铅神经毒性相关microRNA表达谱改变及其作用的初步研究
发布时间：2018-05-02 12:01
本文选题：miRNA + 铅 ； 参考：《第四军医大学》2013年硕士论文
【摘要】：【背景】 铅是危害人体健康的一种常见环境重金属毒物，分布广泛，主要靶器官是中枢神经系统，严重影响儿童的生长发育和智力水平，还会造成大脑损伤、精神发育迟滞、发育缓慢、行为异常和暴力倾向等，重度铅暴露甚至可以引起死亡[1-3]。铅中毒的发生、发展非常隐匿，常为低剂量长期慢性暴露所致，，目前尚无特别有效的治疗方法。铅在人体代谢缓慢，半衰期长，慢性蓄积毒性效应可一直延续到成年甚至老年，引起神经退行性疾病。因此，揭示铅的神经毒性机制尤其是对学习记忆影响的分子生物学机制，提出切实有效的诊疗、防治措施刻不容缓。 miRNA通过在转录后水平负性调节靶mRNA的表达。miRNA在大脑中的表达丰度很高，在神经发育、突触可塑性以及学习记忆方面起重要作用。miRNA具有在树突周围瞬时、区域表达的特点，对外界刺激具有活动依赖的反应模式，且一个miRNA可以同时调节多个靶mRNA，一个mRNA也可以同时接受多个miRNA的调节，使得大脑特异性miRNA成为神经可塑性和记忆形成微调基因表达的可靠候选。因此我们预测miRNA在铅诱导的神经毒性和学习记忆损伤中可能发挥了重要的作用。 【目的】 在建立铅暴露诱导大鼠学习记忆能力和海马神经元损伤模型的基础上，探讨miRNA表达谱的变化；验证miRNA是否参与了铅诱导的神经毒性效应；揭示miRNA在铅诱导的神经毒性中的作用和机制；为铅中毒的预防和治疗提供实验依据。 【方法】 1、通过饮水染铅、血铅监测的方法建立铅暴露动物模型；通过体外培养PC12细胞系染铅的方法建立细胞模型。 2、通过水迷宫实验（Morris Water Maze）监测隐蔽平台潜伏期、去平台目标象限停留时间等指标评价大鼠空间学习记忆能力；运用TUNEL、Western blot和免疫组织化学染色方法检测铅暴露大鼠海马组织损伤状况。 3、运用miRNA芯片（microarry analysis）检测miRNA表达谱的改变并通过qRT-PCR技术加以验证。 4、运用生物信息学方法预测miRNA靶基因并用Western blot方法验证预测结果。 5、运用miRNA抑制剂（miRNA inhibitor）建立“Loss of function”模型，初步探究miRNA参与铅诱导的神经毒性的作用机制。 【结果】 1、铅可以引起SD大鼠空间学习记忆能力降低 铅暴露组（包括100ppm、200ppm和300ppm组）大鼠在8周饮水染铅暴露过程中和对照组大鼠相比，饮水量和体重变化均无统计学差异，而血铅水平随暴露时间延长及暴露剂量增加显著升高。Morris水迷宫实验结果显示染铅大鼠空间学习记忆能力明显受损，Western blot结果显示海马组织学习记忆相关的谷氨酸受体、NMDA受体等表达水平降低。 2、铅可以诱导神经元凋亡 300ppm饮水铅暴露8w大鼠模型和10μmol/L铅暴露48h PC12细胞模型TUNEL检测均显示铅可以分别诱导海马神经元和PC12细胞凋亡增加，提示铅可能是通过诱导神经元凋亡引起学习记忆能力改变。 3、铅诱导海马组织miRNA表达谱发生改变 miRNA芯片结果显示断乳SD大鼠饮水染铅300ppm8w后海马组织与对照组相比，7个miRNA表达水平发生显著变化，其中miR-204、miR-211、miR-448、miR-449a、miR-34c、miR-34b表达水平升高1.5倍以上；miR-494表达降低为原来的1/6。qRT-PCR验证结果与芯片结果一致。筛选其中表达差异较大的三种miRNA（miR-204、miR-211和miR-448），分别对2w、4w、6w和8w的海马组织进行qRT-PCR验证，结果表明三种miRNA表达水平均升高。 4、生物信息学技术分析结果显示差异表达显著的miRNA靶基因与凋亡、LTP/LTD、钙信号通路等相关，且三个miRNA具有共同的靶基因即Bcl-2和Itpr1 应用生物信息学技术（DIANA、TargetScan和miRNA三个网站）分析预测miRNA的靶基因与凋亡、轴突延伸、钙信号通路、LTP/LTD、MAPK信号通路、Notch信号通路、紧密连接和神经退行性疾病相关。其中miR-204、miR-211和miR-448拥有共同靶基因，即与凋亡相关的Bcl-2和与钙通道相关的Itpr1，Western blot和qRT-PCR方法验证的结果（动物和PC12细胞）与预测结果一致。 5、干扰miR-204、miR-211和miR-448表达，能在一定程度上保护细胞免受铅暴露损伤的影响 应用miRNAinhibitor建立“Loss of function”模型，使用MTT、TUNEL等方法验证干扰miRNA后铅对PC12细胞活力和凋亡的影响，结果发现干扰miRNA表达后能在一定程度上保护PC12细胞免受铅暴露损伤的影响，其中干涉miR-448表达后可使细胞活力提高约20%。 【结论】 1、铅暴露可能通过引起海马神经元凋亡影响学习记忆功能。 2、铅暴露可以引起海马miRNA表达谱发生改变，这种改变可能在铅神经毒性作用中发挥着重要作用。 3、差异表达显著的miR-204、miR-211和miR-448可以通过负性调节其共同靶基因与凋亡相关的Bcl-2和与钙离子通道相关的Itpr1影响铅的神经毒性作用。 4、干扰miR-204、miR-211和miR-448表达，能在一定程度上保护细胞免受铅暴露的损伤。
[Abstract]:[background]
Lead is a common environmental heavy metal poison which is harmful to human health and is widely distributed. The main target organ is the central nervous system, which seriously affects the growth and intelligence level of children. It can also cause brain damage, mental retardation, slow development, abnormal behavior and violence, and severe lead exposure can even cause death in [1-3]. lead. The occurrence of poison is very hidden and often caused by low dose chronic chronic exposure. There is no special effective treatment at present. Lead is slow in metabolism, long half-life and chronic accumulative toxic effect can continue to adult and even old age, causing neurodegenerative diseases. It is urgent to recall the molecular biological mechanism of influence and put forward effective diagnosis and treatment.
The expression of miRNA through the post transcriptional level negatively regulates the expression of target mRNA in the brain, the expression of.MiRNA is high in the brain, and plays an important role in neural development, synaptic plasticity and learning and memory..miRNA has the characteristics of transient, regional expression around the dendrites, a reactive pattern of active dependence on external stimuli, and a miRNA can simultaneously be used. To regulate multiple target mRNA, a mRNA can also accept multiple miRNA regulation, making the brain specific miRNA a reliable candidate for neural plasticity and memory formation. Therefore, we predict that miRNA may play an important role in lead induced neurotoxicity and learning memory damage.
[Objective]
On the basis of establishing the learning and memory ability of rats induced by lead exposure and the damage model of hippocampal neurons, the changes of miRNA expression profiles were explored, and whether miRNA was involved in the neurotoxicity induced by lead was verified, and the role and mechanism of miRNA in lead induced neurotoxicity were revealed, and the experimental basis for the prevention and treatment of lead poisoning was provided.
[method]
1, a lead exposure animal model was established by monitoring lead in drinking water and blood lead monitoring. A cell model was established by culturing PC12 cells in vitro.
2, using the water maze test (Morris Water Maze) to monitor the latent period of the hidden platform, and to evaluate the spatial learning and memory ability of the rats by the target quadrant time of the target, and to detect the damage of the hippocampus in the lead exposed rats by TUNEL, Western blot and immunohistochemistry.
3, miRNA chip (microarry analysis) was used to detect the change of miRNA expression profile and verified by qRT-PCR technology.
4, bioinformatics method was used to predict miRNA target genes and Western blot method was used to verify the prediction results.
5, use miRNA inhibitor (miRNA inhibitor) to establish "Loss of function" model, and explore the mechanism of miRNA involved in lead induced neurotoxicity.
[results]
1, lead can reduce the spatial learning and memory ability of SD rats.
The lead exposure group (including 100ppm, 200ppm and 300ppm) had no significant difference in drinking water amount and weight change during the 8 weeks of exposure to lead exposure in drinking water and the control group, while the blood lead level increased significantly with the exposure time and the increase of exposure dose. The results of the.Morris water maze test showed that the spatial learning and memory ability of the lead exposed rats was clear. Western blot results showed that the expression level of learning and memory related glutamate receptors and NMDA receptors decreased in hippocampus.
2, lead can induce neuron apoptosis
300ppm 8W rat model of drinking lead exposure and TUNEL detection of 48h PC12 cell model of 10 mol/L lead showed that lead could induce the increase of apoptosis in hippocampal neurons and PC12 cells respectively, suggesting that lead may cause the change of learning and memory by inducing neuronal apoptosis.
3, lead induced changes in miRNA expression profiles in hippocampal tissue
The results of miRNA chip showed that after drinking lead 300ppm8w in weaning SD rats, the expression level of 7 miRNA was significantly changed compared with the control group, in which the expression level of miR-204, miR-211, miR-448, miR-449a, miR-34c, miR-34b increased more than 1.5 times; miR-494 expression decreased to the original 1/6.qRT-PCR verification results consistent with the chip results. Three different kinds of miRNA (miR-204, miR-211 and miR-448) were selected to perform qRT-PCR verification on the hippocampus of 2W, 4W, 6W and 8W, and the results showed that the expression level of the three miRNA increased.
4, bioinformatics analysis showed that the significant differentially expressed miRNA target genes were related to apoptosis, LTP/LTD, calcium signaling pathway, and the three miRNA had common target genes, Bcl-2 and Itpr1.
Using bioinformatics (DIANA, TargetScan and miRNA three websites) to analyze and predict the target genes of miRNA and apoptosis, axon extension, calcium signaling pathway, LTP/LTD, MAPK signaling pathway, Notch signaling pathway, close connection and neurodegenerative diseases. MiR-204, miR-211 and miR-448 have common target genes, namely Bcl-2 on apoptosis. The results of validation of Itpr1, Western blot and qRT-PCR methods related to calcium channels (animal and PC12 cells) were consistent with the predicted results.
5, interference with miR-204, miR-211 and miR-448 expression can protect cells from exposure to lead in a certain extent.
The "Loss of function" model was established by using miRNAinhibitor and the effects of MTT and TUNEL were used to verify the effect of lead on the vitality and apoptosis of PC12 cells after interference of miRNA. The results showed that the interference of miRNA expression could protect PC12 cells from the damage of lead exposure to a certain extent, and the activity of cell viability could be increased after the interference of miR-448 expression.
[Conclusion]
1, lead exposure may affect learning and memory function by inducing apoptosis of hippocampal neurons.
2, lead exposure can cause changes in hippocampal miRNA expression profile, which may play an important role in lead neurotoxicity.
3, the significant differentially expressed miR-204, miR-211 and miR-448 can affect the neurotoxicity of lead by negatively regulating its common target gene with apoptosis related Bcl-2 and the Itpr1 associated with calcium ion channels.
4, interference with miR-204, miR-211 and miR-448 expression can protect cells from lead exposure to a certain extent.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
【共引文献】
相关期刊论文 前10条
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2 郑伟;周虹;;葡萄糖调节蛋白58的结构和功能[J];医学分子生物学杂志;2007年02期
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4 刘现芳;马海燕;曲宝明;金心;李红;;孕期不同阶段铅暴露对仔鼠脑组织S100B蛋白表达影响[J];青岛大学医学院学报;2013年05期
5 颜崇淮;徐健;李e
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