Effects of intracellular free calcium concentration and its manipulation on function and apoptosis of blood and milk neutrophils of lactating goats

嗜中性球扮演著體內抵禦病原菌之重要角色，因此乳汁中嗜中性球成為抵禦病原菌侵入乳房的第一道防線。嗜中性球的活化可藉由細胞內二級訊息傳遞者 (鈣離子)，而啟動細胞內一系列的訊息傳遞路徑，引發自我免疫反應。本試驗之目的在探討調控泌乳山羊之血液及乳嗜中性球之細胞內鈣儲藏量，提升細胞內鈣濃度 (intracellular free calcium concentration; [Ca2+]i) 及活化蛋白質激酶 C (protein kinase C；PKC)對其功能性及凋亡發生之影響，並與 PKC有效活化物phorbol 12-myristate 13-acetate (PMA) 處理作比較。 試驗結果顯示，取自同一羊隻血液及乳中分離出之嗜中性球，以光學顯微鏡及掃瞄式電子顯微鏡觀察，發現乳嗜中性球之核分葉多為4∼5葉，且於質膜外形上出現許多凸起的皺折 (ruffling)，而血液嗜中性球其核葉絕大多為2∼3葉，而質膜外形上則較為完整平滑。利用鈣螢光螯合呈色劑 (Fura 2-AM) 偵測 [Ca2+]i ，結果發現乳嗜中性球基礎鈣濃度 (88.7 ± 26.2 nM) 略高於血液嗜中性球 (71.4 ± 59.1 nM)，但兩者無顯著差異 (P>0.05)。然而，在基礎鈣濃度持平穩後，大約偵測3分鐘後，注入ionomycin (100 nM) 誘導胞器內鈣儲藏量釋放，在乳嗜中性球 [Ca2+]i (691.2 ± 344.2 nM) 上升明顯低 (P<0.05) 於血液嗜中性球 (2381.7 ± 1097.3 nM)。試圖將乳嗜中性球培養於高鈣環境中 (2.5 mM CaCl2之HBSS)， 用以提升乳嗜中性球細胞內鈣儲藏量之，結果發現，並無法提升乳嗜中性球細胞內鈣儲藏量。 以SOD抑制細胞色素c還原反應原理，偵測呼吸爆發，結果發現乳嗜中性球之基礎O2- 生成量 (0.21 ± 0.16 n moles/1×106 cells)，與血液嗜中性球基礎O2- 生成量 (0.32 ± 0.12 n moles/1×106 cells)，並無顯著差異 (P>0.05)。以ionomycin 誘導升高 [Ca2+]i 時，血液嗜中性球其 O2- 生成量 (0.82 ± 0.37 n moles/1×106 cells) 與乳嗜中性球 O2- 生成量 (0.62 ± 0.18 n moles/1×106 cells)，並無顯著差異 (P>0.05)；血液及乳嗜中性球經ionomycin 刺激後，其O2- 生成量均明顯高於 (P0.05) 差異不大。以PMA刺激後，血液嗜中性球之O2- 生成量 (2.22 ± 0.73 n moles/1×106 cells) 明顯高於 (P0.05)。 以瓊脂凝膠電泳分析細胞凋亡方面，結果發現血液及乳嗜中性球之細胞內鈣儲藏量，經ionomycin處理後提升 [Ca2+]i，分別在10小時及6小時後出現典型凋亡之階梯狀DNA (DNA laddering)。而以PMA處理後則在6小時後出現階梯狀DNA。血液及乳嗜中性球以BAPTA-AM和staurosporine處理後，皆會加速細胞凋亡。 利用西方吸漬法偵測其PKC活化程度，結果發現血液及乳嗜中性球經PMA處理後，PKC βII明顯由細胞質轉移至細胞膜上。提升 [Ca2+]i後，血液嗜中性球之 PKC βII 明顯轉移至細胞膜上，但乳嗜中性球之PKC βII之轉移並不明顯，而血液及乳嗜中性球以BAPTA-AM和staurosporine處理後，PKC βII 皆明顯轉移至細胞膜上。 本試驗結果推論，相較於血液嗜中性球，乳嗜中性球鈣儲藏量低、功能性差及提早發生凋亡，可能是由於乳嗜中性球較為老化，以及可能處於部分活化狀態所導致，而且乳嗜中性球的低鈣儲藏量，無法回復至血液嗜中性球之水準。Neutrophil is one of the major effector cells of defence against bacterial infection. Migration of neutrophil into mammary tissue also provides the first line of barrier of the mammary gland. Neutrophil was ativated by intracellular second any messenger calcium to induce self-immunoreaction mediated by intracellular message pathways. The present study investigated whether intracellular free calcium concentration ([Ca2+]i) and its manipulation affects the on function and apoptosis of blood and milk neutrophils of lactating goat. Neutrophils were isolated from blood and milk of goats at peak lactation. Under light microscope, the nucleus of milk neutrophils were of about almost four or five lobes. In contrast, blood neutrophils were of only two or three lobes. With scanning electron microscope milk neutrophils were displayed more villi-like rufflings than blood neutrophils on the plasma membrane. [Ca2+]i of neutrophils was measured by flurocesence dye Fura 2-AM. There were no significant difference (P>0.05) in basal [Ca2+]i of blood and milk neutrophils. After exposing to ionomycin (100 nM), [Ca2+]i of goat blood and milk neutrophils elevated but that of milk neutrophils were significantly lower than blood neutrophils (P<0.05). The present study also incubated milk neutrophils in high calcium medium to investigate the effects of increase calcium storage of neutrophils function. The data shown indicated that no further increase in intracellular calcium storage of milk neutrophils was achieved. SOD-inhibited cytochrome c reaction was used to estimated respiratory burst. We found that was no significant (P>0.05) in superoxide production of blood and milk neutrophils. Stimulation with ionomycin increase (P>0.05) superoxide production of blood and milk neutrophils althaugh they were higher (P0.05) superoxide production of blood and milk neutrophils. Apoptosis of neutrophils was measured as DNA laddering on electrophoresis. Intracellular calcium storage of milk neutrophils treated with both ionomycin and PMA displayed onset of DNA laddering similarly offer 6 hrs. post exposure, while DNA laddering of blood neutrophils occurred at 10 and 6 hrs. respectively, with the same treatments. Therefore BAPTA-AM and staurosporine treated acceleration of apoptosis in blood and milk neutrophils. PKC activation was measured with western blotting. PKC βII was found translocated from cytosol to membrane in both blood and milk neutrophils after PMA treatment. Elevated of [Ca2+]i increased PKC βII translocation in blood neutrophils but no in milk neutrophils. BAPTA-AM and staurosporine treatment did not induce PKC βII translation in blood and milk neutrophils. It is found that, compared to blood neutrophils, milk neutrophils are much less in intracellular calcium storage and functionality. Besides, they are more sensitive to ionomycin-induced apoptosis. The lack of calcium storage in milk neutrophils can not be restored to the level of blood neutrophils by in vitro supplementation in milk neutrophils. It is suggested that milk neutrophils are aged and partially activated.壹、摘 要 1 貳、前 言 3 參、文獻檢討 4 一、山羊乳腺之構成 4 二、山羊嗜中性白血球之生物特性及生理功能 6 (一) 生物特性 6 (二) 生理功能 6 三、嗜中性球細胞內鈣離子之運輸 7 四、鈣離子與凋亡之關係 8 (一) 何謂細胞凋亡與特徵 8 (二) 凋亡細胞的生化變化 11 (三) 細胞凋亡之基因調控 15 1.Bcl家族 15 2.c-myc基因 16 3.p53基因 16 4.Apo-1/Fas 17 5.ICE基因 17 6.Caspase 18 五、嗜中性球之呼吸爆發 18 (一) 何謂活性氧屬 (reactive oxygen species; ROS) 18 (二) 活性氧屬之種類 18 1. 超氧 19 2. 過氧化氫 19 3. 氫氧自由基 19 4. 單重態氧 19 5. 過氧化自由基 19 6. 過氧化硝基 19 (三) NADPH oxidase之調控 19 六、PKC 之簡介 25 肆、材料與方法 30 一、試驗動物及飼養管理 30 二、試驗藥品 30 三、嗜中性球之分離 32 (一)、血液 32 (二)、乳汁 34 (三)、.純度及存活率 34 四、細胞型態之顯微觀察 35 (一)、光學顯微鏡 35 (二)、掃瞄式電子顯微鏡 35 五、嗜中性球 [Ca2+]i 之測定….…………………………………………………36 (一)、原理 36 (二)、步驟 36 (三)、嗜中性球以細胞內鈣螯合劑 (BAPTA-AM) 處理後，再培養不含鈣之DPBS或高鈣緩衝液 (HBSS + 2.5 mM CaCl2) 不同時間，測定其[Ca2+]i 之變化 38 六、嗜中性球呼吸爆發測定 38 (一)、原理 38 (二)、實驗設計 39 (三)、步驟 39 七、DNA laddering之觀察………………………………………………………….39 (一)、原理 39 (二)、實驗設計 39 (三)、步驟 40 1．體DNA之萃取 40 2．DNA電泳 40 八、PKC 之西方吸漬測定 40 (一)、原理 40 (二)、實驗設計 41 (三)、步驟 41 1. 細胞質液 (cytosol) 與細胞膜 (membrane)分離 41 2.細胞質與細胞膜蛋白質定量 42 (四)、SDS-PAGE 電泳分離細胞質及細胞膜蛋白 42 3. 電泳 42 (五)、西方吸漬法 42 九、統計分析 43 伍、結果 44 一、血液及乳嗜中性球顯微形態之觀察 44 二、血液及乳嗜中性球經ionomycin誘導後[Ca2+]i 之差異……………………44 三、BAPTA-AM 處理後，血液及乳嗜中性球 [Ca2+]i 之變化………………..47 四、血液及乳嗜中性球培養於高鈣環境不同時間觀察其 [Ca2+]i之變化…….....51 五、血液及乳嗜中性球 [Ca2+]i 與呼吸爆發之關係……………………………51 六、PKC 活性與血液及乳嗜中性球呼吸爆發之關係 54 七、血液及乳嗜中性球經老化處理，對其存活率之影響 57 八、調控 [Ca2+]i 對於血液及乳嗜中性球凋亡之影響…………………………57 九、活化PKC，對於血液及乳嗜中性球凋亡之影響 59 十、高鈣環境對乳嗜中性球細胞凋亡之影響 65 十一、血液及乳嗜中性球之PKC 受 [Ca2+]i刺激活化後，由細胞質轉移至細胞膜程度之差異……………………………………………………………………..65 十二、血液及乳嗜中性球之PKC 受PMA刺激活化後，由細胞質轉移至細胞膜程度之差異.. 67 陸、討論 70 柒、結論 78 捌、參考文獻 79 玖、英文摘要 8