铝致神经细胞凋亡机制的体外实验研究

【作者】 张勤丽；

【导师】 牛侨；

【作者基本信息】 山西医科大学 ， 劳动卫生与环境卫生学， 2005， 硕士

【摘要】 目的通过体外培养大鼠神经细胞并用不同浓度氯化铝染毒,在细胞水平上研究铝致神经细胞凋亡的分子机制。方法采用新生1-3天的SD大鼠,在无菌条件下取出脑皮质并用0.25%胰酶消化,以1×10⁵密度接种神经细胞。用AlCl₃·6H₂O分别对神经细胞染毒,分为对照组、低剂量组、中剂量组、高剂量组,使其终浓度为0mM/L、0.5 mM/L、1.0 mM/L、2.0 mM/L, 继续培养48h ,观察细胞的生长状况并测定其生化和分子生物学改变。①光镜、电镜、荧光染色和TUNEL法观察神经细胞的形态学变化,并用流式细胞术定量检测神经细胞的早期、晚期及总凋亡率。②透射电镜观察线粒体的形态学改变并检测线粒体的膜标志酶和ATP酶活力,用western blot 法测定凋亡相关蛋白CtyC和Caspase-3的含量变化。③透射电镜观察内质网的形态学改变并检测内质网的脂质过氧化水平,用western blot 法测定凋亡相关蛋白CtyC和Caspase-3的含量变化。④免疫组化法及RT-PCR法检测神经细胞Bcl-2、Bax 蛋白及基因的含量及表达情况。结果①光镜下可见凋亡细胞体积缩小,染色质致密聚集成斑块状,铝剂量的加大可以明显减少神经元的轴突和树突的数量、缩短突触长度和减少神经元细胞间和细胞与突触间的连接。相差显微镜下可清楚地观察到凋亡细胞的发泡和凋亡小体。透射电镜下也可以见到凋亡细胞的特征性变化,扫描电镜更加直接地观察到凋亡细胞表面泡状突起的形成。吖啶橙（AO）-溴化乙啶（EB）双荧光染色法可以清楚地观察到细胞的早期凋亡和晚期凋亡现象,并随着染铝剂量的加大可见TUNEL法DNA碎片绿染颗粒逐渐增多。流式细胞术定量检测结果显示,细胞的早期、晚期和总凋亡率均随着染铝剂量的加大而升高（p<0.01, r为0.878, 0.927,0.976）。②透射电镜观察线粒体的形态学改变发现,凋亡细胞线粒体的结构变化主要是肿胀,并可伴有线粒体嵴数减少或消失。肿胀重者呈气球样变和空泡化。线粒体的内膜标志酶琥珀酸脱氢酶（SDH）、外膜标志酶单胺氧化酶（MAO）及Ca²⁺-Mg²⁺ATP酶、Na⁺-K⁺ATP酶活力检测结果显示,各酶的活力在低剂量染毒组均有显著升高（p<0.01）,但随着染毒剂量的加大,在中高剂量组逐渐下降,且在高剂量染铝组各酶的活力均较对照组显著降低（p<0.01）。检测线粒体凋亡相关蛋白CtyC和活性caspase-3的含量变化发现,胞浆和线粒体中的活更多还原

【Abstract】 Objective To study the molecular mechanism of aluminum-introduced neurocyte apoptosis on cultured neurocytes of new born rats, which had been treated by aluminum chloride in different concentrations. Methods Cerebellum cortex from 0-3 days pups was dissected, then cells were dissociated and digested by trypsin, planted into cubation tube at a density of 1×10⁵ cells /ml, and incubated for 5 days. After incubation, the cells were treated with aluminum chloride（AlCl₃·6H₂O） at final concentrations: 0 mM/L （control group）, 0.5 mM/L（low-dose group）, 1.0 mM/L（medium-dose group）, and 2.0 mM/L（high-dose group） sepatately, then incubated for 48 hr. The neurocytes living conditions were observed after 48 hours incubation and molecular biological changes were detected at the same time. ①Morphologic characteristics were observed under light microscope, electron microscope, measured with AO-EB fluorescent staining and TUNEL methods, and the early stage apoptosis rate, late stage apoptosis rate and the total one were detected with flow cytometry. ②Morphologic characteristics of mitochondria were observed under transmission electron microscope, levels of mitochondria membrane enzyme marker and contents of apoptosis related protein were detected as well. ③Morphologic characteristics of endocytoplasmic reticulum were observed under transmission electron microscope, and lipid peroxidation levels and apoptosis related protein contents were examined. ④Bcl-2, Bax protein contents and their gene expression levels were measured with immunohistochemical method and reverse transport-polymerase chain reaction （RT-PCR） method. Results ①Nuclei shrinkage or decreament of neurocytes volume were noticed under light microscope,quantity of neurite and dendritic reduced with the rising aluminum concentration. Blebbings on the surface of the cells were clearly observed under contrast phase microscope. Distinctive apoptosis image can be observed under transmission electron microscope, especially blebbings on the cells surface under scanning electron microscope. Under fluorescence light, alive neurocytes showed a bright green color in the cell body, while did red in the dead, so that we can distinguish living conditions of the neurocytes. Numerous fluorescent green staining, which means DNA damage, and nuclear fragmentations could clearly be observed in higher dose groups. Apoptosis rate examination showed the higher early stage, late stage and total apoptosis rates of neurocytes after aluminum treatment, which were confirmed to be dose-dependent （r =0.878, 0.927,0.976）. ②Mitochondria swelling and ridges reducing or even disappearing were observed under transmission electron microscope. Activities of succinate dehydrogenase（SDH）, monoamine oxidase（MAO）, which were enzyme markers of endomembrane and outer membrane, Ca²⁺-Mg²⁺ATP enzyme, and Na⁺-K⁺ATP enzyme all showed a sharp elevation in the low-dose aluminum treated group, and then reduced with the rising aluminum dose. There was a significant decrease of enzyme activities in high-dose group than that in control group（p<0.01）. CtyC, which is localized only in mitochandria, was confirmed to be released into cytoplasm after aluminum treatment. Active caspase-3 contents were significantly increased in both cytoplasm and mitochondria with the rising aluminum concentration （p<0.01, r=0.963, 0.859）. ③Image of endocytoplasmic reticulum under transmission electron microscope showed a typical swell and deformation. Superoxide dismutase（SOD） enzyme activity were found significantly increased in the low-dose group, but reduced in mid and high-dose group（p<0.01）, whereas malondialdehyde （MDA） levels were siginificantly decreased in the low-dose group, but increased in mid and high-dose group（p<0.01）. There were significant higher CtyC contents in control group than that in Al-treated groups. Active caspase-3 contents were significantly increased in Al-treated groups comparing with the control group （p<0.01, r=0.902） , but there was no significant difference among the aluminum treated groups (p>0.05更多还原