02， chocolate cyst group 則為 9.12 ± 3.22 8-OHdG/10- 5dG。並且檢測出 6.2 倍的 lipoperoxide 含量增加。正常人週邊血球值為 0.18±0.02 pmol/μg protein, chocolate cyst group 為 1.66±0.80 pmol/μg protein。此外，於病灶 處標本中檢測出4977 bp, 7599 bp 及兩種新的粒線體基因斷損突變(5335 bp 及 5756 bp 斷損)。此新的斷損突變經 DNA 序列分析，於 5355 bp deletion 其斷損位 置為nucleotide position (np) 8273 至 np13607，斷損兩側有 5’-CCTATAGCAC-3’
的direct repeat。於 5756 bpdeletion 斷損位置為 np 8065 至 np13820，斷損兩側有 5’-CTT-3’的 direct repeat。異位組織其斷損突變的發生(frequency) 為 41﹪，29﹪
及55﹪(n=23，16 及 31)。巧克力囊腫及子宮腺肌瘤的 8-OHdG 氧化性傷害與 mtDNA deletion 呈正相關性，MDA 與 mtDNA mutation 則無明顯相關性。此外，
我們分析glutathione-S-transferase M1 (GSTM1) 和 catechol-O-methyltransferase (COMT) gene 的基因多型性與子宮內膜異位症之間氧化性傷害的關係。患有子宮 內膜異位症婦女中有48.6﹪(n=18)為 GSTM1 null type。統計後，發現子宮內膜異 位症與GSTM1 null type 並無明顯相關性。而患有子宮內膜異位症婦女的 COMT polymorphism 分別為 GG (43%), AG (43%), 及 AA (14%) 。非子宮內膜異位症者 的基因多型性則為GG (78%), AG (22%), 及 AA (0%)。此種低活性 COMTLL 僅於 患有子宮內膜異位症婦女發現。根據實驗結果顯示，子宮內膜異位組織中存在著
高量的氧化傷害，及粒線體DNA 的斷損突變，我們確認氧化性傷害確實與子宮
內膜異位症的形成有關，而氧化性壓力的來源及粒線體功能的缺損與子宮內膜 異位症的形成機轉需進一步探討，以期能提供更有效的療程。
英文摘要
Endometriosis, one of frequent diseases in gynecology, is a considerable threat to the physical, psychological and social integrity of women. Moreover, up to 50% of infertile patients have this disease .The etiology and pathogenesis of this important disease, defined as the ectopic location of the endometrium-like glandular epithelium and stroma outside the uterine cavity, is poorly understood. To date, however, little is known about the pathogenesis of endometriosis. It still remains an open question as to what extent the factors influences the establishment and/or progression of
endometriosis. As a result of such a stress, a sterile, inflammatory reaction with secretion of growth factors, cytokines, and chemokines is generated, which is
deleterious especially to successful reproduction. In this study, the significantly higher amounts of oxidative damages were detected in endometriotic lesions than in
controlled normal endometrium such as the mitochondrial DNA rearrangement, 8-OH deoxyguanosine (8-OHdG), and lipoperoxide contents. Our central hypothesis
proposes that oxidative damages might be anticipated in the pathogensis of endometriosis. The study was conducted to investigate mtDNA mutation and
oxidative damage in endometriotic tissue of women with endometriosis. Large-scale deletion of mtDNA were detected and occurred at a high frequency in the
endometriotic tissue. We detected four types of mtDNA deletions in the endometriotic tissue such as 4977 bp. 7599 bp, and two novel deletions (5335 bp and 5756 bp).
Using primer-shift PCR and DNA sequencing, we identified and characterized
mutiple deletions of mtDNA in the endometriotic tissue. The 5335 bp deletion, which occurred between nucleotide position (np) 8,273 and np 13,607, was flanked by a 10- bp direct repeat of 5’-CCTATAGCAC-3’. The 5756 bp deletion, which occurred between np 8,065 and np 13,820, was flanked by a 3-bp direct repeat of 5’-CTT-3’.
The propotion of mtDNA was found to correlate positively with of the 8-OHdG. The amount of 8-OHdG in chocolate cyst was significantly higher than the control group (9.12 ± 3.22 vs 1.65 ± 0.02 8-OHdG/10-5dG). Moreover, we found that average contents of lipoperoxide in chocolate cyst were higher than the control group
(1.14±0.68 vs 0.18±0.02 pmol/μg protein). Furthermore, we examined glutathione S- transferase M1 (GSTM1) and catechol-O- methyltransferase (COMT) gene
polymorphism. The frequencies of GSTM1 null genotype were 48.6% and 55.6% for women with or without endometriosis, respectively. We found the higher contents of 8-OHdG and MDA were detected in the tissue with GSTM1 null type. The
frequencies of COMT genotype COMTHH, COMTHL, COMTLL were 43%, 43%, and 14% in the endometriotic tissue. On the other hand, the frequency were and 78%, 22% and 0% in women without endometriosis. The frequency of lowly active
COMTLL was increased in the chocolate cyst. GSTM1 null type and COMT activity
may predisposes to increased oxidative damages to the women with endometriosis.
According to these finding, we suggest that endometriosis leads to mtDNA mutations together with oxidative damage to DNA and lipids.
