以流式細胞技術評估肌無力病患之免疫功能

1. 以流式細胞技術評估肌無力病患之免疫功能 • 肌無力症(Myasthenia gravis; MG)為器官專一的自體免疫疾病。在病人體內由於存有直接對抗肌膜上的〝乙醯膽鹼接受體(AchR)〞之抗體，以致神經興奮之乙醯膽鹼無法與受體相結合而出現肌肉無力的症狀。此種乙醯膽鹼接受體抗體 (AchRAb)即為引發肌無力症之致病因子。在85%的廣泛型肌無力症病人體內皆可測得此抗體。因此肌無力症之治療方式之ㄧ是以能降低血中乙醯膽鹼接受體抗體為主要目標。 • 本論文目前分析經由血漿分離術 (plasmapheresis)之20位肌無力症病人 (8男:12女)及19位正常志願者 (10男:9女)，以流式細胞儀檢測其淋巴細胞亞群分布（lymphocyte subpopulations）、細胞吞噬活性（phagocytic activity）、自然殺手細胞毒殺能力（natural killer cell activity）、周邊血液單核細胞（peripheral blood mononuclear cells, PBMC）的細胞凋亡比例（apoptosis percentage）以及細胞週期（cell cycle）分布情形，同時和正常人作比對。以淋巴細胞免疫分型法（immunophenotyping）進行淋巴球亞群分布分析，發現肌無力症病患的自然殺手細胞（natural killer cell, NK）高於正常人約1.3倍 (p=0.052) ，特別是男性肌無力病患的自然殺手細胞高於女性約1.61倍 (p<0.050) ，然而男性肌無力病患B細胞 (B cells)較正常人低0.72倍 (p<0.050)。在幫助性T細胞 (T helper cells, Th)方面，單純肌無力無胸腺增生 (hyperplasia)也無胸腺瘤 (thymoma)較正常人、肌無力合併胸腺增生及肌無力合併胸腺瘤分別高1.26倍 (p <0.050) 、1.46倍 (p <0.050) 及1.49倍 (p <0.050) ;在胸腺瘤開刀後較無開刀的病人低0.64倍 (p <0.050) 。有使用藥物的病患較沒有使用藥物的病患在總T細胞(total T cells; T)下降約0.81倍 (p <0.050)。然而在單核球（monocytes）的吞噬活性分析，肌無力病人有較低的趨勢;但在顆粒球（granulocytes）的吞噬活性方面並無明顯差異。在肌無力症具有胸腺增生者較正常人、單純肌無力無胸腺增生也無胸腺瘤病患及肌無力合併胸腺瘤者，分別低0.97倍 (p <0.050) 、0.96倍 (p <0.050) 及0.96倍 (p <0.050)。女性病患、年齡超過40歲、病程小於兩年或非肌眼型的患者，NK細胞活性比正常人皆有上升之趨勢，然而若病患使用免疫抑制藥物之後NK細胞活性則明顯下降(p <0.050)。使用亞二倍體法（hypodiploidy analysis）分析肌無力病患PBMC的細胞凋亡比例較正常人高2.25倍 (p <0.050) ，特別是肌眼型病患較正常人高2.72倍 (p <0.050)。PBMC的細胞週期，S期在肌無力病患上升約3.24倍 (p <0.050) ，特別是肌無力女性病患較男性高3.14倍 (p <0.050)。而使用免疫抑制藥物之後肌無力病患的S期較正常人及未使用藥物病患分別高4.39倍 (p <0.050) 及2.73倍 (p <0.050) ; G0/G1期則較未使用藥物病患低0.92倍(p <0.050)。 • 利用血漿分離術治療肌無力病患，以降低病人體內乙醯膽鹼接受體抗體含量，特別是具有「呼吸窘迫之危象」的病患，可使病患在臨床症狀上快速獲得改善。肌無力病患在血漿分離術治療後，不但 T細胞、幫助性T細胞及Th/Ts ratio下降，而且B細胞、抑制性T細胞及NK細胞上升，但無顯著差異。單核球及顆粒球的吞噬活性方面則無明顯差異。在血漿分離術治療後，NK細胞毒殺力明顯降低。周邊血液單核細胞之細胞凋亡比例肌無力患者血漿分離術治療後明顯上升約2倍(p=0.021) ，以女性患者細胞凋亡比例上升較有統計意義。G0/G1 phase在治療後有下降之趨勢，S phase及G2/M phase則有上升之趨勢，女性病患細胞週期改變較為明顯，但無顯著差異。 • 綜合以上結果，利用流式細胞儀可快速評估肌無力病人的免疫功能。

2. Immune Function Assessment of Myasthenia Gravis Patients Using Flow Cytometry • Myasthenia gravis (MG) is an organ-specific autoimmune disease generally mediated by antibodies against the acetylcholine receptor (AChR) of skeletal muscle, characterized by fatiguable muscle weakness. This impairment is attributed to the presence of autoantibodies directed against the AChR. In 85% of MG patients had raised levels of AChR-specific antibodies by this approach. Therefore, it is an important issue to decrease AChR antibodies for treatment of MG patients.To assess the immune functions of MG patients after plasmapheresis, a total of 20 MG patients (M8: F12) and 19 normal volunteers (M10: F9) was collected and subjected for analysis of their lymphocyte subpopution, phagocytic activity, Natural killer cell activity, apoptosis percentage and cell cycle distribution of peripheral blood mononuclear cell(PBMC) using flow cytometry. Results of lymphocyte subset indicated that the NK cell proportion of MG patients was increased about 1.3 folds (p=0.052) while comparing with normal controls. Specially, MG male NK cells were higher than that of female for about 1.61 fold (p<0.050). In contrast, B cells of MG male were lower than that normal volunteers for about 0.72 fold. For T helper cells, the MG patients without hyperplasia and thymoma were higher than those of normal volunteers, MG patients with hyperplasia, MG patients with thymoma for about 1.26- (p<0.050), 1.46- (p<0.050), and 1.49-folds (p<0.050), respectively. Th cells proportion in MG patients with thymoma operation observed for 0.64 fold (p<0.050) lower than that without operation. A decrease of 0.81 fold (p<0.050) was observed in MG patients treated with drug than that without drug administration. Otherwise, the phagocytic activity of monocyte excerted a trend of decrease than the normal control. NK cell activity of MG patients with female, age over 40, duration two years or without ocular was likely higher than the normal controls but no statistical significantly. Nevertheless, the NK cell activity of immunosuppression- treated patients were significantly decreased than that without drug treatment. The PBMC apoptotic percentage of MG patients was 2.25 folds higher than that of normal controls (p<0.050), especially the ocular-MG patients were 2.72 folds higher than normal controls (p<0.050). For cell cycle analysis, there was a trend that the S phase was 3.24 fold (p<0.050) elevated in MG patients, especially the MG females were high than males for about 3.14 folds (p<0.050). The S phase of drug-treated MG patients was higher than that of the normal controls and the ones without drug treatment for about 4.39-(p<0.050), 2.73-folds (p<0.050). In addition, the G0/G1 phase in MG patients treated with drug was lower than the ones without drug treatment for about 0.92 fold (p<0.050).Double filtration plasmapheresis (DFP) is a useful methoud for reducing the amount of AChR antibodies which is able to improved the MG clinical condition. For lymphocyte subpopulation assay, the total T cells, Th cells, and Th/Ts ratio were decreased, whereas the B cells and NK cells were increased after DFP without statistical significance. After DFP, B cells (p<0.001), Th cells (p<0.010) and Th/Ts ratio (p<0.001) of healthy volunteers were increased, whereas Ts cells (p<0.010), NK cells and total T cells were decreased. Moreover, the apoptotic percentage of PBMC was increased for about two fold (p=0.021) in MG patients after DFP, especially in female patients. For analysis of cell cycle, G0/G1 phase decreased but S phase and G2/M phase increased in MG female.In conclusion, this study established a flow cytometric method which provides a verified assessment for the immune functions of MG patients.