ALA-PDT诱导C6胶质瘤细胞自噬和胞内Ca^2＋浓度变化的研究

目的探讨5-氨基乙酰丙酸介导的光动力治疗（ALA—PDT）诱导大鼠C6胶质瘤细胞发生的自噬与细胞内游离钙的关系，及自噬特异抑制剂3-甲基腺嘌呤（3-MA）对自噬和胞内游离钙浓度的影响。方法C6细胞经ALA—PDT后不同时间用单丹磺酰戊二胺（MDC）染色和电镜观察其胞内自噬现象，流式细胞仪检测细胞的凋亡率，并测定胞内游离钙离子浓度的变化。结果ALA—PDT诱导C6胶质瘤细胞发生了自噬被观察到，且ALA—PDT后C6胶质瘤细胞凋亡率明显高于对照组（P〈0．05）。与此同时胞内钙离子浓度较对照组明显升高，以ALA—PDT后20min和6h最为明显（P〈0．05）；3-MA可显著抑制ALA—PDT所致的胞内Ca2^浓度升高并能降低凋亡细胞比例（P〈0．05）。结论在体外条件下ALA—PDT可诱导C6胶质瘤细胞发生自噬＋，其发生可能与ALA-PDT导致细胞内游离钙升高有关；3-MA可能通过抑制胞内游离钙的升高而抑制ALA-PDT诱导的细胞自噬。

Effects of 5-aminolaevuliulc Acid-mediated Photodynamic Theraphy on Autophagy and Intracellular Ca2+ Concentration in Vitro In C6 Glioma Cells

Objectives To study the change in the autophagy and intracellular Ca2 concentration induced by 5-aminolaevulinic acid-mediated photodynamic therapy (ALA-PDT), and the effect of 3-methyladenine (3-MA) , a special inhibitor of autophagy on the autophagy and the intracellular Ca2 concentration in C6 glioma cells of rats. Methods The autophagy were observed by fluorescent light microscope (mondonsyl cardeverine staining) and electron microscope. The apoptosis of C6 glioma cells induced by ALA-PDT was determined by flow cytometry and the intracellular Ca2 concentration was measured by confocal laser scanning microscope and flow cytometry 20 minutes, and 1, 3, 6, 12 and 24 hours after ALA-PDT in C6 glioma cells. Results The autophagy occurred in C6 glioma cells after ALA-PDT and did not in the control group. The intracellular Ca2 concentrations in the C6 gliomas cells 20 minutes, and 1, 3, 6, 12 and 24 hours, especially 20 min and 6 hours after ALA-PDT were significantly higher than those in control group (P<0.05). The apoptosis rate of C6 gliomas cells after ALA-PDT was significantly higher than that in control group (P<0.05). The intracellular Ca2 concentration and the apoptostis rate of C6 glioma cells were significantly lower in the treatment group with ALA-PDT plus 3-MA than those in the treatment group with ALA-PDT (P<0.05). Conclusions That the autophagy can be induced by ALA-PDT in vitro may be related to an increase in the intracellular Ca2 concentration induced by ALA-PDT in glioma C6 cells. The ALA-PDT-induced autophagy can be significantly reducec by 3-MA, which may reduced the Ca2 concentration in C6 glioma cells.