凋亡诱导因子在慢性氟中毒大鼠脑组织神经细胞凋亡中的作用

发布时间：2018-05-23 13:41

本文选题：慢性氟中毒 + 神经细胞　；参考：《贵阳医学院》2015年硕士论文

【摘要】：目的:通过检测慢性氟中毒大鼠脑组织中神经细胞凋亡率、凋亡诱导因子(apoptosis-inducing factor,AIF)和相关凋亡因子caspase在蛋白表达的水平,了解AIF介导的非caspase依赖性细胞凋亡途径是否参与慢性氟中毒大鼠大脑神经细胞凋亡过程,并探讨其发生机制。方法:实验用Sprague Dawley(SD)大鼠60只,雌雄各半,随机分为两组,即正常对照组:自由饮用自来水,饮水中含氟量0.5 mg/L;染氟组:饮水中加入氟化钠,使氟含量为50 mg/L。两组均饲以标准饲料(氟含量小于0.5mg/kg),实验期为10个月。实验结束时,观察大鼠氟斑牙发生情况;用氟离子选择电极法测定大鼠尿氟、骨氟含量;HE染色观察大鼠大脑神经组织学改变,尼氏染色观察大鼠脑组织神经细胞内尼氏小体形态学改变;免疫组化检测大脑皮质及海马AIF分布;蛋白印迹(Western blotting)检测大脑皮质及海马AIF、caspase-3及caspase-9的蛋白表达水平;流式细胞术(flow cytometr,FCM)检测大鼠大脑皮质及海马细胞凋亡率。结果:染氟成年大鼠出现不同程度的氟斑牙,尿氟及骨氟含量明显高于对照组(P0.05);HE染色病理形态学检查显示,各组大鼠脑组织未见明显异常改变,尼氏染色发现染氟组大鼠脑组织神经细胞中尼氏体数量较对照组颜色变浅,数量减少;免疫组化结果显示,氟中毒组海马及皮质顶叶神经细胞AIF阳性表达率与染色程度明显高于对照组,差异有统计学意义(P0.05),但在额叶和枕叶差异无统计学意义(P0.05);蛋白印迹结果显示,染氟组大鼠脑组织神经细胞线粒体内AIF(57-k Da)的蛋白表达水平明显低于对照组(P0.01),而细胞核中AIF的蛋白表达水平明显高于对照组(P0.01);染氟组大鼠脑组织神经细胞含半胱氨酸的天冬氨酸蛋白水解酶(cysteinyl aspartate specific proteinase,Caspase)-3的蛋白表达水平明显高于对照组(P0.01),caspase-9的蛋白表达水平高于对照组(P0.05);流式细胞术结果显示,染氟组大鼠海马和皮质神经细胞凋亡率与对照组相比显著增高,差异有统计学意义(P0.05)。结论:以AIF为主的非caspase依赖性凋亡途径与通过激活caspase介导的经典途径共同参与慢性氟中毒大鼠脑组织神经细胞凋亡,是慢性氟中毒引起的脑损伤发生机制之一。
[Abstract]:Objective: to investigate the expression of apoptosis-inducing factor (AIFF) and apoptosis-inducing factor (caspase) in the brain of rats with chronic fluorosis. Objective: to investigate whether AIF mediated caspase dependent cell apoptosis is involved in the process of neurocyte apoptosis in rats with chronic fluorosis and to explore its mechanism. Methods: sixty male and female Sprague Dawley SD rats were randomly divided into two groups: normal control group: drinking tap water freely, fluorine content in drinking water 0.5 mg / L, fluoride group: adding sodium fluoride into drinking water, making fluorine content 50 mg / L. Both groups were fed with standard diet (fluoride content less than 0.5 mg / kg 路kg ~ (-1) for 10 months. At the end of the experiment, the occurrence of dental fluorosis in rats was observed, the urine fluoride was determined by fluorine ion selective electrode method, and the histological changes of brain nerve were observed by bone fluorine content and HE staining. Nissl's staining was used to observe the morphological changes of neuronal Nissl corpuscles in rat brain, the distribution of AIF in cerebral cortex and hippocampus was detected by immunohistochemistry, and the expression of AIF-caspase-3 and caspase-9 in cerebral cortex and hippocampus were detected by Western blotting (Western blotting). Apoptosis rate of cerebral cortex and hippocampal cells in rats was detected by flow cytometry (FCM). Results: dental fluorosis of different degrees was found in adult rats exposed to fluoride. The contents of urine fluoride and bone fluorine in adult rats were significantly higher than those in control group (P 0.05) and HE staining showed that there were no obvious abnormal changes in brain tissue of rats in each group. Nissl staining showed that the number of Nissl bodies in brain tissue of fluoride group was lighter than that of control group. The positive expression rate and staining degree of AIF in hippocampus and parietal cortex in fluorosis group were significantly higher than those in control group (P 0.05), but there was no significant difference in frontal lobe and occipital lobe (P 0.05). The expression level of AIF(57-k Daa in brain mitochondria of rats exposed to fluoride was significantly lower than that of control group (P 0.01), while the expression of AIF protein in nucleus was significantly higher than that in control group, and cysteine was found in nerve cells of rats exposed to fluoride. The protein expression level of cysteinyl aspartate specific proteinase Caspase- 3 was significantly higher than that of control group (P 0.01), and the expression of caspase-9 was significantly higher than that of control group (P 0.05), and the results of flow cytometry showed that the expression of caspase-9 was significantly higher than that of control group (P < 0.05), and the expression of caspase-9 was significantly higher than that of control group. The apoptosis rate of hippocampal and cortical neurons in fluoride group was significantly higher than that in control group (P 0.05). Conclusion: the AIF independent caspase dependent apoptosis pathway and the classical pathway mediated by caspase are involved in the neuronal apoptosis in the brain tissue of rats with chronic fluorosis, which is one of the mechanisms of brain injury induced by chronic fluorosis.
【学位授予单位】：贵阳医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R599.1

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