【作者】 吕占云；

【导师】 唐北沙； 郭纪锋；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 研究背景：帕金森病(Parkinson’s disease, PD)又称震颤麻痹(Paralysis agitans),是发病率仅次于阿尔兹海默病的第二大神经退行性疾病。近年来,人们发现基因单核苷酸多态性(single nucleotide polymorphism,SNP)与PD具有相关性,且基因SNPs在群体中的分布具有明显的种族特异性。PLA2G6基因,也被称作PARK14,是2009年Paisan-Ruiz等在一个常染色体隐性遗传性早发性帕金森综合征家系中发现的,但是近年来关于PLA2G6基因的SNPs与PD发病风险是否具有相关性尚无文献报道。研究目的：本研究旨在探讨四个PLA2G6的SNPs与华中地区汉族人群散发性PD是否具有相关性。研究方法：此部分实验主要采用了DNA直接测序的方法,检测了中国华中地区汉族人群531例散发性帕金森病患者和561例健康对照者的PLA2G6基因的四个SNPs:rs4375、 rs2267369、rs132985和rs2284063。实验结果：1. PLA2G6基因rs4375、rs2267369、rs132985、rs2284063在中国华中地区汉族人群中是常见的单核苷酸多态位点,不支持这四个SNPs是帕金森病的危险因素。2.将散发性PD组和正常对照组分为≤50岁人群、>50岁人群两组进行分析,结果发现PLA2G6基因rs4375、rs2267369、 rs132985、rs2284063在无论基因型还是等位基因分析均无统计学差别。3.对SNPs进行性别分层后分析发现：男性PD病人与男性正常对照组之间rs132985在基因型和等位基因方面分析都具有明显统计学意义,而两组人群根据性别进行分层后分析发现rs4375、 rs2267369、rs2284063的基因型和等位基因均无统计学差别。4.单体型rs132985A-rs2284063C在散发性PD组和正常对照组之间具有统计学意义,而其余单体型在散发性PD组和正常对照组之间均无统计学意义。结论：PLA2G6基因并不是中国汉族人群PD发病的风险基因。研究背景：PLA2G6基因位于22q13.1,其编码的蛋白质称为iPLA2VIA或iPLA2β,此基因导致PD的发病机制尚无报道。iPLA2β是磷脂酶A2(PLA2)的一种,对于维持膜的流动性具有重要的作用。研究目的：本部分实验主要研究iPLA2β酶活性下降是否通过损伤线粒体而导致细胞凋亡的发生。研究方法：本部分主要运用了MTT实验、流式细胞术,电镜观察、Western Blots实验等检测了与线粒体及细胞凋亡相关的指标。实验结果：1.MTT实验发现不同浓度S-BEL预孵育SH-SY5Y细胞可导致细胞活性出现不同程度的下降。2.运用流式细胞仪检测发现不同浓度S-BEL预孵育SH-SY5Y细胞可以导致线粒体膜电位下降。3.运用电镜检测发现不同浓度S-BEL预孵育SH-SY5Y细胞可以导致线粒体形态发生改变及细胞核固缩等改变。4. Western Blots检测发现不同浓度S-BEL预孵育细胞可以导致SH-SY5Y细胞线粒体细胞色素C释放入细胞浆内。5.Western Blots检测发现不同浓度S-BEL预孵育细胞可以导致SH-SY5Y细胞caspase9、caspase3活化。6.运用Annexin-PI检测发现不同浓度S-BEL预孵育细胞均可以导致SH-SY5Y细胞发生细胞凋亡。结论：iPLA2β酶活性下降可以通过线粒体途径导致细胞凋亡。当然本部分实验仅是细胞水平的研究,进一步的动物模型仍在构建中。研究背景：PLA2G6基因突变可导致婴儿神经轴索变性病(INAD,infantile neuroaxonal dystrophy)及常染色体隐性遗传性PD,因为部分INAD病人和常染色体隐性遗传性早发性帕金森综合征患者在MRI上脑内苍白球区域可见铁离子沉积,因此也被称作脑内铁离子沉积性疾病-2型(Neurodegenerative disorders with brain iron accumulation,NBIA-2)。散发性PD病人脑内黑质(SN)区域的铁水平增高已被证实,且已被证明为散发性PD的重要发病原因之一。铁离子增高可以导致细胞内活性氧水平增高、加重线粒体的损伤从而导致细胞凋亡的发生。研究目的：本部分主要研究iPLA2β酶活性下降是否会导致铁离子沉积、铁离子代谢障碍从而导致细胞凋亡。研究方法：本部分主要运用Perls铁染色方法观察是否存在铁离子沉积、运用Western Blots方法观察铁离子代谢相关蛋白的变化。研究结果：1.运用Perls铁染色方法发现不同浓度S-BEL孵育SH-SY5Y细胞并不能导致细胞内铁离子沉积。2.对SH-SY5Y细胞预孵育铁离子螯合剂去铁敏,并不能降低S-BEL孵育所导致的caspase3活化带的表达。3.不同浓度S-BEL孵育SH-SY5Y细胞并不能导致铁离子转运相关蛋白的表达调控发生障碍。4.不同浓度S-BEL孵育Neuro2a细胞并不能导致细胞内铁离子沉积。5.对Neuro2a细胞预孵育铁离子螯合剂去铁敏,并不能改变S-BEL孵育所导致的细胞存活率下降。6.运用不同浓度S-BEL孵育Neuro2a细胞发现铁离子代谢相关蛋白的表达变化未出现障碍。结论：iPLA2β酶活性下降不能通过导致铁离子沉积途径导致细胞凋亡的发生。当然本部分实验仅是细胞水平的研究,进一步的动物模型仍在构建中。更多还原

【Abstract】 Background:Parkinson’s disease (PD), also known as paralysis agitans, is one of the most common neurodegenerative diseases. In recent years, the relationship between single nucleotide polymorphisms (SNP) and the risk of Parkinson’s disease becomes a central issue, and the single nucleotide polymorphisms in the population distribution have obvious racial specificity. The PLA2G6gene, also called PARK14, was found in2009when Paisan-Ruiz et al studying the autosomal recessive hereditary PD pedigrees, but until now there is still no study about the PLA2G6gene single nucleotide polymorphisms and the risk of PD. Objective:This part of study aims at exploring the relationship between the four of selected PLA2G6single nucleotide polymorphic loci and sporadic Parkinson’s disease. Methods:In this study, direct DNA sequencing methods were used to detect the531patients with sporadic Parkinson’s disease and561healthy people of Han population, four SNPs of PLA2G6gene were selected:rs4375, rs2267369, rs132985and rs2284063. Results:The results of the association study of4tag SNPs in Chinese PD patients and unrelated controls are as follows:1. The SNPs (rs4375, rs2267369, rs132985, rs2284063) are common single nucleotide polymorphic loci in Han population in China, which are not risk factors of Parkinson’s disease.2. Genotype and allele frequencies of all SNPs in patients with either early-onset (EOPD,≤50years) or late-onset (>50years) PD revealed no statistically significant differences from controls.3. The rs132985allele frequency showed a difference in male patients but not in female patients after adjustment for age; Genotype and allele frequencies of rs4375、rs2267369、rs2284063in patients with either male or female PD revealed no statistically significant differences from controls.4. The haplotype rs132985A—2284063C was associated with the increased risk of developing PD in our case control sample set. Conclusion:The results showed that the four single nucleotide polymorphic loci selected were common in Chinese Han population and were not the risk factors in Parkinson’s disease. Background:The PLA2G6gene located at22q13.1, encodes a protein called iPLA2VIA or iPLA2β, there is still no paper reported about this gene in the pathogenesis of Parkinson’s. IPLA2β is one of the phospholipase A2(PLA2), which plays an important role in maintaining the membrane fluidity. Objective:This part aimed at study the relationship between the reduction of iPLA2βactivity and apoptosis. Methods:The specific inhibitor S-BEL was used to pre-incubate SH-SY5Y cells, the activity of iPLA2β enzyme was detected, and MTT, Western Blots, transmission electronic microscopy, Flow Cytometry were used to detect the indicators of related mitochondrial and apoptosis. Results:1. Different concentrations of S-BEL pre-incubation can lead to the decrease of SH-SY5Y cells activity.2. Different concentrations of S-BEL pre-incubation of SH-SY5Y cells can lead to decline in mitochondrial membrane potential.3. Different concentrations of S-BEL pre-incubation can result in cytochrome C release from mitochondrial into the cytoplasm in SH-SY5Y cells.4. Different concentrations of S-BEL pre-incubation can result in caspase9, caspase3activation in SH-SY5Y cells.5. Different concentrations of S-BEL pre-incubation can result in SH-SY5Y cell apoptosis using Annexin-PI detection. Conclusion:The decrease of iPLA2(3activity may cause mitochondrial dysfunction which leads to apoptosis. Of course, our experiments were done on the cellular level and animal model is on the agenda. Background:The mutation of PLA2G6gene can lead to infantile neuroaxonal dystrophy (INAD) and autosomal recessive genetic PD, because in the globus pallidus region of part of the INAD and autosomal recessive PD patients there were MRI visible iron deposition, and therefore the disease of the mutation of PLA2G6gene is classified as the brain iron deposition disease—2(neurodegenerative disorders with brain iron accumulation, NBIA—2). In sporadic patients of Parkinson’s disease, the iron levels have been confirmed to be increased in substantia nigra (SN), and the increased iron proved to be one of the important causes of Parkinson’s disease. Objective:Whether the decrease of the iPLA2β activity may cause the disturbance of iron metabolism then leading to apoptosis become the third part of our study. Methods:This section used Perls iron staining; Western Blots to observe whether there are changes in the deposition of iron and iron metabolism related proteins. Results:The results are as follows:1. There was no intracellular iron deposition in SH-SY5Y cells incubated with different concentrations of S-BEL.2. SH-SY5Y cells were pre-incubated iron chelator deferoxamine, but the protein level of caspase3caused by S-BEL incubation can not be reduced.3. There was no disorder for the expression of iron transport protein for SH-SY5Y cells.4. There was no intracellular iron deposition in Neuro2a cells incubated with different concentrations of S-BEL.5. Neuro2a cells were pre-incubated iron chelator deferoxamine, but cells activity caused by S-BEL incubation can not be rescued.6. There was no disorder for the expression of iron transport protein for Neuro2a cells. Conclusion:The decrease of iPLA2P activity may not cause iron deposition. Of course, our experiments were done on the cellular level and animal model is on the agenda.更多还原