粒線體基因與甲狀腺疾病之相關性研究

甲狀腺腫（ goiter ）及良性甲狀腺疾病是普遍盛行的疾病，根據資料顯示，目前約有百分之四的人口有甲狀腺疾病的問題。但是引起甲狀腺腫及甲狀腺增生 (thyroid hyperplasia) 的病因目前仍然不明。粒線體 DNA 被認為比細胞核 DNA 的演化速率快。這其中有許多的原因，包括了粒線體 DNA 是處於很多超氧化物 (superoxide) 存在的環境下增加了氧化損傷及 DNA 突變的機率。由文獻證實粒線體 DNA 突變的累積，與人類的老化及某些癌症的生成有關。而甲狀腺是人體內重要的能量與熱量新陳代謝的主要器官，在粒線體製造能量的氧化還原反應過程中產生大量的超氧化物及游離基，更容易造成甲狀腺內粒線體 DNA 的氧化損傷及粒線體 DNA 的突變產生，而且甲狀腺荷爾蒙 (T3) 可以直接或間接調控粒線體及粒線體 DNA 的表現。所以本論文中我們從署立新竹醫院收集了 40 位甲狀腺腫患者在手術後切下的甲狀腺病灶組織及其手術前和手術後血液和等檢體，以 long extension PCR 、 primer-shift PCR 等方法分析粒線體 DNA 的變異。並從瓊膠作 DNA 擷取 (gel extraction) 分析斷損突變斷損處的 DNA 序列 (DNA sequence) 。並且以 real time PCR 的方法分析 mtDNA 的基因拷貝數（ mtDNA copy number ）的變化。此外，我們並且收集及分析患者的臨床 TSH 、 T3 或 Free T4 的數據。根據實驗結果顯示，在甲狀腺病灶組織中含有三種斷損突變型的粒線體DNA 如 4977 bp 或 7599 bp 或 5335 bp 的粒線體 DNA 斷損突變。而且粒線體 DNA 斷損突變的發生率在甲狀腺乳突性肉瘤的發生率達 67.3 ％，在結節性甲狀腺增生、擴散性甲狀腺增生及惡性乳突性肉瘤分別是 20.0 ％、 37.5 ％及 67.3 ％。我們發現在這些良性甲狀腺增生及甲狀腺癌症患者的手術前和手術後血液檢體中的粒線體 DNA 並無 deletion 的情形。再與病人血液中的甲狀腺荷爾蒙的濃度比對分析後，我們發現粒線體 DNA 的斷損與甲狀腺荷爾蒙的濃度不正常有關。 TSH 異常者 7599 bp deletion 發生率達 62.5 ％， 5335 bp deletion 發生率達 37.5 ％， 4977 bp deletion 發生率達 25.0 ％。 T4 異常者 7599 bp deletion 發生率達 50.0 ％， 5335 bp deletion 發生率達 66.6 ％， 4977 bp deletion發生率達 33.3 ％。 T3 異常者 7599 bp deletion 發生率達 50.0 ％， 5335 bp deletion 發生率達 100.0 ％， 4977 bp deletion 但發生率達 50.0 ％。我們也用 Real-time PCR 的技術來定量偵測粒線體 DNA 的 copy number ，結果顯示病患組織中的粒線體 DNA 的 copy number 比正常血液對照組約減少 52 ％。而且我們也發現，開刀後血液檢體中的粒線體 DNA 的 copy numbers ，比開刀前的約增加了 10.3 ％。顯示開刀治療後，病患的粒線體 DNA 的 copy number 有逐漸恢復的趨勢。但甲狀腺荷爾蒙如何影響粒線體 DNA 斷損的機轉需進一步探討。期望藉由研究良性甲狀腺增生、甲狀腺腫的病人 mt DNA deletion 的結果能有助於了解粒線體 genome 於甲狀腺增生、甲狀腺腫的病理形成機制。

Mitochondrial DNA Mutation in Thyroid Disease

Goiter and benign thyroid disease are a common problem with prevalence about 4 ％ in the general population. The pathogenesis of goiter and gland hyperplasia is still unclear. Human mtDNA is recognized to evolve 10–100 times faster than nuclear DNA with a number of reasons. Those most often reported are the aggressive environment rich in ROS to mitochondrlia and caused oxidative damage of Mitochondrial DNA . Accumlation of human mtDNA mutations has been demonstrated to be an important factor of human aging and degenerative disease. Thyroid hormone exert profound effects on the energy metabolis- m thus extremely vulnerable to oxidative damage by ROS and other free radicals generated as by products in the electron transport chain. It is well defined that thyroid hormone could contribute in the regulation of the expression of mtDNA replication and nucleus encoded mitochondrial protein. Forty patients with thyroid hyperplasia patient from Shin-Chu Hospital were enrolled in this study. We also collected the clinical data of TSH 、 T3 and free T4. The large-scale mtDNA deletion were identified by long-rang PCR, primer -shift PCR and semi-quantitative PCR. Thyroid tissue and blood samples were collected and applied to the analysis of mtDNA mutation .There were three types of mtDNA deletion identified in this study including 4977bp, 7599bp and 5335 bp deletions. The highest frequencies of occurrence in mtDNA deletion were found in the papillary carcinoma is (67.3%). The occurrence of mtDNA deletion was 67.3%, 37.5% and 20.0% in papillary carcinoma, Nodular hyperplasia, and diffuse hyperplasia, respectively. Abnormal hormone level were correlated with the occurrence of mtDNA deletion. TSH level abnormal bearing with 7599bp deletion was about 62.5%, with 5335bp deletion was about 37.5%, with 4977bp deletion was about 25.0%. T4 level abnormal bearing with 7599bp deletion was about 80.0%, with 5335bp deletion was about80.0%, with 4977bp deletion was about 60.0%. The mtDNA copy-number were detected by real-time PCR and we finned that mtDNA copy-number were decreased in tissue sample of patient (52%). The mtDNA copy-number in the blood sample of patient after operation was increase about 10 ％ than patient before operation. It reveals that patient after operation treatment may increase there mtDNA copy-number. We suggest that mutations and/or depletion of mtDNA might play some important role in the progression and pathogenesis of thyroid hyperplasia.