甲醇对人神经母细胞瘤细胞增殖及凋亡的影响

Effect of Methanol on Proliferation and Apoptosis of SK-N-SH Cells

目的探讨甲醇对人神经母细胞瘤细胞(SK-N-SH)的增殖及细胞凋亡的影响。方法设置不同浓度梯度甲醇溶液(0、250、500、750、1000、1250、1500、1750、2000mmol·L^-1)染毒SK-N-SH细胞24、48h后,用CCK-8法测出各组光密度(OD)值,并绘制甲醇染毒对SK-N-SH细胞活力影响曲线,得出半数染毒剂量(IC50),确定后续实验的甲醇染毒浓度为0、250、750、1250mmol·L^-1,分析各浓度甲醇对SK-N-SH细胞活力和形态的影响;采用流式细胞术检测细胞凋亡情况。结果甲醇对SK-N-SH细胞的半数染毒浓度IC50为1292.96mmol·L^-1。与对照组比较,250、750、1250mmol·L^-1甲醇作用SK-N-SH细胞24、48h后,均对细胞活力有影响(P均<0.01);且随甲醇浓度增加和作用时间的延长,SK-N-SH细胞活力明显下降。对照组SK-N-SH细胞形态为梭形或多边形,有较长的突起,细胞之间存在细胞连接,为单层呈扁平贴壁生长的神经胶质瘤细胞。250mmol·L^-1甲醇组有个别细胞变为圆形,随着甲醇浓度增高,多数细胞变圆缩小、细胞漂浮失去贴壁能力,细胞间隙增大,折光性变强,细胞周围有明显透明圈,培养液中有大量变形细胞漂浮,只有少量正常细胞贴壁。不同浓度(0、250、750、1250mmol·L^-1)、不同时间(0、6、12、24、48h)甲醇染毒SK-N-SH细胞凋亡率差异均有统计学意义(P均<0.001),SK-N-SH细胞甲醇染毒与细胞凋亡率之间存在时间和浓度依赖关系,并且时间和浓度之间存在交互作用(P<0.001)。随着染毒浓度增加,不同时间甲醇染毒组细胞凋亡率变化趋势一致,浓度越高、时间越长、细胞凋亡率越高。结论甲醇可抑制SK-N-SH细胞增殖活力,促进其凋亡。

Objective To investigate the effects of methanol on the proliferation and apoptosis of SK-N-SH cells.Methods The optical density(OD)values of SK-N-SH cells were measured by CCK-8 Method 24 and 48 hours after exposure to 0,250,500,750,1000,1250,1500,1750 and 2000mmol·L^-1 with different concentration gradients of methanol.The influence curve of methanol exposure on the viability of SK-N-SH cells was drawn,and half of the exposure dose(IC50)was obtained.The concentration of methanol in subsequent experiments was determined to be 0,250,750 and 1250mmol·L^-1.The effects of methanol on the viability and morphology of SK-N-SH cells were analyzed,and the apoptosis of SK-N-SH cells was detected by flow cytometry.Results The IC50 of methanol to SK-N-SH cells was 1292.96 mmol·L^-1.After treatment with 250,750 and 1250mmol·L^-1 methanol to SK-N-SH cells for 24 and 48 hours,there was a significant effect on cell viability compared with the control group(P all<0.01);with the increase of methanol concentration and the prolongation of treatment time,the activity of SK-N-SH cells decreased significantly.The SK-N-SH cells in the control group were fusiform or polygonal in shape,with long protrusions,and there were cell junctions between the cells,which were squamous cells with flattened monolayer adherent growth.In the 250mmol·L^-1 methanol group,individual cells became round.As the concentration of methanol increased,most of the cells became round and narrow,the cells floated and lost their adherence ability,the cell gap increased,the refractive index became stronger,and there was a clear transparent circle around the cells.There were a large number of deformed cells floating,and only a small number of normal cells were attached.The apoptosis rate of SK-N-SH cells in different concentrations of methanol was compared.With the increase of methanol concentration,the apoptosis rate of SK-N-SH cells was higher(P<0.001).At different time periods(0,6,12,24,48h),with the prolongation of methanol exposure time,the apoptosis rate of SK-N-SH cells increased gradually(P<0.001);the apoptosis rate of SK-N-SH cells had time and dose effects.However,there was an interaction between time and concentration(P<0.001).With the increase of methanol concentration,the apoptosis rate of methanol exposure group was consistent at different times.The higher the concentration,the longer the time,the higher the apoptosis rate.Conclusion Methanol can inhibit the proliferation of SK-N-SH cells and promote their apoptosis.