- 學位論文
探討硬皮症相關之自體免疫抗體anti-CENP B和anti-TOPO1對血管內皮細胞之病理作用
Study on the pathological effects of scleroderma-associated autoantibodies, anti-CENP B and anti-TOPO 1, on vascular endothelial cells
摘要
全身性硬化症是一種系統性的自體免疫疾病,其特點包括過度活化的免疫反 應、血管損傷、發炎反應和皮膚及許多體內器官的纖維化,而雷諾氏現象則常見 於此疾病的早發時期。 雷諾氏現象在臨床上是肢端微血管損傷的表現之一,此種疾病會使得病人在 遇到低溫時引起指尖微血管不正常的收縮。它是經由免疫系統作為媒介所造成血 管內皮細胞損傷,諸如淋巴細胞和單核球細胞的單核細胞則會被吸引到結締組 織,造成纖維母細胞的活化、膠原蛋白的沉積所致,固血管收縮或堵塞或引起組 織缺氧及壞死。 雖然系統性硬化症的初始原因目前仍為未知,其產生多種辨認核內抗原的自 體抗體則為此疾病的一個重要特性。在臨床診斷上,系統性硬化症伴隨著雷諾氏 症病人血液中的抗染色體中心結蛋白及拓樸異構酶自體抗體會明顯增加。而這兩 種自體抗體甚至會造成指尖皮膚的壞死。但這些自體抗體引起血管內皮細胞損傷 的相關機制目前仍不明確。 本篇論文以小牛肺動脈內皮細胞來研究這兩種自體抗體對血管生物學的影 響。我們發現當細胞與抗染色體中心節蛋白培養時,生物能量腺苷三磷酸的製造 會減少,同時,細胞產生的乳酸脫氫酶產生則會受到抗體的作用而增加。由結果 得知,細胞與自己抗體共同培養會改變細胞活性和引起細胞壞死。更進一步,我 們以流式細胞儀偵測 Annexin V/propidium iodide (PI)雙染的螢光訊號,顯示以 抗體處裡過的細胞其細胞凋亡的百分比也會增加。同時我們也用β-半乳糖苷酶染 色法和偵測端粒 DNA 序列的長度發現到受抗體處裡過的細胞會變的更容易產生老 化現象。最重要的是,我們發現當細胞受到抗體的處理過後會引起細胞內 p53 蛋 白的表現。這些結果確認了抗染色體中心節蛋白及拓樸異構酶這兩種自體抗體在 系統性硬化症的細胞毒殺角色。同時,這些自體抗體也會引起內皮細胞中 p53 相 關路徑的細胞凋亡及細胞老化現象。
並列摘要
Systemic sclerosis (SSc) is a systemic autoimmune disorder characterized by immune activation, vascular injury, inflammation, and fibrosis in the skin and various internal organs. Raynaud’s phenomenon (RP) is the most common clinical manifestation in the early stage of the disease. RP is a clinical reflection of digital microvascular damage that causing insufficient blood flow in the end of digital and toes in systemic sclerosis (SSc). When the patients suffer from RP, abnormal digital vasoconstriction in response to coldness occurs. It is conceivable that RP is elicited by the immune-mediated vascular endothelial cell injury. Histologically, mononuclear cells such as lymphocytes and monocytes are recruited into the connective tissues, leading to fibroblast activation, collagen accumulation, and tissue hypoxia. Although the initial event leading to the development of SSc is still poorly understood, an important characteristic feature of the disease is the production of a variety of autoantibodies to nuclear antigens. In clinical setting, two unique autoantibodies, anti-centromere (anti-CENP B) and anti-topoisomerase1 (anti-TOPO1), are found elevated in SSc sera concomitantly with RP or even digital skin necrosis. However, the real mechanism of the correlation among autoantibodies and vascular endothelial cell damage remains unclear. Here, we use the calf pulmonary artery endothelial cells (CPAE) as a model to clarify the affections of these two autoantibodies on vascular biology. We found that the adenosine triphosphate(ATP) production was significantly decreased when cells were incubated with anti-CENP B. Besides, the amount of lactate dehydrogenase (LDH) was increased under treatment of these antibodies. These results suggested that the incubation of autoantibodies with CPAE would destroy cell function and induce cell necrosis. Moreover, the flow cytometrical analysis by annexin V/PI double staining demonstrated that the percentage of apoptotic cell was increased upon antibody treatment. Also, we noticed that the antibody-treated cells tended to become senescence as detected by the senescence-associated β-gal staining and telomere DNA measurement. For elucidating the molecular basis of cell damage, we noted that the antibodies would induce p53 expression in the cells. In conclusion, our data revealed the cytotoxic roles of anti-CENP B and anti-TOPO 1 antibodies on vascular endothelial of patients with SSc through p53 related signaling pathway.