英文缩略词表(Abbreviations and acronyms) 缩写英文全称中文全称
DA Dopamine 多巴胺
GFP green fluorescent protein 绿色荧光蛋白
MOI multiple of infection 感染倍数
MPP+1-methyl-4- phenyl-pyridine ion 甲基苯基四氢吡啶离子mRNA Message ribomicleic acid 信使核糖核酸
PBS Phosphate-burrered saline 磷酸盐缓冲液疯狂猜成语2
PCR Polymera chain reaction 聚合酶链式反应中国文明网留言
PD Parkinson Dia 帕金森病
PGC-1αperoxisome proliferator-activated receptor 1
alpha 过氧化物酶体增殖物激活受体γ辅激活因子1α平行线的性质教案
PMSF Phenylmethanesulfonyl fluoride 苯甲基磺酰氟RNAi RNA interference RNA干扰siRNA Small interfering RNA 小干扰RNA TH tyrosine hydroxyla 酪氨酸羟化酶
siRNA-PGC-1α基因对MPP+诱导SH-SY5Y
细胞损伤的影响
中文摘要
【背景】
帕金森病(Parkinson dia，PD)是一种中老年人常见的中枢神经系统退行性疾病，线粒体功能障碍在PD发病过程中发挥着重要作用，在与PD发病相关的众多环境与遗传因素中，线粒体功能障碍是一个共同的作用点。过氧化物酶体增殖物激活受体γ辅激活因子1α(peroxisome proliferator-activated receptor 1 alpha，PGC-1α) 是一个多功能转录调节因子，广泛参与线粒体生物合成、糖脂代谢以及抗氧化通路的调节。近年来普遍认为PGC-1α有可能成为早期干预PD的重要治疗靶点，但在PD的发病机制中PGC-1α信号传导对线粒体生物活性的影响尚不明确。目前虽然已对PGC-1α有了大量研究，但目前尚未发现它的特异性抑制剂。RNAi技术，即RNA 干扰，能利用特定序列的小双链RNA高效、特异的阻断体内特定基因表达，促使mRNA降解，诱使细胞表现出特定基因缺失的表型，广泛用于基因分子生物学研究。
【目的】
1、通过腺病毒介导siRNA对SH-SY5Y细胞PGC-1α基因沉默，并筛选出最有效的干扰序列。
2、探讨siRNA-PGC-1α对MPP+诱导SH-SY5Y细胞模型的生长及线粒体功能、能量的影响。
刷牙歌儿歌
【方法】
1、通过免疫组化鉴定SH-SY5Y细胞酪氨酸羟化酶（TH）的表达情况。
2、用携带绿色荧光蛋白的腺病毒(Ad-GFP)转染SH-SY5Y细胞，培养24h后在荧光显微镜下观察荧光细胞的数目判断转染率，确定合适的感染倍数（MOI）。
3、用腺病毒分别介导四种针对PGC-1α基因的干扰序列PGC-1、PGC-2、PGC-3、PGC-4和一个阴性对照序列Ad，感染SH-SY5Y细胞；再用real-time PCR和Western-blot检测各组细胞PGC-1α的mRNA和蛋白质的表达情况。
4、用MTT法检测不同浓度MPP+(0、250、500、1000、1500、2000umol/L)对SH-SY5Y细胞存活率的影响，以制备PD细胞模型。
5、用最有效的干扰序列PGC-1感染PD细胞模型，分组为PBS组、PBS+MPP+组、Ad+PBS组、Ad+MPP+、PGC-1+PBS、PGC-1+MPP+，用MTT法检测各组细胞增殖情况；流式细胞仪分析线粒体膜电位来间接反应线粒体整体的功能状态；通过荧光素酶发光法检测ATP水平了解线粒体产能能力；利用荧光分析法检测H2O2水平来反应线粒体产生氧化物情况。
【结果】
1、免疫组化鉴定显示，实验所用SH-SY5Y细胞TH表达阳性。
2、MOI为50时，腺病毒对SH-SY5Y细胞有较高的转染率，且对细胞损伤较少。
3、通过real-time PCR和Western-blot检测：与阴性对照Ad组相比，PGC-1、PGC-2、PGC-3、PGC-4各组PGC-1α的mRNA和蛋白的表达均下调（P<0.01）；其中PGC-1组PGC-1α的mRNA和蛋白的相对表达量分别为30.74％、15.56％，下调最为显著。
4、MTT 检测发现，通过MPP+处理SH-SY5Y细胞存活率明显下降，且存在剂量依赖关系，在MPP+(1000μΜ/L)时，细胞存活率比对照组降低35.23%（P<0.01），该浓度可作为细胞造模浓度。
5、沉默PGC-1α基因可显著引起正常SH-SY5Y细胞和PD模型细胞的损伤、线粒体膜电位降低、减少ATP的产生以及增加H2O2生成。
【结论】
1、腺病毒对SH-SY5Y细胞有较高的转染率，且筛选出了高沉默效率的干扰序列PGC-1，为进一步探讨沉默PGC-1α基因在PD细胞模型的作用提供了实验依据。
偶成
2、沉默PGC-1α基因可引起PD细胞模型的线粒体功能障碍，并降低细胞存活率。【关键词】
帕金森病，SH-SY5Y细胞，PGC-1α，腺病毒，RNA干扰
Effects of siRNA targeting PGC-1αgene on
SH-SH5Y cell induced by MPP+
Abstract
Background
Parkinson dia（PD）is a high incidence of neurodegenerative dias in elderly people,and attracted widespread attention becau of the high incidence and lack of effective treatment. Mitochondrial dysfunction plays an important role in PD pathogenesis mechanism. Peroxisome prolif
erator-activated receptor 1 alpha (PGC-1α) is a multifunctional transcription factor and widely involved in mitochondrial biogenesis, glucolipid metabolism as well as antioxidant pathways regulating. In recent years, it is generally considered the PGC-1αmay become an important therapeutic target for early intervention in PD. But it is not clear that PGC-1α signal transduction in the pathogenesis of PD activity of mitochondrial biogenesis mechanism. RNA interference (RNAi) is an evolutionary method that effects gene post-transcriptional regulation and expression. RNAi，having highly specific quence and inhibiting target gene expression accordingly ,has developed as a powerful tool for experimental application.
Objective
In this study,the adenovirus vector-bad siRNA targeting PGC-1α gene was transfected into SH-SY5Y cell. The most efficient of interference quence was chon out, further transfected into SH-SY5Y cell and assd the impact of down-expresd of PGC-1αgene on SH-SH5Y cell induced by MPP+.
Methods
Immunohistochemistry was ud to identify the expression of tyrosine hydroxyla. SH-SY5Y cells tr
eated with MPP+ were ud as the in vitro cell model. A MTT assay was ud to investigate the effects of MPP+. The SH-SY5Y cell were transfected with adenovirus mediating green fluorescent protein（Ad-GFP）parately at 1,10,50,100,200 moltiplicity of infection(MOI).The suitable MOI was determine after transfer efficiency was tested by fluorescence microscope. PGC-1α mRNA and protein level were detected
by real-time PCR and Western blot. mitochondrial membrane potential level were detected by flow cytometry(FCM),ATP level were detected by lucifera driven bioluminescence,H2O2level level were detected by fluorescence analysis (Amplex red hydrogen peroxide assay kit).
Results两界穿越做富商
The SH-SY5Y Cells contain tyrosine hydroxyla. After the treat of MPP+, cell viability was significantly decread. .Results indicated that the transfer efficiency of the adenovirus to SH-SY5Y was high, and 50MOI was the suitable MOI．Four pairs of PGC-1αspecific siRNA can regulate down the PGC-1αgene mRNA and protein expression in SH-SY5Y cell. The most efficient interference quence was chon out, which further provide an experimental basis for PGC-1αgene silencing. siRNA interference targeting PGC-1αgene promoted the MPP+-induced mitochondrial damages，including mitochondrial membrane potential, ATP content,H2O2 generation. Conclusion
1. Adenovirus is a high transfection rate of SH-SY5Y cells. The most efficient interference quence was chon out , which further provide an experimental basis for PGC-1α gene silencing in targeting therapy of PD.
2. siRNA interference targeting PGC-1αgene may cau mitochondrial dysfunction of SH-SY5Y cell, and induced the cell apoptosis.
Key words：
减函数的定义Parkinson dia; SH-SY5Y cell; PGC-1α;adenovirus;RNA interference
三方合作协议合同范本
