【作者】 吴凤霞；

【导师】 刘树伟； 潘伟生；

【作者基本信息】 山东大学 ， 人体解剖与组织胚胎学， 2009， 博士

【摘要】 帕金森病（Parkinson’s disease,PD）是最早由James Parkinson医生于1817年报告并因此而得名的一种神经系统疾病。其临床症状有静止性震颤、肌肉强直、随意运动减少和正常的姿势平衡反射丧失等。PD在60岁以上人群中患病率约为1%～2%,是神经系统中的第二大变性疾病。随着社会老龄化进程的推进,患病率还将继续升高。因此如何有效的预防和治疗帕金森病日益引起人们的关注。PD的病因尚不清楚,目前认为与线粒体功能异常、氧化应激等因素密切相关。Schapira等首次报道PD病人的黑质内线粒体呼吸链复合物Ⅰ活性降低,之后,大量的文献报道PD病人的血小板、淋巴细胞及肌肉组织内都存在线粒体呼吸链复合物Ⅰ活性降低。因此PD的发病伴随着线粒体功能异常。而且用复合物I的抑制剂鱼藤酮处理后,动物表现出PD样行为学改变,并且黑质纹状体内出现类似PD的病理变化。研究表明PD病人细胞内的氧化产物如活性氧簇（reactive oxygen species,ROS）增多,而抗氧化物如谷胱苷肽（glutathione,GSH）水平降低,引起PD病人的黑质内多不饱和脂肪酸的浓度下降、脂质氧化的标志物丙二醛（malondialdehyde,MDA）和4-羟基壬烯醛（4-hydroxy-2-nonenal,HNE）浓度升高、可溶性蛋白的羰基修饰水平明显提高并且PD病人脑内细胞核DNA和线粒体DNA中的脱氧鸟苷被氧化成8-羟脱氧鸟苷（8-hydroxydeoxyguanosine,8-OHdG）,这表明PD病人脑内的脂质、蛋白质及DNA都处于氧化应激状态。PD的主要病理变化包括黑质内多巴胺神经元死亡和残留神经元胞浆内蛋白积聚出现嗜酸性包涵体即路易小体（Lewy bodies,LB）。组织学发现LB的纤维成分主要由泛素、神经丝和α-突触核蛋白（α-synuclein,α-syn）组成。α-突触核蛋白是一种突触前神经末梢蛋白,其生理作用尚不明确。据报道它可能参与调节细胞的生长和分化、突触可塑性和递质的释放及参与某些信号传导通路的调节。但α-突触核蛋白基因SNCA的错义点突变A53T、A30P、E46K都可以导致家族性帕金森病症状。另外SNCA的二倍重复突变体和三倍重复突变体也可以引起早发性遗传性帕金森症状,而且在三倍重复突变体家族比二倍重复突变体家族发病年龄早、进程快,这说明α-突触核蛋白表达量也与PD密切相关。许多文献报道非家族性PD病人的脑内也出现α-突触核蛋白表达升高及蛋白的异常积聚。体内和体外的研究也表明,过度表达α-突触核蛋白会造成神经元死亡。这些都提示α-突触核蛋白在PD的发病中起重要作用,抑制其表达可能会阻止或延缓多巴胺能神经元的死亡。RNA干扰（RNA interference,RNAi）是一种在转录后水平有效抑制基因表达的方法。化学合成的小干扰RNA（small interference RNA,siRNA）是一种常用的介导RNAi的双链RNA,与载体携带的短发夹RNA结构（short hairpin RNA,shRNA）相比作用时间短暂、转染率低。Fountaine等利用化学合成的siRNA可以减少α-突触核蛋白表达并能抵抗1-甲基-4-苯基吡啶离子（1-methyl-4-phenylpyridinium ion,MPP⁺）引起的细胞活性下降,但是否影响了细胞线粒体的功能、凋亡调控因子表达及细胞内氧化应激水平未见报道。本研究用采用了分子克隆、基因沉默和细胞培养等技术,构建出靶向α-突触核蛋白的shRNA重组质粒载体,转染细胞并筛选出稳定转染的细胞。利用MPP⁺制作PD细胞模型,利用RT-PCR、MTT分析、流式细胞分析和Westernblotting、DCFH-DA法等方法探讨了干扰α-突触核蛋白表达对帕金森病细胞模型的形态改变、线粒体的功能、Bcl-2/Bax表达及细胞内的ROS和GSH水平的影响。以期为PD的基因治疗提供可靠的实验依据,为PD的治疗提供新思路。本研究分三部分,摘要如下:第一部分RNAi表达载体的构建及沉默效应的鉴定目的:应用基因工程技术构建靶向α-突触核蛋白基因的shRNA表达载体,并将其转染SH-SY5Y细胞系,鉴定其沉默基因的效果。方法:1.构建靶向α-突触核蛋白基因的shRNA表达载体。根据表达载体的要求和文献报道的有效siRNA的序列,设计合成插入载体的shRNA序列,并以一对无关序列的寡核苷酸作为阴性对照。将单链DNA退火连接,并将退火片段与线性化的质粒载体pGenesil-2.2连接,形成重组质粒pGenesil-α-syn-shRNA与pGenesil-scrambled shRNA。将重组质粒转化感受态大肠杆菌DH5α,筛选卡那霉素抗性（kan¹）的阳性克隆,提取质粒。对构建的表达载体质粒pGenesil-α-syn-shRNA与pGenesil-scrambled shRNA分别经Sall酶切电泳鉴定及DNA测序。2.重组质粒转染SH-SY5Y细胞系,鉴定基因沉默效果。将pGenesil-α-syn-shRNA与pGenesil-scrambled shRNA通过阳离子脂质体(Lipofectamin^TM 2000)的介导转ASH-SY5Y细胞内,利用G418抗性筛选稳定转染的细胞。转染了pGenesil-α-syn-shRNA的细胞为干扰组,转染了pGenesil-scrambled shRNA的细胞作为载体对照组,未转染任何载体的细胞为正常对照组。提取各组细胞蛋白,利用Western blotting检测各组细胞中α-突触核蛋白的表达水平。结果:1.构建的表达载体pGenesil-α-syn-shRNA与pGenesil-scrambled shRNA经Sall酶切、琼脂糖凝胶电泳后观察到一条约620bp的条带。DNA测序结果证实目的序列准确无误,与设计结果完全相符。2.经G418筛选,可获得稳定转染的pGenesil-α-syn-shRNA与pGenesil-scrambledshRNA的阳性细胞。经Western blotting检测,载体对照组的细胞与正常对照细胞α-突触核蛋白的表达相似,而干扰组细胞内表达的α-突触核蛋白明显降低。结论:靶向α-突触核蛋白的表达载体pGenesil-α-syn-shRNA经酶切鉴定及DNA测序证实构建成功,转染细胞后可以稳定地干扰SH-SY5Y细胞内α-突触核蛋白的表达。第二部分RNA干扰α-突触核蛋白对MPP⁺引起的SH-SY5Y细胞线粒体功能障碍的抑制作用目的:研究RNA干扰α-突触核蛋白对MPP⁺引起的SH-SY5Y细胞凋亡、线粒体功能障碍和Bcl-2/Bax下降的作用。方法:1.采用MTT法,检测三组细胞:正常未转染的细胞（正常对照组）、转染pGenesil-scrambled shRNA的细胞（载体对照组）及转染pGenesil-α-syn-shRNA的细胞（干扰组）在不含MPP⁺或含500μM MPP⁺的培养基中孵育24 h后的细胞活力。2.用Hoechst 33258对细胞进行染色,观察正常对照组或干扰组细胞在MPP⁺处理后细胞核的形态变化。3.用流式细胞技术检测正常对照组或干扰组的细胞在MPP⁺处理后线粒体膜电位的变化。4.Western blotting检测MPP⁺处理后正常对照组或干扰组细胞浆内细胞色素c（cytochrome c）和细胞内Bcl-2、Bax蛋白表达。结果:1.MPP⁺处理24 h后正常对照组、载体对照组和干扰组细胞的活力都显著降低,与MPP⁺处理的正常组细胞相比,经MPP⁺处理的载体对照组无显著差异,而干扰组细胞能显著抵抗MPP⁺引起的细胞活力下降（p＜0.05）。2.Hoechst 33258染色显示正常对照组细胞在MPP⁺处理24 h后有30.2±1.6%细胞核呈凋亡状念,而干扰组细胞与MPP⁺共孵育后仅有14.2±1.3%细胞凋亡,与前者相比有明显差异（p＜0.05）。3.MPP⁺作用后,正常对照组低线粒体膜电位的细胞占41.0±1.5%,在MPP⁺处理的干扰组此比例仅为13.6±1.2%,两者有显著差异（p＜0.05）。4.MPP⁺引起细胞质中的细胞色素c明显升高,在干扰组细胞中这种升高明显减弱（p＜0.05）。5.正常对照细胞在MPP⁺处理后Bcl-2/Bax比值仅为原来的35.5±3.8%,而干扰组细胞在MPP⁺处理后Bcl-2/Bax比值约为85.2±3.0%,比前者明显升高（p＜0.05）。结论:干扰α-突触核蛋白表达可能通过上调Bcl-2/Bax比值、抵抗线粒体膜电位下降、抑制线粒体内细胞色素c释放进胞浆,从而保护线粒体的正常功能,抵抗MPP⁺引起的细胞凋亡。第三部分RNA干扰α-突触核蛋白对MPP⁺引起的SH-SY5Y细胞内氧化应激的抑制作用目的:研究RNA干扰α-突触核蛋白对MPP⁺引起的SH-SY5Y细胞内ROS和GSH水平变化的影响作用。方法:1.将DCFH-DA装载到细胞,利用多功能酶标仪检测荧光强度来显示细胞内的ROS水平。2.用GSH分析试剂盒来检测细胞内的GSH水平。结果:1.MPP⁺处理后,SH-SY5Y细胞内ROS水平升高至正常对照组的234.7±3.8%,干扰组细胞经MPP⁺处理后,ROS水平升高至149.2±8.2%,但与MPP⁺处理的正常对照细胞相比,ROS水平显著降低,差异有统计学意义（p＜0.05）。2.SH-SY5Y细胞经500μM MPP⁺处理后,细胞内的GSH水平降低为正常对照细胞内的56.0±3.5%。干扰组细胞在MPP⁺处理后,细胞内的GSH水平为61.1±3.2%,与MPP⁺处理的正常对照细胞相比,GSH水平显著升高（p＜0.05）。结论:干扰α-突触核蛋白表达能抑制MPP⁺引起的ROS水平升高和GSH水平降低。这可能是干扰α-突触核蛋白表达能抑制MPP⁺细胞毒性的一个原因。小结:本课题应用基因工程技术构建的shRNA表达质粒能有效地干扰SH-SY5Y细胞内α-突触核蛋白表达并可抑制MPP⁺引起的细胞凋亡和形态学改变。其机理可能是通过上调Bcl-2/Bax比值、抵抗线粒体膜电位下降、抑制线粒体内细胞色素c释放进胞浆、拮抗细胞内ROS水平升高及GSH水平下降,从而保护线粒体的正常功能及维持细胞内正常的氧化水平,进而抵抗MPP⁺引起的细胞凋亡。更多还原

【Abstract】 Parkinson’s disease （PD） is named after Dr. James Parkinson, who provided a detailed description of this disorder in 1817. The clinical symptoms of PD include resting tremor, rigidity, gait disturbance, postural instability and bradykinesia. Parkinson’s disease is the second most common form of neurodegenerative disease, affecting about 1%- 2% of the population older than 60. As the populatin of the elders is growing, more and more attention is attracted to how to prevent or treat PD.Although the etiopathology of PD remains elusive, mitochondrial dysfunction and oxidative stress may be involved in disease pathogenesis. The deficit of Complex I in the mitochondrial respiratory chain was firstly reported by Schapira and his colleagues, and then the deficit of Complex I in platelets, lymphocytes and muscles was found. Consistent with this, after chronic exposure to rotenone, a specific Complex I inhibitor, rats showed anatomical, neurochemical, behavioral and neuropathological changes similar to human PD. Postmortem researches showed that in the substantia nigra of patients with PD the cellular reactive oxygen species （ROS） level increased and the cellular glutathione （GSH） level decreased, which led to oxidative damage to lipid, protein and mitochondrial and genomic DNA.PD is characterized pathologically by the selective loss of dopaminergic neurons in substantia nigra and the presence of intracytoplasmic protein inclusions called Lewy bodies （LB）. Histological research showed thatα- synuclein is one of the main components of LB.α- Synuclein is a presynaptic protein of unknown function. It is reported that it may be involved in regulating cell growth and differentiation, synaptic plasticity, and the release of neurotransmitters and some signal transduction pathway. The point mutations, A53T, A30P, and E46K, in the SNCA gene which encodesα- synuclein cause family PD. And the duplications or triplications of the normal wild-type allele of SNCA lead to autosomal dominant PD, which suggests that the quantity ofα- synuclein expression without point mutations can also be the reason for PD. Furthermore, patients with sporadic forms of the disease present with abnormalα- synuclein accumulation and aggregates in a subset of central and peripheral neurons. Moreover it’s reported that increasedα- synuclein levels can be neurotoxic both in vitro and in vivo. The involvement ofα- synuclein in PD points to the possibility that strategies aimed at suppressingα-synuclein may potentially halt or slow down the progression of dopaminergic cell death in PD.RNA interference （RNAi） is one method to suppress gene expression. It refers to the specific degradation of homologous mRNA induced by double-stranded RNA （dsRNA）. It silences gene on post- transcriptional level. Because of its high efficiency and specificity, it provides a new tool to investigate the gene function and gene therapy. In mammals, there are several strategies to knock down the expression of mRNA. And shRNA expression vector is the most stable and durable method.It’s reported that 1- methyl- 4- phenylpyridinium ion （MPP⁺） could induce mitochondrial dysfunction and oxidative stress in SH-SY5Y cells, while α-synuclein knockdown protected SH- SY5Y cells against MPP⁺. However, whether the mitochondrial dysfunction and the oxidative stress in MPP⁺ treatedα- synuclein knockdown cells are attenuated is unknown.In the present study, the shRNA expression plasmid targetingα- synuclein was constructed and transfected to SH- SY5Y cells using gene engineering, RNA interference and cell culture. After that, stable transfected cells were selected by G418. Then the PD in vitro models was made by MPP⁺ treatment. And then RT-PCR, MTT assay, DCFH- DA assay, flow cytometry and Western blotting were used to explore the effect ofα-synuclein knockdown on mitochondrial function, the Bcl-2/ Bax expression and the oxidative stress. The experiments were divided into 3 parts.PART I THE CONSTRUCTION OF THE RNAi EXPRESSION VECTOR AND ITS SILENCING EFFECTS ONα- SYNUCLEIN EXPRESSION IN SH-SY5Y CELLSObjective: To construct the RNAi expression vector targetingα- synuclein gene （SNCA）, and study its silencing effect onα- synuclein expression in SH-SY5Y cells.Methods:1. The construction of RNAi expression vector targeting SNCA: Design and construction of siRNA oligonucleotides according to the pGenesil vector and the siRNA sequence were confirmed to be valid by a previous report. The whole oligonucleotides template chain is as follows: 5’-TTGGACCAGTTGGGCAAGAA TTTCAAGACGATTCTTGCCCAACT GGTCCTTTTTTG-3’. The sequence of the negative control is 5’-GATCCGACTTCATAAGGCGCATGCTTCAAGACGGCAT GCGCCTTATGAAGTCTTTTTTGTCGACA-3’, which bears no homology to any sequences in the human genome database. Therefore, the transcript- hairpin siRNA is expected to have no interference on human genes. Then they were annealed and ligated into a linear pGenesil- 2.2 vector. The recombinant plasmid pGenesil-α-syn- shRNA and pGenesil-scrambled shRNA were transformed respectively into E coli. DH5a. After screening kanamycin- resistant （kan^r） clones, the plasmids were collected. And then they were identified by PCR and DNA sequenee analysis.2. Investigate the silencing effect onα-synuclein gene: pGenesil-α- syn-shRNA （α- syn- shRNA） and pGenesil-scrambled shRNA （control vector） were transfected into SH- SY5Y cells using Lipofectamine^TM 2000 according to the manufacturers’ instructions. Stably transfected cells were selected by G418 （400μg/ml） 24 h after transfection. The expression level ofα- synuclein was analyzed by Western blotting.Results:1.α- Syn- shRNA and control vector were digested by Sal I enzyme and prepared for agarose gel electrophoresis. A 620bp DNA band was observed under ultraviolet. The DNA sequencing results showed that the inserted sequences were the same with the designed shRNA fragments.2. Stable transfected cells were obtained with G418 resistance. The results obtained from Western blotting showed that compared with control,α- syn- shRNA inhibitedα- synuclein protein expression significantly, while pGenesil- scrambled shRNA had no effect onα- synuclein protein level.Conclusion: The interfering sequence was successfully cloned into the vector.α- syn- shRNA transfection is an effective and long term silencing method of endogenousα-synuclein. PART IIα- SYNUCLEIN KNOCKDOWN SUPPRESSED MPP⁺- INDUCED MITOCHONDRAL DYSFUNCTION OF SH- SY5Y CELLSObjective:1. To evaluated the effect ofα- synuclein knockdown on cell apoptosis and mitochondrial function in MPP⁺- induced PD in vitro model.2. To evaluated the effect ofα- synuclein knockdown on Bcl- 2/ Bax expression.Methods:1. After the cells were incubated in MPP⁺- free medium or medium containing 500μM MPP⁺, MTT assay were used to evaluate the cell viability.2. After incubating in MPP⁺- free medium or medium containing 500μM MPP⁺ for 24 h, cells were washed twice with PBS and incubated with 10μg/ml Hoechst 33258 to show the nuclear morphology.3. The mitochondrial membrane potential was detected by flow cytometry.4. Western blotting was used to detecte the expression of Bcl- 2, Bax and Cytosolic cytochrome c in control orα- syn- shRNA transfected cells.Results:1. After treatment with MPP⁺ for 24 h, the cell viability of control cells, control vector transfected cells andα- syn- shRNA transfected cells was decreased to 67.7±2.9%, 6.5±3.3% and 89.6±2.7%, respectively. The difference between MPP⁺-treated control cells andα-synuclein knockdown is significant （p <0.05）.2. Very few control cells or cells transfected withα- syn- shRNA showed apoptotic nucleus （0.89±0.4% and 1.03±0.6%, respectively）. After 500μM MPP⁺ treatment for 24 h, 30.2±1.6% of cells exhibited apoptotic nucleus. However the percentage was 14.2±1.3 % in cells transfected withα-syn-shRNA. The difference between them is significant （p<0.05）. 3. The results showed that after exposure to 500μM MPP⁺ for 24 h, about 41.0±1.5% control cells showed low mitochondrial membrane potential. However, the percentage was 13.6±1.2% in MPP⁺ treatedα- synuclein knockdown cells. The difference is significant （p <0.05）.4. MPP⁺ induced cytochrome c release significantly, which was about 3.1- fold compared with that of control. However, inα- synuclein knockdown cells, the release of cytochrome c was blocked, which was about 1.4- fold compared with that of control. It’s significantly lower than the former （p <0.05）. The Bcl- 2/ Bax ratio of SH- SY5Y cells reduced to 35.5±3.8% after MPP⁺ treatment, and this ratio was 85.2±3.0% in MPP⁺ treatedα- synuclein knockdown cells. It’s significantly higher than the former （p <0.05）.Conclusion:α-Synuclein knockdown suppressed the MPP - induced apoptosis and protected mitochondrial normal function.α- Synuclein knockdown may afford significant neuroprotection against MPP⁺- induced injury via up-regulation of Bcl-2/ Bax ratio, attenuating the depression of mitochondrial membrane potential, inhibiting cytochrome c release to cytosole and preventing apoptosis.PART IIIα- SYNUCLEIN KNOCKDOWN ATTENUATED MPP⁺- INDUCED OXIDATIVE STRESS OF SH- SY5Y CELLSObjective: To study the effect ofα- synuclein knockdown on ROS and GSH level in MPP⁺- induced PD in vitro model.Methods: DCFH- DA was introduced into cells and intracellular ROS level is measured using multi- detection microreader plate. The intracellular GSH level is tested by GSH assay kit.Results: Compared to the control group, the intracellular ROS level of SH-SY5Y cells increased to 234.7±3.8% after MPP⁺ treatment. Inα- syn- shRNA transfected cells, the ROS level was 149.2±8.2% after MPP⁺ treatment, which is significantly lower than that of the MPP⁺- treated normal cells （ p<0.05）. The intracellular GSH level in MPP⁺- treated normal cells was 56.0±3.5% of the normal control group. Inα- syn- shRNA transfected cells, the percentage is 61.1±3.2%, which is much higher than that of the MPP⁺- treated normal cells （p<0.05）.Conclusion:α- Synuclein knockdown attenuated MPP⁺- induced elevated ROS level and GSH depletion, which may be one of the reasons whyα- synuclein knockdown suppressed the neurotoxicity of MPP⁺.Summary:The shRNA expressing plasmid constructed by gene engineering could downregulate theα- synuclein expression and suppress MPP⁺- induced apoptosis and nuclear morphological changes in SH-SY5Y cells.α- Synuclein knockdown may afford significant neuroprotection against MPP⁺- induced injury via upregulation of Bcl-2/ Bax ratio, attenuating the depression of mitochondrial membrane potential, inhibiting cytochrome c release to cytosole, preventing cellular ROS elevation and GSH depletion, thus protecting mitochondrial normal function and maintaining normal oxidative stress.更多还原