以流式細胞技術評估人類周邊血液白血球之免疫功能
Assessment of Human Immune Functions for Peripheral Blood Leukocytes Using Flow Cytometry
中文摘要
白血球（leukocytes）於人體防禦機制上扮演重要的角色，也與生理調控、發 育及免疫反應有關。在台灣地區，白血球免疫功能的評估方法及參考值
（reference values）仍無定論。為提供給臨床及研究單位準確評估國人的免 疫功能，本論文使用具有標準化、便利且低成本的流式細胞技術（flow
cytometry）平台開發系統性評估周邊血液白血球（peripheral blood leukocytes）免疫功能的方法。
本論文共分析北台灣地區212 位自願者的周邊靜脈血液，經剔除白血球數異常
及帶有疾病的個案後，共收納141 位 7 到 83 歲健康受測，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。以淋巴細 胞免疫分型法（immunophenotyping）進行淋巴球亞群分布分析，發現總 T 淋巴球（total T cells）、輔助性 T 細胞（T helper cells, Th）、抑制性 T 細胞
（T suppressor cells, Ts）、B 淋巴球（B cells）及自然殺手細胞（natural killer cells, NK）的相對比例（relative counts）依序為 56.0-85.4%、21.0- 53.3%、13.7-42.4%、4.6-23.0%及 4.7-31.0%；其絕對計數（absolute counts）分別為 660-2263 /mL、358-1151 /mL、213-981 /mL、59- 435 /mL 及 66-714 /mL，而 Th/Ts 的比值為 0.7-2.7。以標示螢光的大腸桿菌 K12 菌株進行嗜中性白血球（neutrophils）及單核球（monocytes）的吞噬 活性分析，結果分別為88.5- 99.8%及 64.3- 97.2%。以 K562 細胞株測試 NK 細胞活性，在作用細胞與標靶細胞比例為 20:1 時的毒殺力為 2.2-33.9%。
使用亞二倍體法（hypodiploidy analysis）分析 PBMC 的細胞凋亡比例為 0.2-6.5%。分析 PBMC 的細胞週期，G0/G1、S 及 G2/M 期所占的比例依序為 93.9-100.0%、0-4.5%及 0-2.0%。各項實驗結果以 SPSS 軟體進行數值運算，
並以單因子變異數分析（one-way ANOVA analysis）、無母數統計分析法
（non-parametric statistical analysis）比較年齡及性別對白血球免疫功能 所造成的差異，以雙向邏輯式迴歸分析（binary logistic regression test）
比較高危險群與健康對照組的差別。
依統計結果分析，女性在Th 細胞分布相對值（p = 0.004）、Th/Ts 比值（p
= 0.021）、嗜中性白血球的吞噬能力（p = 0.032）較男性為高，具有統計學 上的意義；隨著年齡增加，NK 細胞相對值（p = 0.001）、PBMC 細胞週期中 的G0/G1 期呈現顯著的增加（p = 0.004），而 S 期則顯著的下降（p =
0.007）；自然殺手細胞的毒殺力、PBMC 的細胞凋亡情形則不受性別及年齡的 影響。利用本實驗所整合建立的免疫細胞評估方法，以雙向邏輯式迴歸試驗分析 21 名白血球數異常及已知帶有痼疾的的受測者，探討健康個體與危險群之間的 模式分析，發現本實驗方法可有效評估健康人與危險群的差別，做為評估人類 周邊血液白血球免疫功能的篩檢工具。此外，將本論文結果與其他已發表的報告 相比較，顯示白血球的免疫功能與種族、性別、年齡及環境因素有密切關係。這 些結果或許可以作為評估國人白血球免疫功能的參考值。
英文摘要
Leukocytes play a key role in host defense, and are associated with physical regulation, development, and immune response. However, the reference values of immune functions for leukocytes remain inconclusive in Taiwan. In this study, we established a series of flow cytometric methods with the characteristics of
standardization, convenience, and low cost to assess human immune functions for peripheral blood leukocytes.
Peripheral whole blood samples from 212 volunteers were obtained by venipuncture in north Taiwan. Abnormal WBC counts and concomitant medications of subjects were excluded from further analysis. Here, a total of 141 healthy subjects with age ranging from 7 to 83 years old were studied for measurement of their lymphocyte subpopulations distribution, phagocytic activity, natural killer cell activity, apoptotic percentage and cell cycle distribution of peripheral blood mononuclear
cells（PBMC）. The lymphocyte immunophenotyping was employed to determine the relative counts of lymphocyte subpopulations, total T cells, T helper cells（Th）, T suppressor cells（Ts）, B cells, and natural killer cells（NK）were 56.0-85.4%, 21.0-53.3%, 13.7-42.4%, 4.6-23.0%, and 4.7-31.0%, respectively, as revealed by immunophenotyping. The absolute counts were 660-2263/mL, 358-1151/mL, 213- 981/mL, 59-435/mL, and 66-714/mL, respectively. Accordingly, the ratio was calculated as 0.7-2.7%. Based on the ingestive capacity of fluorescien-labeled E.coli K12. The phagocytic activity of neutrophils and monocytes were 88.5-99.8 % and 64.3-97.2 %, respectively. In addition, K562 cells were used as target cells for
determing the NK cell activity. The results indicated the cytotoxic activity of NK cells was 2.2-33.9% by the effector cells to target cells ratio of 20:1. On the other hand, using hypodiploidy analysis with PI staining, the apoptotic content of PBMC was 0.2- 6.5 %. The percentage of G0/G1, S, and G2/M phase were 93.9-100.0%, 0-4.5%, and 0-2.0%, respectively, as revealed by PI staining and ModFit analysis. Statistical analysis was made using the SPSS package. One-way ANOVA analysis and non- parametric statistical analysis was used to determine the differences among age and sex groups. Using binary logistic regression test to discriminate the at-risk and healthy
subjects.
According to the results, females had significantly higher percentage of Th cell subset（p = 0.004）, Th/Ts ratio（p = 0.021） and the phagocytic activity of
neutrophils（p = 0.032） than males. The percentage of NK cell subset（p = 0.001）
and the G0/G1 phase of PBMC’s cell cycle（p = 0.004） were showed to increase significantly with age. In contrast, the S phase was found to decline with age（p = 0.007）. However, there were no age- and sex-related significant differences in NK cell activity and apoptosis of PBMC. To investigate the differences between healthy and high risky populations, 21 blood samples obtained from patients with abnormal WBC counts suffering from several diseases were analyzed using binary logistic regression test. These results demonstrated that the established flow cytometric methods in this study could be served as a useful diagnostic tool to assess the immune functions of PBMC. This observation supports the notion that race, sex, age and environment could be responsible factors contributing to the immune functions in healthy humans while comparison with reports. Taken together, the results of this thesis might serve as reference values of immune status for the Taiwanese population.
