帕金森病相关的神经毒素对MEF2DmRNA的影响及其作用的研究

发布时间：2018-04-04 02:19

本文选题：帕金森病　切入点：肌细胞增强因子MEF2D　出处：《第四军医大学》2014年硕士论文

【摘要】：背景及目的：帕金森病是一种严重影响运动功能的神经退行性疾病[1]。该病在65岁以上老年人中的发病率大概在1-2%，影响着全世界数百万老龄人群的生活和健康。帕金森病的临床症状主要是静止性震颤，肌张力增高和运动迟缓，还伴有植物神经系统的症状和情绪情感的变化。严重者生活不能自理，为家庭和社会增添了沉重的经济负担和精神心理负担。目前的治疗手段主要在于改善临床症状，，但是都无法改变该病的进行性进展的步伐。弄清帕金森病的病因以及探索其背后的发病机制对于家庭和社会来说具有重大意义。帕金森病的病因目前仍然不是十分清楚，研究显示，衰老与帕金森病的发病密切相关。帕金森病的主要病理特点是多巴胺能神经元的死亡。研究显示，一些神经毒素能够特异性的损伤黑质致密部的多巴胺能神经元。比如，MPTP，6-OHDA，鱼藤酮和百草枯等[2,3,4,5,6]。针对神经毒素导致帕金森病的分子机制，人们进行了大量的研究工作。有研究证实，自噬和帕金森病的发病密切相关。帕金森病相关的毒剂能够影响自噬的过程。例如，MPTP，作为线粒体复合物Ⅰ的抑制剂，可以抑制自噬[7]。6-OHDA能够上调黑质神经元中的LC-3,从而促进大自噬[4,8]。百草枯可以使自噬囊泡在胞内堆积[9,10]。所以这些神经毒素常用于帕金森病细胞和动物模型的建立。肌细胞增强因子MEF2D是一种转录因子，是自噬的底物之一。有研究证实，MEF2D是神经元存活的重要调节因子，和帕金森病的发病密切相关[11,12]。MEF2D作为转录因子，可以参与细胞核和线粒体的基因转录表达，若MEF2D的水平下降，则导致神经元死亡，所以MEF2D在多巴胺能神经元的存活中起着关键性的作用。前期研究发现毒素应激可通过磷酸化修饰MEF2D加速其降解[13,14]。但是，前人的研究主要集中在神经毒素对MEF2D的翻译后修饰水平的研究。是否存在神经毒素对MEF2D其他调节途径目前尚无报道。神经毒素能否通过影响MEF2D mRNA的水平而影响其蛋白的表达水平呢？因此，我们进行了本实验，以探究神经毒素对MEF2DmRNA表达水平的影响，并进一步探究神经毒素对MEF2D mRNA稳定性的影响和MEF2D mRNA的变化对细胞存活死亡的作用。方法：本实验首先在多巴胺能神经元细胞系SN4741细胞中，采用神经毒素MPP+处理，通过实时定量PCR技术检测不同时间点的MEF2D mRNA的变化。然后利用体内帕金森病模型进一步验证细胞学实验结果。在C57小鼠中，腹腔注射神经毒素MPTP造成小鼠亚急性帕金森病模型，通过激光捕获显微切割的方法选择性的切取酪氨酸羟化酶（TH）阳性的神经元，然后通过实时定量PCR技术特异性地检测多巴胺能神经元中MEF2D mRNA的变化。进一步，通过放线菌素D抑制基因转录实验，探索神经毒素对MEF2D mRNA稳定性的影响。随后，通过慢病毒感染的方法将细胞中的MEF2D mRNA降低，通过流式细胞仪检测和蛋白电泳观察MEF2D mRNA的降低对神经元存活死亡的作用。结果：实验一：帕金森病相关的神经毒素对MEF2D mRNA水平的影响（1）在SN4741细胞中，加入50uM的神经毒素MPP+后12小时，24小时和36小时分别收集细胞检测，免疫细胞荧光和蛋白电泳结果显示，和对照组相比，MPP+组的MEF2D蛋白水平下降。然后，提取分离细胞中的mRNA，实时定量PCR结果显示，和对照组相比，MPP+组的MEF2D mRNA水平也下降，和蛋白水平的下降趋势相一致。（2）C57小鼠腹腔注射MPTP构建帕金森病亚急性模型。第一次腹腔注射MPTP后3天和5天处死小鼠，取脑切片染色进行相关实验。免疫组织荧光染色结果显示，和对照组相比，TH阳性的神经元的数目明显下降，表明小鼠亚急性帕金森模型构建成功。采用快速染色和激光捕获显微切割特异性地切取TH阳性的神经元后，提取RNA，实时定量PCR的结果显示，和对照组相比，MPTP组的MEF2D mRNA显著下降(Р0.01)。实验二：帕金森病相关的神经毒素对MEF2D mRNA稳定性的影响以及MEF2DmRNA水平的变化对神经元存活的作用（1）放线菌素D能够快速有效的抑制基因转录，常用于mRNA半衰期的检测。通过放线菌素D抑制基因转录实验，我们发现，放线菌素D单独处理组的MEF2DmRNA半衰期为1.5小时，而同放线菌素D和MPP+联合处理组的MEF2D mRNA半衰期为0.6小时，两组之间有显著差异，Р0.01。表明MPP+影响了MEF2D mRNA的稳定性。（2）慢病毒shRNA-MEF2D感染后，通过半定量PCR证实了慢病毒的干扰效果。随后，通过流式细胞仪检测发现，对照组的凋亡细胞比例为0.4%，空病毒载体FUGW组的凋亡细胞比例为0.5%，而shRNA-MEF2D组的凋亡细胞比例为14%。这说明，单独干扰MEF2D mRNA水平就能够诱导细胞凋亡。（3）在神经毒素MPP+处理条件下，活化的caspase3的水平显示，和正常对照组和FUGW组相比，shRNA-MEF2D组的活化的caspase3的量明显增高。这说明，降低MEF2D mRNA能够使细胞对MPP+的毒性更加敏感。结论：本实验主要的创新点在于发现了神经毒素调控MEF2D的新途径，即在转录水平上对MEF2D进行调控。另外，运用了当今先进的激光捕获显微切割技术，能够特异性地获取目的细胞，排除了周围细胞的干扰，确保了实验结果的准确性。（1）在SN4741细胞系和C57小鼠帕金森病模型中，帕金森病相关的神经毒素能够使MEF2D mRNA降低。MEF2D mRNA的降低是和其蛋白水平的降低相一致的。（2）帕金森病相关的神经毒素通过影响MEF2D mRNA的稳定性，导致MEF2DmRNA降低。（3）单独降低MEF2D mRNA，能够诱导多巴胺能神经元的凋亡。（4）降低MEF2D mRNA能够加重帕金森病相关的神经毒素诱导的多巴胺能神经元的死亡。
[Abstract]:Background and Purpose :

Parkinson ' s disease is a neurodegenerative disease that severely affects exercise . The morbidity of Parkinson ' s disease is about 1 - 2 % , affecting the lives and health of millions of elderly people all over the world . The clinical symptoms of Parkinson ' s disease are mainly characterized by static tremor , increased muscle tone and slow motion .

It is found that MEF2D is an important regulatory factor for neuronal survival and is closely related to the pathogenesis of Parkinson ' s disease .

Method :

In this experiment , the changes of MEF2D mRNA in dopaminergic neurons were detected by means of real - time quantitative PCR . The effect of neurotoxin on MEF2D mRNA stability was investigated by means of laser capture microdissection .

Results :

The Effect of Neurotoxin Associated with Parkinson ' s Disease on MEF2D mRNA Level

( 1 ) In SN4741 cells , the levels of MEF2D protein in MPP + group decreased after adding 50uM neurotoxin MPP + 12 hours , 24 hours and 36 hours respectively .

( 2 ) The subacute model of Parkinson ' s disease was constructed by intraperitoneal injection of MPTP in C57 mice . The mice were sacrificed 3 days and 5 days after the first intraperitoneal injection of MPTP . The results showed that the number of TH - positive neurons decreased significantly after the first intraperitoneal injection of MPTP . The results showed that the expression of MEF2D mRNA in MPTP group was significantly decreased compared with the control group ( P0.01 ) .

The effects of neurotoxins associated with Parkinson ' s disease on the stability of MEF2D mRNA and the effect of the changes of MEF2DmRNA level on the survival of neurons

( 1 ) actinomycin D can effectively inhibit the transcription of mRNA and is often used for the detection of mRNA half - life . Through actinomycin D , we find that the half - life of MEF2 DmRNA of actinomycin D alone is 1.5 hours , while the half - life of MEF2D mRNA in actinomycin D and MPP + combined treatment group is 0.6 hours , and there is a significant difference between the two groups .

( 2 ) After the slow virus shRNA - MEF2D infection , the interference effect of the lentivirus was confirmed by semi - quantitative PCR . The percentage of apoptotic cells in the control group was 0.4 % and 0.5 % in the control group , while the percentage of apoptotic cells in the shRNA - MEF2D group was 14 % .

( 3 ) Under the condition of MPP + treatment , the level of activated caspase3 showed that the amount of caspase - e3 activated in shRNA - MEF2D group was significantly higher than that of normal control group and FUGW group .

Conclusion :

The main innovation point of this experiment is to find a new way to regulate MEF2D by neurotoxin , that is to regulate the MEF2D at the transcriptional level . In addition , the advanced laser capture microdissection technique is used to obtain the target cell specifically , the interference of peripheral cells is eliminated , and the accuracy of the experimental results is ensured .

( 1 ) In the SN4741 cell line and the Parkinson ' s disease model of C57 mice , the neurotoxins associated with Parkinson ' s disease were able to lower the MEF2D mRNA . The decrease in MEF2D mRNA was consistent with the reduction of its protein level .

( 2 ) Neurotoxin associated with Parkinson ' s disease , by affecting the stability of MEF2D mRNA , resulted in a decrease in MEF2DmRNA .

( 3 ) MEF2D mRNA alone can induce apoptosis of dopaminergic neurons .

( 4 ) The decrease of MEF2D mRNA can aggravate the death of dopaminergic neurons induced by neurotoxins associated with Parkinson ' s disease .

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

【共引文献】