臺灣博碩士論文加值系統

水解磷酸脂包括水解磷脂酸（lysophophatidic acid，LPA）和鞘胺醇1-磷酸鹽（sphingosine 1-phosphate，S1P）等，為具有細胞多樣性作用之脂質衍生物。本實驗室之前的結果顯示水解磷酸脂能減少牛腎上腺嗜鉻細胞中的鈣離子電流，在此報告中，我們將重點放在水解磷酸脂對於鈣離子倚賴性鉀離子（BK）通道以及胞吐胞吞作用的影響。首先針對BK通道不同選擇性剪接異形體設計專一性引子，利用反轉錄聚合酶連鎖反應，結果發現嗜鉻細胞中，至少有四種異形體。將嗜鉻細胞以穿孔性細胞膜組態，將電位箝制在-70 mV，隨後紀錄去極化至+80 mV時的向外鉀離子電流。結果顯示54.1 ± 5.3%的向外電流可被BK通道專一性抑制劑iberiotoxin所抑制，但在S1P或LPA一小時前處理後，能被抑制的電流分別降為27.7 ± 10.7%和26.3 ± 7.6%。改以全細胞組態，直接提高胞內鈣離子緩衝容量，會影響BK通道電流大小，但不改變BK電流比例；若直接將胞內鈣離子濃度上升至1 μM，所引發的向外電流則不會被水解磷酸脂所影響。為進一步瞭解水解磷酸脂是否也會影響胞吐胞吞作用，以穿孔性膜箝制技術，紀錄細胞電容值以代表胞吐胞吞的變化，並利用伏安法（voltammetry)偵測分泌膜囊釋放的動態性質，以水解磷酸脂前處理一小時的細胞，突波形狀較為狹窄且面積減小，顯示水解磷酸脂減少囊泡釋放容量；而clathrin的分佈，在水解磷酸脂處理後，較少的clathrin凝集光點分布於細胞膜周邊區域。本研究結果顯示水解磷酸脂藉由抑制鈣離子電流而間接調控BK通道活性，亦會影響胞吐胞吞作用。

Lysophospholipids（LPLs）, including lysophosphatidic acid（LPA） and sphingosine 1-phosphate（S1P）, are lipid derivatives with versatile cell functions. Our previous results have shown that LPLs attenuated the Ca2+ currents in bovine chromaffin cells. In this report, we are focused on their effects on Ca2+-activated K+ （BK）channels and exo/endocytosis. Using specific primers against BK channel alternative-splicing isoforms, four isoforms were identified using RT-PCR. Chromaffin cells were voltage-clamp in perforated patch configuration and the outward K+ currents at +80 mV were recorded. 54.1 ± 5.3% of the outward current was inhibited by BK channel specific inhibitor, iberiotoxin. The toxin sensitive currents were reduced to 27.7 ± 10.7 and 26.3 ± 7.6% of the total outward current by S1P and LPA treatment, respectively. By patching the cell in whole-cell mode, increasing Ca2+ buffering capacity affected BK current amplitude, but not the ratio to total current. However, BK evoked by elevating the [Ca2+]i to 1 μM was not affected by S1P treatment. To characterize the roles of LPLs on exo/endocytosis, cells were voltage-clamped in perforated patch mode and the membrane capacitance was recorded to reflect the changes in the exo/endocytosis. To monitor the release kinetics from each secretary vesicle, voltammetry was applied. The results showed that LPL treatment sharpens the spikes and reduces spike area, indicating that LPLs decrease vesicle quantal content. The distribution of clathrin at the subplasmalemma region was less in LPL treated groups. These results suggest that LPLs indirectly modulate BK channel activity by attenuating the Ca2+ currents and modify the exo/endocytosis kinetics.

摘要 4
Abstract 5
目錄 6
1. 前言 8
1.1水解磷酸脂之一般細胞生理功能 8
1.2水解磷酸脂對於細胞離子通道的影響 10
1.3水解磷酸脂對於細胞內鈣離子濃度的調控 11
1.4鈣離子倚賴性鉀離子通道 12
1.5嗜鉻細胞胞吐胞吞作用 14
1.6實驗目標 15
2. 材料與方法 17
2.1溶液與化學藥品 17
2.2初級牛腎上腺嗜鉻細胞培養 18
2.3藥物配製與處理 19
2.4電生理 19
2.5 RNA萃取與cDNA製備 21
2.6聚合酶連鎖反應 21
2.7細胞免疫螢光染色 22
2.8資料分析 22
3. 結果 23
3.1 Cd2+抑制向外鉀離子電流 23
3.2提高胞外鈣離子濃度增加向外鉀離子電流 23
3.3向外鉀離子電流大部分由BK通道貢獻 24
3.4嗜鉻細胞中至少表現四種BK通道a subunit剪接異變體 24
3.5 S1P減弱BK電流 25
3.6 LPA減弱BK電流 25
3.7以BAPTA提高胞內鈣離子緩衝容量影響BK通道電流 26
3.8在全細胞膜箝制組態下LPL不影響BK通道活化 27
3.9 S1P與LPA影響細胞胞吐胞吞作用 28
3.10 S1P和LPA影響細胞鈣離子內流之動力學性質 29
3.11 S1P與LPA減小囊泡釋放之量子容量 29
3.12 S1P與LPA抑制細胞胞吞作用進行 30
4. 討論 32
4.1牛腎上腺嗜鉻細胞中的BK電流成分 32
4.2 LPL對於離子通道的調控 33
4.3 LPL對於細胞胞吐胞吞作用的調控 35
4.4 LPL作用對於嗜鉻細胞的生理意義 37
5. 參考文獻 39
6. 表 46
7. 圖解 48
8. 圖 50

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