細胞質磷脂水解酵素A2 媒介氧化低密度脂蛋白導致之足細胞凋亡現象

Cytosolic PLA2 Mediates the Apoptosis of Podocytes Induced by Oxidized LDL

鄭玉琪(Yu-Chi Cheng)

指導教授 ： 陳鴻鈞

高雄醫學大學/醫學院/醫學研究所/碩士(2006年)

https://doi.org/10.6832/KMU.2006.00034

摘要

局部節段性腎絲球硬化症（FSGS）已被認為是足細胞受傷害引起，而氧化低密度脂蛋白（OxLDL）被認為是引起FSGS的原因之一，OxLDL及細胞質磷脂水解酵素A2（cPLA2）都會導致細胞凋亡，過去研究發現以OxLDL刺激鼠腎絲球間質細胞後，cPLA2表現會增加。因此，我們欲了解cPLA2是否媒介OxLDL導致之足細胞凋亡現象？本論文分三部份來探討。
首先，由FGSG、微小變化症（MCD）病人及正常腎組織（NC）之腎臟切片，經免疫組織化學染色，結果顯示FSGS病人腎絲球凋亡現象及cPLA2磷酸化情形明顯高於NC，且OxLDL及cPLA2表現也明顯高於MCD病人及NC。此外，依腎絲球細胞型態發現，陽性染色結果同時也分佈於足細胞位置。
其次，以OxLDL刺激鼠足細胞株，發現刺激16小時後，經西方點墨法及流式細胞儀分析，結果顯示鼠足細胞株凋亡增加，cPLA2表現及磷酸化情形也明顯增高。
接著，OxLDL刺激鼠足細胞株前先加入AACOCF3（cPLA2抑制劑）前處理，發現凋亡情況被抑制，所以我們認為OxLDL導致鼠足細胞株凋亡是由cPLA2所媒介。探討其可能機轉，由本研究結果顯示OxLDL刺激鼠足細胞株後，細胞內活性氧族（ROS）增加，粒腺體膜電位下降，並透過粒線體路徑，釋放出cytochrome c進而誘發凋亡現象。另一方面也藉由活化MAPKs中ERK及p38，引起一連串細胞訊息傳導，可能包括細胞存活及凋亡。
結論：綜合以上結果，我們認為確實cPLA2媒介OxLDL導致之足細胞凋亡現象，且OxLDL刺激鼠足細胞株可透過粒線體路徑，釋放出cytochrome c進而誘發細胞凋亡。然而cPLA2在OxLDL刺激導致足細胞凋亡的過程中，扮演何種角色仍有待更進一步實驗來證明。

關鍵字

局部節段性腎絲球硬化症 ； 細胞質磷脂水解酵素A2 ； 足細胞

並列摘要

It has been well established that podocyte injury is the first event in the initiation of focal segmental glomerulosclerosis (FSGS), and oxidized low density lipoprotein (OxLDL) has been identified in the lesions of FSGS. Both OxLDL and cPLA2 have been reported to cause apoptosis. It was described that there is an augmentation of PLA2 activity in mesangial cells by OxLDL. Therefore, we investigated whether OxLDL induce apoptosis and whether cPLA2 mediates the apoptosis of podocytes induced by OxLDL. We have investigated these effects in three different levels.
First, we have demonstrated by immunohistochemistry the presence of BrdU, OxLDL, cPLA2 and phospho-cPLA2 in the renal biopsy samples of patients of FSGS, minimal change disease (MCD) and normal controls. We found that the apoptosis and cPLA2 phosphorylation in the glomeruli of patients with FSGS and MCD were higher than those of normal controls, and the glomerular expression of OxLDL and cPLA2 were significantly higher in patients with FSGS than those of MCD and normal controls.
Second, we stimulated mouse podocyte with OxLDL for 16 hours and then analyzed by Western blot and flowcytometry. We found that there is a significant increase in the apoptosis and cPLA2 phosphorylation level.
Third, there was significant increase of apoptosis when mouse podocytes were pretreated with cPLA2 inhibitor, the AACOCF3. Therefore, we have further investigated whether cPLA2 mediates the apoptosis of podocytes induced by OxLDL. We found that there is significant increase in the intracellular ROS generation, and a significant decrease in mitochondria membrane potential, cytochrome c release, and apoptosis through the mitochondrial pathway. In addition, OxLDL-stimulated mouse podocyte exhibited an increased phosphorylation of ERK and p38, which mediate signal tranductions including survival and apoptosis.
In conclusion, we have demonstrated that cPLA2 mediates the apoptosis of podocytes induced by OxLDL. OxLDL stimulated mouse podocyte induced apoptosis through the mitochondrial pathway.

並列關鍵字

FSGS ； cPLA2 ； podocyte

參考文獻

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