血卟啉甲醚声动力疗法诱导体外C6胶质瘤细胞凋亡作用

目的 探讨血卟啉甲醚声动力疗法(SDT)对体外C6胶质瘤细胞的促凋亡作用及机制.方法 取处于对数生长期的C6胶质瘤细胞接种于96孔、6孔培养板上.采用噻唑蓝(MTT)比色法观察在不同时间超声波辐照后细胞存活率;流式细胞仪检测24 h后对照组、血卟啉甲醚组(HMME)、单纯超声组、超声血卟啉组(SDT)C6胶质瘤细胞凋亡率、活性氧(ROS)产生、线粒体膜电位(MMP)的改变;Western blot检测凋亡蛋白bcl-2、bax、Caspase-3、-9的表达及细胞色素C(Cyt-C)的释放.结果 当超声频率为1.0 mHz,声强为1.0 W/cm2,MTT筛选60 s为最佳辐照时间.与对照组、HMME及单纯超声组比较,SDT组凋亡率(34.7±1.9)%明显增高(P＜0.05),同时伴有ROS(36.4±3.1)%的产生和MMP(44.9±3.4)%的降低.SDT组Cyt-C的释放增加;凋亡蛋白bax、Caspase-3、-9表达水平增高,凋亡蛋白bcl-2表达水平减低.结论 血卟啉甲醚声动力诱导C6胶质瘤细胞凋亡过程中伴有ROS的产生和MMP的减低,线粒体途径对C6胶质瘤细胞凋亡起着重要作用.

Abstract：

Objective To study apoptotic effect and mechanisms of sonodynamic therapy (SDT) with hematoporphyrin monomethyl ether (HMME) on C6 glioma cells in vitro. Methods Time of ultrasound irradiation was optimized by methyl thiazol tetrazolium (MTT) . Apoptotic rate( AR) generation of reactive oxygen species (ROS) ,mitochondrial membrane potential (MMP) were detected by flow cytometry ,and expressions of apoptosis proteins were measured by Western blotting after SDT treatment. Results 60 s is the optimal time irradiation by MTT when the ultrasound frequency was maintained in 1 mHz and the average intensity was kept in 1. 0 W/cm2. To compare with the control,HMME and ultrasound group,treatment of C6 glioma cells with SDT resulted in the occurrence of apoptosis(34. 7 ±1.9)% .which was associated with the production of ROS ( 36. 4 ± 3. 1) % and loss of MMP (44. 9 ± 3. 4) %. The results showed that significantly increased expression in bax,Caspase-9 ,-3 , decreased in bcl-2 after SDT treatment. The results also showed apparent release of cytochome c in the SDT group versus to the other three groups. Conclusion Our results suggested that SDT mediated by HMME could induce apoptosis in C6 glioma cells.The mitochondrial signal pathway may play a pivotal role in the apoptosis.

Apoptosis induced by sonodynamic therapy with hematoporphyrin monomethyl ether in C6 glioma cells in vitro

Objective To study apoptotic effect and mechanisms of sonodynamic therapy (SDT) with hematoporphyrin monomethyl ether (HMME) on C6 glioma cells in vitro. Methods Time of ultrasound irradiation was optimized by methyl thiazol tetrazolium (MTT) . Apoptotic rate( AR) generation of reactive oxygen species (ROS) ,mitochondrial membrane potential (MMP) were detected by flow cytometry ,and expressions of apoptosis proteins were measured by Western blotting after SDT treatment. Results 60 s is the optimal time irradiation by MTT when the ultrasound frequency was maintained in 1 mHz and the average intensity was kept in 1. 0 W/cm2. To compare with the control,HMME and ultrasound group,treatment of C6 glioma cells with SDT resulted in the occurrence of apoptosis(34. 7 ±1.9)% .which was associated with the production of ROS ( 36. 4 ± 3. 1) % and loss of MMP (44. 9 ± 3. 4) %. The results showed that significantly increased expression in bax,Caspase-9 ,-3 , decreased in bcl-2 after SDT treatment. The results also showed apparent release of cytochome c in the SDT group versus to the other three groups. Conclusion Our results suggested that SDT mediated by HMME could induce apoptosis in C6 glioma cells.The mitochondrial signal pathway may play a pivotal role in the apoptosis.