ATP诱导大鼠原代培养海马神经元神经毒性及对GAP-43表达的影响

ATP inuced neurotoxicity and influence on the expression of GAP-43 of the cultured rat hippocampal neurons

目的:观察不同浓度ATP对大鼠原代培养海马神经元的损伤作用及GAP-43表达的影响,并探讨其可能机制。方法:原代培养新生SD大鼠海马神经元至第8 d,MTT法检测不同浓度ATP作用下海马神经元的存活率,倒置相差显微镜观察海马神经元的形态学改变,免疫荧光细胞化学法观察损伤后海马神经元GAP-43的表达变化,钙离子荧光染料Flou-4/AM检测细胞内钙离子的浓度变化。结果:低浓度ATP对海马神经元无明显影响,高浓度ATP对海马神经元有毒性损伤作用,且呈剂量依赖性。P2X受体拮抗剂可阻断ATP介导的细胞毒性作用,但P2X7受体拮抗剂的阻断作用不明显。高浓度ATP作用海马神经元4 h后,GAP-43的表达水平明显上调,且表达部位发生改变,主要表达于胞体,突起表达减少甚至消失。高浓度ATP组荧光衰减速率明显减慢,说明高浓度ATP参与了海马神经元细胞内钙调节。结论:高浓度ATP对海马神经元有毒性损伤作用,损伤后GAP-43代偿性表达增多,且与细胞内钙离子浓度变化有关,P2X7受体可能不是介导ATP此作用的主要受体亚型。

Objective： To study the effects of ATP on primary cultured hippocampal neurons and influence on the expression of GAP-43,and the possible mechanism.Methods： The primary hippocampal neurons from neonatal SD rats were cultured in vitro,and MTT assay was used to study the cell survival rate after various concentration ATP treatment at 8 day.The morphological changes in hippocampal neurons were observed under the phase-contrast microscope.Immunofluorescence staining was used to detect the GAP-43 expressional change.Calcium indicator Fluo-4/AM was used to detect the change of intracellular calcium concentration.Results： The lower concentration of ATP had no effects on cell survival rate,and the higher concentration of ATP mediated cytotoxic damage in primary cultured hippocampal neurons in dose-dependent.P2X receptor antagonist blocked ATP-induced cytotoxicity,but P2X7 receptor antagonist did not.The level of GAP-43 expression was elevated significantly after exposure to higher concentration of ATP for 4 hour.GAP-43 was mainly expressed on soma,and the numbers of neuritis were reduced even disappeared.The rate of fluorescence attenuation of calcium in the higher concentration of ATP group was decelerated significantly,indicated that the higher concentration of ATP mediated regulation of intracelluar calcium level.Conclusion： The higher concentration of ATP mediated the cytotoxic damage in primary cultured hippocampal neurons,and the level of GAP-43 expression was elevated significantly after injury by regulating intracellular calcium concentration by P2X7 receptor independent probably.