血卟啉甲醚-PDT诱导C6胶质瘤细胞凋亡机制的研究

C6 Glioma Cell Apoptosis Induced by Hematoporphyrin Monomethyl Ether-PDT and Mechanism

目的探讨血卟啉甲醚-光动力学疗法(PDT)诱导C6胶质瘤细胞的凋亡机制。方法常规传代培养C6胶质瘤细胞系,取处于对数生长期细胞按实验要求分组接种于96孔培养板上,应用光敏剂血卟啉甲醚,以不同照光剂量(40、80、120、160、200、240、280和320J/cm2)处理C6胶质瘤细胞。MTT法检测不同照光剂量的细胞死亡率;电镜观察PDT后C6细胞形态学变化;以Fluo-3/AM为细胞内钙离子荧光指示剂,激光共聚焦显微镜测定不同照光剂量作用后C6胶质瘤细胞内[Ca2+]i。结果PDT组对C6胶质瘤细胞死亡率影响显著,照光剂量与细胞死亡率正相关,随照光剂量增加,细胞死亡率逐渐增大,当达到能量密度大于200J/cm2时,细胞死亡率不再明显增加。PDT作用后早期出现一定数量凋亡的C6胶质瘤细胞和多数线粒体、内质网等亚细胞结构肿胀。PDT作用晚期可见大量坏死细胞和部分凋亡细胞。PDT作用后C6胶质瘤细胞内游离钙明显升高。结论血卟啉甲醚-PDT诱导C6胶质瘤细胞坏死和凋亡,钙超载在广泛超微结构损伤中起重要作用。

Objective To investigate apoptosis mechanism of C6 glioma treated by hematoporphyrin monomethyl ether（HMME）-photodynamic therapy（PDT）.Methods The C6 glioma cell was subcultivated serially in routine way.On the basis of experiment regulation,the cells were inoculated during exponential phase of growth to 96 hole culture plate,and then treated by photosensitizer-HMME mediated PDT with different doses.Cell death rate was detected through MTT at different light doses and morphology change in C6 glioma cell treated by PDT was observed with electron microscope.Making Fluo-3/AM as fluorescent indicator of intracellular Ca^2＋,to determine [Ca^2＋] of C6 glioma cell by confocal laser scanning microscope.Results In experiment group,cell death rate of C6 glioma cell by light influence was significant;the light dose positivety correlated to cell death rate.As light dose increased,death rate raised up gradually.When the light dose added to 200J/cm2,the cell death rate no longer increased significantly.In initial stage of PDT,there were some apoptotic C6 glioma cell and most of subcellular structures such as mitochondria and endocytoplasmic reticulum swollen.In advanced stage,it was thus clear that a great of dead cell and some apoptosis cell were seen;meanwhile free Ca^2＋ of C6 glioma cell steped up significantly.Conclusions HMME-PDT can induce C6 glioma cell death and apoptosis.The calcium overloaded plays an important role in mechanism of extensive subcellular structure injury.