尼莫地平对HL-60细胞凋亡的影响及其机制

目的研究尼莫地平(NMDP)对HL-60细胞的作用及其机制。方法取对数生长期HL-60细胞并调整细胞浓度为5×108/L,分为对照组和实验组,置24孔板培养,每孔加入HL-60细胞培养液1.5 mL,实验组各孔再加NMDP 0.01 g/L。分别培养0、8、16、24 h,通过倒置显微镜观察细胞的动态变化;采用Giemsa染色光学显微镜下观察细胞的形态学变化;琼脂糖凝胶电泳检测DNA的碎片;流式细胞仪检测DNA含量;细胞免疫组化方法检测细胞凋亡相关基因bcl-2、bax的蛋白表达。结果实验组细胞从培养8 h起,可见不同程度的变形、出泡,细胞和小泡仍保持膜的完整性,随着培养时间延长,出泡细胞增加,细胞体积变小,部分细胞裂解;对照组细胞一直无形态学变化。Giemsa染色可见实验组细胞核膜裂解,染色质呈紫红色或蓝紫色,分割成块状,浓缩、靠近核膜,呈边集现象,并有典型的凋亡小体形成;对照组细胞保持大小一致,胞内染色质呈紫红色或蓝紫色,分布均匀。DNA凝胶电泳显示,实验组在8、16、24 h出现典型的梯形带。实验组细胞的凋亡率随时间延长而增加,自8 h起与对照组差异有显著意义(t=15.06~20.75,P<0.01)。实验组Bcl-2蛋白表达下降,16、24 h与对照组比较差异有显著意义(t=3.66、5.62,P<0.05);Bax蛋白表达升高,与对照组比较,162、4 h有显著性差异(t=4.13、7.55,P<0.01)。Bcl-2/Bax蛋白表达比值逐渐下降,与对照组比较,8、16、24 h差异皆有显著性(t=5.32~7.76,P<0.05)。结论NMDP可诱导HL-60细胞凋亡。Bcl-2/Bax蛋白比值下降,可能是NMDP诱导HL-60细胞凋亡的机制之一。

EFFECT OF NIMODIPINE ON HL-60 CELL APOPTOSIS AND ITS MECHANISM

ObjectiveTo study the effect of Nimodipine on HL-60 and its mechanism.MethodsThe standard HL-60 cells were divided into control and experimental groups.Nimodipine(0.01 g/L) was added into the experimental group. The dynamic changes were observed through an inverted microscope and morphological changes through a microscope with Giemsa method.The DNA fragments were detected through agarose gel electrophoresis.The content of DNA was analyzed through a flow cytometer.The expression of apoptosis-related genes bcl-2 and bax were investigated using immunohistochemical technique.Results(In experimental) group: from after eight hours of cell culture,the cells showed deforming,bubbling;while the membrane of the cells and bubbles remained integrity.With the extension of the culture time,the number of bubbled cell increased,cell capacity decreased,some cells split;no morphological changes were noted in the control group.With Giemsa staining,the split of cell membrane was seen and the chromatin appeared as purplish red,and concentrated into pieces,approaching to nuclear membrane,gathering to the edge and forming typical apoptosis bodies.DNA ladders on agarose gel were clearly seen at 8 h,16 h and 24 h in the experimental group.The apoptosis rate increased in the experimental group with time elongating,being markedly statistically significant from 8 h compared with the control one.The optical density(OD) values of Bcl-2 protein expression obviously decreased in the experimental group,being different(t=3.66,5.62;P<0.05) by 16 h and 24 h compared with the control group.The OD values of Bax obviously elevated,being different(t=4.13,P<0.05) by 16 h and significantly different(t=7.55,P<0.01) by 24 h compared with the control group.The rate of bcl-2/Bax protein descended and showed a significant difference(t=5.32-7.76,P<0.01) by 8 h,16 h and 24 h compared with the control group.ConclusionNimodipine could effectively induce apoptosis of HL-60 cells.The decrease of the proportionality of Bcl-2/Bax protein might be one of the mechanisms inducing apoptosis of HL-60.