School of Forensic Medicine,Kunming Medical University,NHC Key Laboratory of Drug Addiction Medicine, Kunming 650500,China
目的 探讨甲基苯丙胺(methamphetamine,METH)损伤不同类型神经细胞双链DNA 的情况。 方法 体外实验,分别培养原代皮质神经元、HT-22 细胞、BV2 细胞、HMC3 细胞和U-87 MG 细胞,分别给予METH 后,观察细胞形态和用Western blot 检测各组细胞表达γ-H2AX 的水平。体内实验,建立METH 腹腔注射小鼠模型,应用旷场实验进行小鼠行为学分析。HE 染色观察小鼠前额叶皮质和海马内神经细胞的形态变化,用IF 观察两个脑区内双链DNA 损伤情况,用Western blot 检测两个脑区表达γ-H2AX 的水平。 结果 体外实验,给予METH 后,原代皮质神经元、HT-22 细胞、BV2 细胞、HMC3 细胞和U-87 MG 细胞形态发生明显变化,细胞突触变短或消失,胞体皱缩,间隙增宽。各组细胞表达γ-H2AX 水平明显升高。体内实验,给予METH 后,与生理盐水组相比,小鼠运动极其活跃,运动轨迹和路程明显增加。HE 染色结果显示前额叶皮质和海马内神经元明显水肿,嗜酸性增强,部分神经元变性改变。IF结果显示,与生理盐水组相比,METH 组前额叶皮质和海马内神经细胞发生双链DNA 损伤的数量明显增多,且荧光强度明显增强。Western blot结果显示,与生理盐水组相比,METH 组前额叶皮质和海马组织表达γ-H2AX 水平明显升高。 结论 METH 可损伤神经系统双链DNA。METH 可诱导原代皮质神经元、HT-22 细胞,BV2细胞、HMC3 细胞和U-87 MG 细胞以及小鼠前额叶皮质和海马组织表达γ-H2AX 水平明显增高。该研究可为阐明METH 诱导神经毒性作用机制提供理论依据。
Objective To investigate damage of double-stranded DNA by methamphetamine (METH) in various types of nerve cells. Methods For in vitro experiments, primary cortical neurons and HT-22, BV2, HMC3 and U-87 MG cells were treated with METH. Cell morphology was observed and γ-H2AX expression was detected by Western blot. For in vivo experiments, METH was administered to mice by intraperitoneal injection, and behavioral analysis was performed by the open field test. Morphological changes of neurons in the prefrontal cortex and hippocampus of were observed by HE staining. Double-stranded DNA damage was observed by immunofluorescence, and γ-H2AX expression was detected by western blotting. Results In vitro, after treatment with METH, the morphology of primary cortical neurons, and HT-22, BV2, HMC3 and U-87 MG cells had obviously changed, the synapses of cells were shortened or disappeared, cell bodies had shrunk, and gaps were wider. γ-H2AX expression was significantly increased. In vivo, after administration of METH, compared with the saline group, the movement trajectory and distance were significantly increased in the METH group. HE staining showed that neurons in the prefrontal cortex and hippocampus were markedly edematous and eosinophilic, and some neurons had degenerated. Immunofluorescence showed that double-stranded DNA damage in neurons in the prefrontal cortex and hippocampus of the METH group was significantly increased, and the fluorescence intensity was significantly enhanced compared with the saline group. Western blot showed that γ-H2AX expression in the prefrontal cortex and hippocampus of the METH group was significantly increased compared with the saline group. Conclusions METH damaged doublestranded DNA in the nervous system. METH induced a significant increase in γ-H2AX expression in primary cortical neurons HT-22, BV2, HMC3 and U-87 MG cells, the prefrontal cortex, and hippocampus. This study provides a theoretical basis to elucidate the mechanism of METH-induced neurotoxicity.
MIAO Lin, PENG Yanxia, WANG Feng, LI Yi, WANG Haowei, LI Lihua, ZENG Xiaofeng, YANG Genmeng.甲基苯丙胺损伤神经系统双链DNA 的作用研究[J].中国比较医学杂志,2023,33(7):1~8.复制