Papadopoulos M C, Koumenis I L, Xu L, Giffard R G
Department of Anaesthesia, Stanford University School of Medicine, CA 94305, USA.
Eur J Neurosci. 1998 Apr;10(4):1252-60. doi: 10.1046/j.1460-9568.1998.00134.x.
Overexpression of the proto-oncogene bcl-2 has been shown to protect a variety of cell types from oxidative and non-oxidative injury, blocking apoptotic and necrotic types of cell death. Retroviral vectors were used to stably overexpress bcl-2 in primary murine astrocyte cultures with more than 95% efficiency. Compared to beta-galactosidase-expressing and uninfected control cells, bcl-2 overexpressing astrocytes suffered < 40% injury after 24 h glucose deprivation, while controls were essentially completely injured. After exposure to 0.2 mM hydrogen peroxide, the bcl-2 overexpressing astrocytes suffered < 40% the injury seen in controls. In contrast, when the cultures were injured by combined oxygen-glucose deprivation, no difference in the extent or time course of injury was found between cells overexpressing bcl-2 and those expressing beta-galactosidase. To investigate one possible mechanism of bcl-2 protection, we measured the levels of glutathione and three antioxidant enzymes. Astrocytes overexpressing bcl-2 had elevated glutathione levels (130-200%), increased superoxide dismutase (170%) and glutathione peroxidase (140%) activities compared with beta-galactosidase-expressing controls. Bcl-2 overexpressing astrocytes suffered less lipid peroxidation after glucose deprivation, as assessed by cis-parinaric acid fluorescence, and demonstrated more rapid removal of hydrogen peroxide from the medium. When glutathione levels were decreased 80% by pretreatment with buthionine sulfoximine, the extent of protection from glucose deprivation of bcl-2 overexpressing cells was decreased by about half. Increased antioxidant defence contributes to protection from glucose deprivation in bcl-2 overexpressing astrocytes.
原癌基因bcl-2的过表达已被证明可保护多种细胞类型免受氧化和非氧化损伤，阻止细胞凋亡和坏死性细胞死亡。逆转录病毒载体用于在原代小鼠星形胶质细胞培养物中稳定过表达bcl-2，效率超过95%。与表达β-半乳糖苷酶的对照细胞和未感染的对照细胞相比，过表达bcl-2的星形胶质细胞在24小时葡萄糖剥夺后损伤小于40%，而对照细胞基本完全受损。暴露于0.2 mM过氧化氢后，过表达bcl-2的星形胶质细胞的损伤程度仅为对照细胞的40%。相反，当培养物受到联合氧-葡萄糖剥夺损伤时，过表达bcl-2的细胞与表达β-半乳糖苷酶的细胞在损伤程度或时间进程上没有差异。为了研究bcl-2保护作用的一种可能机制，我们测量了谷胱甘肽水平和三种抗氧化酶。与表达β-半乳糖苷酶的对照细胞相比，过表达bcl-2的星形胶质细胞的谷胱甘肽水平升高（130-200%），超氧化物歧化酶（170%）和谷胱甘肽过氧化物酶（140%）活性增加。通过顺式-紫黄质酸荧光评估，过表达bcl-2的星形胶质细胞在葡萄糖剥夺后脂质过氧化程度较低，并且从培养基中去除过氧化氢的速度更快。当用丁硫氨酸亚砜胺预处理使谷胱甘肽水平降低80%时，过表达bcl-2的细胞对葡萄糖剥夺的保护程度降低约一半。抗氧化防御增强有助于过表达bcl-2的星形胶质细胞免受葡萄糖剥夺的损伤。
