6-羟基多巴胺诱导PC12细胞凋亡及其可能分子机制

6-Hydroxydopamine-induced apoptosis in PC12 cells and its probable molecular mechanism

探讨神经毒素 6 羟基多巴胺 (6 OHDA)诱导PC12细胞死亡本质及其可能分子机制。不同剂量 6 OHDA处理PC12细胞 2 4h后 ,分别用流式细胞仪、透射电镜、TUNEL染色和DNA电泳检测细胞凋亡 ;5 0 μmol/L 6 OH DA处理PC12细胞 2 0h后 ,用RT PCR检测caspase 3表达 ;用westernblot检测Caspase 3p32蛋白酶原的切割。结果如下 :①流式细胞仪检测PC12细胞凋亡百分率 ,对照组为 1.10 %± 1.14% ,30 ,4 0 ,5 0 μmol/L 6 OHDA处理组分别为 4 .73 %± 1.0 6% ,10 .15 %± 1.2 1%和 19.94 %± 2 .5 1% ,较对照组均有显著性差异 (P <0 .0 1)。 4 0 ,5 0 μmol/L 6 OHDA处理组透射电镜、TUNEL染色均证实存在大量凋亡细胞 ;5 0 μmol/L 6 OHDA处理组DNA电泳呈现一定间隔的“梯状”条带。② 5 0 μmol/L 6 OHDA处理组caspase 3mRNA水平增高约一倍 ,并且检测到Cas pase 3p2 0活性片段。本研究提示 6 OHDA能诱导PC12细胞凋亡 ,并呈剂量依赖性 ;激活Caspase 3蛋白酶可能参与 6

The probable molecular mechanism of 6 Hydroxydopamine(6 OHDA) induced PC12 cell apoptosis was investigated in the present study. Using flow cytometry, electronmicroscopy, TUNEL, and DNA electrophoresis, we detected the apoptosis in PC12 cells after a 24 h exposure to 6 OHDA. RT PCR and Western blot were applied to measure the expression of caspase 3 gene and the cleavage of Caspase 3 p32 proteases after a 20 h exposure. The results showed that at the exposure to 6 OHDA 20 μmol/L, 40 μmol/L, and 50 μmol/L, induction of apoptotic rates in PC12 cells were 4.73 %±1.06%,10.15 %±1.21%, and 19.94 %±2.51% respectively. Whereas the apoptotic rate in PC12 cells with no 6 OHDA exposure was 1.10 %±1.14%, which was regarded as the control group. Morphologically, many apoptotic cells were found at the exposure to 6 OHDA 40 μmol/L and 50 μmol/L, and DNA fragmentation was apparent at the exposure to 6 OHDA 50 μmol/L as well. Caspase 3 mRNA levels were elevated by one fold in 6 OHDA exposure group than in the control group. Caspase 3p20 fragments were observed only in 6 OHDA exposure group, but not in the control group. Our study indicates that 6 OHDA, a neurotoxin used as establishing an animal model of Parkinsonism, induces PC12 cell apoptosis in a dose dependent manner. The activation of Caspase 3 protease may contribute to the mechanism by which 6 OHDA induces PC12 cell apoptosis.\[