CAJ | 학술논문

目的：探讨外源性三磷酸腺苷（ ATP）诱导PC12细胞的膜孔形成及关键分子靶标。方法：用不同浓度的ATP处理培养的PC12细胞，采用倒置相差显微镜观察形态，CCK-8法检测细胞存活率，YO-PRO-1染色检测细胞膜通透性，Western blotting和real-time PCR检测P2X7受体和pannexin 1（Panx1）表达的变化。结果：（1）ATP（1 mmol/L、3 mmol/L、5 mmol/L）作用3 h，可见随着ATP浓度升高，PC12细胞变圆，脱壁细胞增多；当ATP浓度为3 mmol/L或5 mmol/L时，PC12细胞活力较对照组显著下降（P＜0．05）。（2）不同浓度的ATP（0、1、3、5 mmol/L）作用1 h，PC12细胞摄入YO-PRO-1的荧光强度随着浓度增加而增加；同一浓度的ATP作用不同时间（15、30、60 min），随着时间的增加，胞内的荧光强度也增加。（3）亮蓝G（P2X7受体的抑制剂）预处理可明显拮抗ATP引起的细胞活力下降和胞内荧光强度增强（P＜0．05），而生胃酮（Panx1的抑制剂）预处理不改变细胞活力和胞内的荧光强度（P＞0．05）。（4）ATP作用3 h使PC12细胞 P2X7受体的mRNA和蛋白表达明显升高（P＜0．05），而Panx1 mRNA和蛋白表达变化不大（P＞0．05）。结论：胞外高浓度ATP引起PC12细胞的膜孔形成可能主要与P2X7受体的表达和激活有关。
목적：탐토외원성삼린산선감（ ATP）유도PC12세포적막공형성급관건분자파표。방법：용불동농도적ATP처리배양적PC12세포，채용도치상차현미경관찰형태，CCK-8법검측세포존활솔，YO-PRO-1염색검측세포막통투성，Western blotting화real-time PCR검측P2X7수체화pannexin 1（Panx1）표체적변화。결과：（1）ATP（1 mmol/L、3 mmol/L、5 mmol/L）작용3 h，가견수착ATP농도승고，PC12세포변원，탈벽세포증다；당ATP농도위3 mmol/L혹5 mmol/L시，PC12세포활력교대조조현저하강（P＜0．05）。（2）불동농도적ATP（0、1、3、5 mmol/L）작용1 h，PC12세포섭입YO-PRO-1적형광강도수착농도증가이증가；동일농도적ATP작용불동시간（15、30、60 min），수착시간적증가，포내적형광강도야증가。（3）량람G（P2X7수체적억제제）예처리가명현길항ATP인기적세포활력하강화포내형광강도증강（P＜0．05），이생위동（Panx1적억제제）예처리불개변세포활력화포내적형광강도（P＞0．05）。（4）ATP작용3 h사PC12세포 P2X7수체적mRNA화단백표체명현승고（P＜0．05），이Panx1 mRNA화단백표체변화불대（P＞0．05）。결론：포외고농도ATP인기PC12세포적막공형성가능주요여P2X7수체적표체화격활유관。
AIM:To investigate the formation of membrane pore in PC 12 cells induced by exogenous adenosine triphosphate ( ATP) and to identify the key molecular targets .METHODS:PC12 cells were treated with different concen-trations of ATP to establish the injury model .The morphological change was observed under an inverted phase -contrast mi-croscope.The viability of the PC12 cells was measured by CCK-8 assay.Fluorescent dye YO-PRO-1 was used to detect the membrane permeability.The expression of P2X7 receptor and pannexin 1 (Panx1) at mRNA and protein levels was as-sessed by real-time PCR and Western blotting .RESULTS:After exposed to ATP (1 mmol/L, 3 mmol/L and 5 mmol/L) for 3 h, the PC12 cells became edematous , and the number of adherent cells decreased gradually in a dose-dependent man-ner .The cell viabilities in 3 mmol/L ATP group and 5 mmol/L ATP group were significantly decreased compared with con-trol group (P<0.05).YO-PRO-1 uptake in the PC12 cells exposed to ATP (0, 1, 3 and 5 mmol/L) for 15 min, 30 min and 60 min increased in a dose-dependent and time-dependent manner .The cell viability increased and the intracellular fluorescence intensity induced by ATP were significantly antagonized in brilliant blue G ( a P2X7 receptor inhibitor ) pre-treatment group (P<0.05), whereas it did not change in carbenoxolone (a Panx1 inhibitor) pretreatment group (P>0. 05).The expression of P2X7 receptor at mRNA and protein levels was significantly increased (P<0.05), but the expres-sion of Panx1 was not changed ( P>0.05) when PC12 cells were exposed to ATP for 3 h.CONCLUSION:Extracellular ATP at high concentration may induce membrane pore formation with the expression and activation of P 2X7 receptor in PC12 cells.