1,2-二氯乙烷对SH-SY5Y细胞毒性作用机制研究

Mechanism of 1,2-dichloroethane-induced toxicity in SH-SY5Y Cells

目的建立1,2-二氯乙烷(1,2-DCE)对离体培养的人神经母细胞瘤细胞系SH-SY5Y细胞的染毒模型,探讨1,2-DCE对SH-SY5Y细胞的毒性作用及机制。方法取对数生长期的SH-SY5Y细胞,分别以终浓度为0、10、20、30、40、50、60、70、80 mmol/L的1,2-DCE染毒并培养24 h后,观察细胞形态,以CCK-8法检测细胞存活率,采用化学比色法检测细胞培养上清液中乳酸脱氢酶(LDH)活力,细胞内丙二醛(MDA)水平和超氧化物歧化酶(SOD)、三磷酸腺苷(ATP)酶的活力。结果随着1,2-DCE染毒剂量的增加,SH-SY5Y细胞细胞密度逐渐降低,突触变短,胞膜破裂,胞质浓缩,胞质内容物外溢逐渐增多。随着1,2-DCE染毒剂量的增加,细胞存活率呈下降趋势(P<0.01),细胞培养上清液中LDH活力呈升高趋势(P<0.01),均呈剂量-效应关系;而细胞内MDA水平和SOD、钠-钾ATP酶、钙-镁ATP酶、总ATP酶的活力总体上均呈先升高后下降的趋势。细胞培养上清液中LDH活力与细胞存活率呈负相关(相关系数为-0.907,P<0.01)。结论 1,2-DCE对SH-SY5Y细胞有增殖抑制作用,其机制可能与1,2-DCE导致的细胞膜通透性改变,自由基对细胞损害加强,细胞对自由基清除能力的下降,ATP酶活力下降以及钙超载有关。SH-SY5Y细胞可作为1,2-DCE细胞毒性分析的常用细胞系。

Objective To establish the cell model of human neuroblastoma cell（ SH-SY5Y cell） exposed to1,2-dichloroethane（ 1,2-DCE） in vitro and to explore the mechanism of 1,2-DCE-induced toxicity in SH-SY5Y cells.Methods SH-SY5Y cells were collected in their logarithmic growth phase and cultured in complete medium that had final concentrations of 1,2-DCE in 0,10,20,30,40,50,60,70 and 80 mmol / L for 24 hours. Cell morphology was observed and cell survival rate was examined by CCK-8 assay. Using chemical colorimetric method, the activity of lactic dehydrogenase（ LDH） in the cell culture supernatant,and the intracellular level of malondialdehyde（ MDA）,the intracellular activities of superoxide dismutase（ SOD） and adenosine triphosphate（ ATP） enzymes were detected. Results With the increasing exposure concentrations of 1,2-DCE,the cell density of SH-SY5Y cells gradually decreased,the synapse became shorter,the membrane ruptured,cytoplasm condensed and cytoplasmic contents overflowed increased.With the increasing concentration of 1,2-DCE,the cell survival rate decreased（ P〈0. 01）,the activity of LDH in the cell culture supernatant increased（ P〈0. 01）. These changes had a dose-effect correlation. Intracellular MDA level,and activities of SOD,Na^＋-K^＋-ATP enzyme,Ca^2＋-Mg^2＋-ATP enzyme and total ATP enzyme increased at first and then decreased. The activity of LDH in the cell culture supernatant and cell survival rate was negatively correlated（ the correlation coefficient is- 0. 907,P〈0. 01）. Conclusion 1,2-DCE could inhibit the proliferation of SH-SY5Y cells.The mechanism may be related to the permeability change of cell membrane,cellular damage from excessive free radicals,the decrease of free radical scavenging capacity,ATP enzyme activity and calcium overloading. SH-SY5Y cells can be used as a common cell line for 1,2-DCE cytotoxicity analysis.