摘要
背景與目的：後天免疫系統的功能會因為急性的劇烈運動和重度低氧而受到暫時性缺損的影響。相反地，慢性的常壓運動卻能藉由改善淋巴球的相關免疫功能而達到減少傳染病的危險性。然而，目前有關長期低氧運動訓練對於淋巴球功能的影響仍處於尚待瞭解的階段。本研究的目的便是去觀察低氧運動訓練如何去影響淋巴球凋亡與再分佈的情況，且進一步去瞭解其背後的機制。
方法：收取六十位健康坐式生活的健康男性，將他們分為五組: 1.絕對作功量組（15%的氧濃度下，運動至60%的最大作功量，60% maximal workload），2.相對作功量組（15%的氧濃度下，運動至60%的最大心跳儲備量，60% maximal heart rate reserve）；3.低氧訓練組 (約海拔3000公尺，15%氧濃度)，4.運動訓練組 (運動至60%的最大心跳儲備量，60% maximal heart rate reserve )，5.控制組。訓練為期四週，每週五天，一天30分鐘。訓練的前後各做一次最大運動測試，並於測試的前、後、及後兩個小時抽血離出淋巴球做檢測。除基礎值外，於淋巴球中會加入雙氧水以模擬病態的氧化壓力環境。最後使用流式細胞儀來檢測以下參數：淋巴球磷脂質外翻（phosphotidyl serine）、粒腺體膜電位的變化、凋亡蛋白酵素（caspase）3、8、9的活化、硫醇類的消耗（Thiol）、淋巴球（CD3、CD4、CD8）的再分佈、淋巴球表面蛋白（CD28、CD45RA、CD45RO、CD62L、CD11a、CD57、KLRG1）的表現狀況。
結果：劇烈運動過後，淋巴球無論是在基礎值或是在加入雙氧水的病態環境下，膜外翻的情況會增加、粒腺體穿膜電位流失變大、凋亡蛋白酵素的活化提高、硫醇類消耗提高、CD4/CD8降低、老化的T細胞增多。低氧運動訓練過後，在淋巴球凋亡的結果方面，細胞的存活度在基礎值及加入雙氧水的病態環境下都有提昇的現象，淋巴球的凋亡壞死程度則有下降的情況，且T細胞中代表功能性分子的CD28有提昇情形，代表老化分子的KLRG1出現下降的現象。中度運動訓練過後老化的T細胞雖然有增多的情形，但不影響其淋巴球存亡。低氧訓練組與控制組在訓練前後沒有明顯變化。
結論: 本實驗所使用的低氧運動訓練劑量、中度運動劑量、低氧訓練劑量並不會對劇烈運動刺激的後天免疫產生免疫壓抑的影響，且在低氧運動訓練組還有免疫功能改善的情形，使用劑量較輕的相對作功強度組能達到與絕對作功強度組的相同效果。
臨床意義：本篇研究預期能決定一個合適的低氧運動訓練處方，且使其能有效地去減輕氧化壓力所誘發的後天免疫壓抑的狀況。

Abstract
Background and Purpose: Various adaptive immune cell functions are temporarily impaired following acute bouts of intense exercise and severe hypoxia. Conversely, chronic exercise under normoxic condition can reduce the risk of infective disease by improving lymphocyte-related immune function. However, the effect of the hypoxic exercise training on lymphocyte function remains unclear. This study investigates how hypoxic exercise training affects oxidative stress-induced lymphocyte apoptosis and further explores its underlying mechanisms.
Methods: Healthy sedentary young men were trained on a bicycle ergometer, engaging five groups in a normobaric hypoxia chamber: two hypoxic exercise training group (i.e., 15% O2 with 60%VO2max and 60%HRR), hypoxic training group (i.e., 15% O2), exercise training group (60%VO2max), and control group. Duration of training is 30 minutes per day, 5 days per week for 4 weeks. Sublethal oxidative stress was administered by treating the lymphocyte with H2O2, to closely approximate in vivo pro-oxidative conditions. Lymphocyte apoptotic events that included caspase 3, 8 and 9 activation, mitochondrial transmembrane potential (MTP) change and phosphotidyl serine (PS) exposure, as well as the expressions of CD marker on the lymphocytes(CD3, CD4, CD8) that included CD28, CD45RO, CD45RA, CD62L, CD11a, CD57, KLRG1 were evaluated by flow cytometry. Results: Before the training, strenuous, acute exercise enhanced H2O2-induced PS exposure of lymphocyte, which was accompanied by a decline in MTP and an increase in active-form caspase 3, 8 and 9 contents in lymphocyte. Moreover, there is more aged T cell circulating in the peripheral blood stream. However, 4 weeks of hypoxic exercise training attenuated H2O2-induced lymphocyte apoptotic response during strenuous, acute exercise. Furthermore, the expressions of CD marker represent that there will be more activated and less aged T cell circulating in the peripheral blood stream followed by the 4 weeks of the hypoxic exercise training. Although 4 weeks of moderate exercise training recruit more aged T cell, the lymphocyte survival and apoptosis are not affected. After 4 weeks of hypoxia training and control group, there is no difference of lymphocyte apoptosis and the expressions of CD marker.
Conclusion: The regimen of the hypoxic exercise training, moderate exercise training and hypoxia training don’t harm the adapted immune function. There is even an improvement of lymphocyte function by hypoxic exercise training. And choosing the more light dose of the relative workload could achieve the same effect of the absolute workload.
Clinical Relevance: These experimental findings help to determine a hypoxic exercise regimen that modulates lymphocyte function, effectively depress the exercise induced adaptive immunosuppression under oxidative stress.
Key Words: hypoxia, exercise, training, redox status, apoptosis, lymphocyte, phosphatidylserine, mitochondrial transmembrane potential, caspase, thiol, CD marker

目錄
指導教授推薦書
口試委員會審定書
長庚大學博碩士論文著作授權書 ii
誌 謝 iv
中文摘要 vi
英文摘要 viii
目錄 x
第一章 緒論 (Introduction) - 1 -
第一節 研究背景及目的 - 1 -
第二節 研究假設 - 3 -
第二章 文獻回顧 (Literature Review) - 4 -
第一節 壓力 - 4 -
第二節 壓力賀爾蒙 - 4 -
第三節 後天性（專一性）免疫系統 - 5 -
第四節 運動對後天免疫的影響 - 7 -
A. 氧化壓力造成細胞凋亡 - 8 -
B. 壓力賀爾蒙導致細胞再分佈 - 12 -
a. 分化後的T細胞分佈與比例 - 12 -
b. T細胞表面分子的表現 - 13 -
c. T細胞的歸巢（homing） - 14 -
C. 運動時的抗發炎反應 - 16 -
第五節 低氧對後天免疫的影響 - 18 -
第三章 實驗設計 - 20 -
第一節 研究對象(Subject) - 20 -
第二節 研究設計(Study design) - 20 -
第三節 研究器材(Study equipments) - 21 -
A. 試劑（Reagents） - 21 -
B. 儀器（Instruments） - 23 -
第四節 研究步驟(Study protocol) - 24 -
純化淋巴球 (Lymphocyte Isolation) - 25 -
偵測粒線體膜電位 - 26 -
偵測細胞膜phosphatidylserine residues (PS) 外翻試驗- 26 -
Active caspase-3, 8, 9 試驗 - 27 -
偵測細胞所表現的Thiol - 28 -
模擬病態高氧化壓力環境下，淋巴球細胞凋亡的試驗 - 28 -
偵測細胞表面CD marker的試驗 - 29 -
第五節 資料擷取與統計分析 - 30 -
第四章 結果 - 32 -
第一節 受測者身體測量、健康狀態及活動情形調查 - 32 -
第二節 不同訓練介入對最大運動表現的變化 - 33 -
第二節 不同訓練介入對於淋巴球的變化 - 33 -
第三節 不同訓練介入對於淋巴球硫醇含量的影響 - 34 -
第四節 不同訓練介入對於淋巴球凋亡的變化 - 34 -
第五節 不同訓練介入對於淋巴球中粒線體膜電位的變化 - 36 -
第六節 不同訓練介入對於淋巴球中凋亡蛋白3、8、9的活性變化- 36 -
第七節 不同訓練介入對於淋巴球族群再分佈的影響 - 39 -
第八節 不同訓練介入後對於淋巴球表面CD marker的表現變化- 40 -
第五章 討論 - 46 -
不同訓練介入下的淋巴球數目變化情形 - 46 -
不同訓練介入下的淋巴球硫醇類的變化情形 - 47 -
不同訓練介入下的淋巴球凋亡的現象 - 49 -
不同訓練介入下的淋巴球再分布的情形 - 52 -
第六章 結論 - 55 -
圖表附錄 - 57 -
參考文獻 (References) - 92 -
附錄 - 102 -

圖表目錄
表一 在不同的低氧運動劑量介入中，發現在60%最大攝氧量的運動合併15％氧濃度的低氧環境，能使後天免疫壓抑的現象降到最低。 - 57 -
表二 五組受試者基本資料。 - 58 -
表三 受試者於不同訓練介入前、後之最大運動測試結果。 - 59 -
表四 不同訓練介入對劇烈運動之淋巴球數目、T細胞(CD3、CD4、CD8total、CD8bright)數目、CD4/CD8變化的影響。 - 60 -
表五 不同訓練介入對劇烈運動之淋巴球硫醇含量變化的影響 - 61 -
表六 不同訓練介入對劇烈運動之淋巴球存活、凋亡(early apoptosis)、壞死(later apoptosis)的影響。 - 62 -
表七 不同訓練介入對劇烈運動之淋巴球粒線體膜電位變化的影響。- 63 -
表八 不同訓練介入對劇烈運動之淋巴球凋亡蛋白3、8、9含量變化的影響- 64 -
表九 不同訓練介入對劇烈運動之T細胞CD28含量變化的影響。 - 65 -
表十 不同訓練介入對劇烈運動之T細胞CD45RO含量變化的影響。- 66 -
表十一 不同訓練介入對劇烈運動之T細胞CD45RA含量變化的影響。- 67 -
表十二 不同訓練介入對劇烈運動之T細胞CD62L含量變化的影響。- 68 -
表十三 不同訓練介入對劇烈運動之T細胞CD11a含量變化的影響。- 69 -
表十四 不同訓練介入對劇烈運動之T細胞CD57含量變化的影響。- 70 -
表十五 不同訓練介入對劇烈運動之T細胞KLRG1含量變化的影響。- 71 -
表十六 劇烈運動後，淋巴球凋亡程度變化的總表。 - 72 -
表十七 不同訓練介入後對劇烈運動之淋巴球凋亡程度變化的總表。- 73 -
表十八 劇烈運動後，淋巴球所表現CDmarker變化情形的總表。- 74 -
表十九 不同訓練介入對劇烈運動之T細胞表現CDmarker變化情形的總表 - 75 -
圖一 低氧造成體內平衡破壞的機制 - 76 -
圖二 暴露在低氧環境的急性期與慢性期相對應的壓力賀爾蒙 - 77 -
圖三 J型曲線及倒J型曲線解釋運動強度對免疫功能及致病率的關係- 78 -
圖四 劇烈運動會造成免疫壓抑,中度運動則產生輕微免疫刺激。- 79 -
圖五 活性氧藉由離子、基因、酵素等機制來影響細胞的基本表現，特別是在細胞的生存/死亡週期而言。 - 80 -
圖六 粒腺體產生及代謝活性氧的過程。 - 81 -
圖七 細胞凋亡與壞死之型態變化比較。 - 82 -
圖八 劇烈運動過後所引發細胞凋亡的路徑，分為內在路徑與外在路徑，劇烈運動時GSH產量下降，而中度運動時會產生GSH抵抗氧化壓力。- 83 -
圖九 TN從胸腺釋放出來後，與TCM會migrate到小腸淋巴結(peyers patch)、脾臟、腸繫膜淋巴結、週邊淋巴結去儲存。而TEFF、TEM與部分的TCM則由次級淋巴組織釋放到血液中，在血液中循環，等待抗原與其反應。 - 84 -
圖十 老鼠運動後肺部、血液、Peyer’s Patch、骨髓中有明顯增加的現象，而運動後T淋巴球在脾臟則有分佈減少的狀況。 - 85 -
圖十一 當淋巴球在血管中預備要穿出內皮細胞時的移行動作。- 86 -
圖十二 運動時所產生的細胞激素變化，IL-6會上升，隨後會誘發IL-10及IL-1ra等抗發炎激素。 - 87 -
圖十三 運動會影響壓力賀爾蒙及促發炎激素形成，導致抗發炎激素產生以及TH細胞激素的變化，進而影響身體免疫及發炎功能。 - 88 -
圖十四 低氧情況下造成的淋巴球數目的變化有相類似運動的影響。- 89 -
圖十五 低氧加上運動可能對免疫有增強刺激的結果。 - 90 -
圖十六 實驗流程設計。 - 91 -

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