GAPDH基因5’非编码区突变及过表达突变在帕金森病中的作用研究

发布时间：2019-07-02 11:15

【摘要】：第一部分帕金森病基因多态性研究第一节GAPDH基因多态性与PD关联分析目的：采用病例对照研究,对PD患者与正常对照组中GAPDH基因进行遗传学分析,并探讨GAPDH基因与PD发病风险及相关性。方法：收集265例原发性PD患者和性别、年龄匹配的269例正常人外周血,采用外周血DNA提取试剂盒,提取基因组DNA。采用SNaPshot的方法检测目的基因并分析结果。结果：所测GAPDH基因中其5’非编码区点rs1136666基因多态性,与PD发病相关。其中G为保护碱基,且此点男性病例对照研究统计学有明显差异性,而女性研究无统计学意义。此点在早发组病例对照研究中等位基因频率研究有统计学意义,但基因型研究无统计学意义。在晚发组等位基因频率研究及基因型研究均有统计学意义。结论：GAPDH基因5’非编码区点rs1136666多态性与PD发病相关,且其相关性与性别、年龄有关。男性、老龄是该基因与PD相关发病的危险因素。第二节NCAPD2、GAPDH家族、PKP2P1、PPM1H、CD9、ETV6基因多态性与PD相关性分析目的：采用病例对照研究,对PD患者与正常对照组中基因组其他入组基因进行遗传学分析,并探讨这些基因与PD发病风险及相关性。方法：收集265例原发性PD患者和性别、年龄匹配的269例正常人外周血,采用外周血DNA提取试剂盒,提取基因组DNA。采用SNaPshot的方法检测目的基因并分析结果。结果：GAPDH基因上游NCAPD2基因中rs7311174、rs2072374位点基因多态性在PD病例对照研究中有统计学意义,而rs740850位点基因多态性在PD病例对照研究中无统计学意义。GAPDH家族位点中rs2029721、s4806173、rs12984928基因多态性在PD病例对照研究中没有统计学意义。rs10492243在此次研究中与PD有强烈的关联性。rs2008134、rs342169、rs740839、rs732868在此次病例对照研究中没有统计学意义。结论：NCAPD2基因与PD发病相关。GAPDH家族中位点所纳入此次研究的三个点未发现与PD相关联。第二部分GAPDH基因突变与PD关系研究第一节鱼藤酮处理SH-SY5Y细胞的氧化水平研究目的：观察鱼藤酮干预SH-SY5Y细胞后细胞内氧化水平的变化。方法：进行SH-SY5Y细胞培养。将细胞按浓度梯度分组,MTT法摸索最佳干预浓度。光镜下观察各浓度梯度细胞形态学变化。使用DCFH-DA处理细胞后流式细胞仪观察细胞内氧化水平变化；采用SOD Assay kit检测细胞内SOD活性水平；采用TBARS Assay kit检测细胞内脂质过氧化水平。结果：MTT检测细胞活力摸索出细胞最适干预浓度为500nnM,且细胞活力与干预浓度呈浓度依赖性。光镜下观察细胞发现经不同浓度处理的细胞,200nM少量细胞出现形态学改变,随着浓度的改变,细胞形态学及细胞数量减少的更为明显,2μM的鱼藤酮加入培养基后细胞几乎停止生长,突起消失变圆。使用DCFH-DA处理500nnM鱼藤酮处理后的细胞发现细胞内ROS水平升高；SOD Assay kit检测细胞内SOD活性水平降低；采用TBARS Assay kit检测细胞内脂质过氧化增强。结论：鱼藤酮干预细胞后能使细胞内氧化水平升高,SOD水平降低,脂质过氧化增强。第二节GAPDH基因5,非编码区突变对细胞功能的影响目的：构建rs1136666点突变载体,探讨GAPDH基因突变与PD的关系。方法：进行SH-SY5Y细胞培养,克隆点rs1136666点所在片段。根据分组采用鱼滕湒或载体单个或同时进行干预细胞。摸索载体转染细胞的合适浓度及转染时间以及检验时间。使用实时荧光定量PCR观察各组中GAPDH基因表达情况。使用western blot观察各组细胞中GAPDH表达的情况及观察各组中凋亡的情况。使用SOD Assay kit检测细胞内SOD活性水平；采用TBARS Assay kit检测细胞内脂质过氧化水平。使用流式细胞仪对各组细胞中神经元凋亡进行检测,比较各组细胞经干预后细胞凋亡的情况；使用DCFH-DA处理细胞后在流式细胞仪中检测,对各组细胞中神经元中氧化应激水平作出比较。结果：实时荧光定量PCR示GAPDH基因5’非编码区突变引起细胞内mRNA表达降低;western blot示GAPDH基因5’非编码区突变可引起细胞内凋亡蛋白表达增高,抗凋亡蛋白表达降低,GAPDH表达增高。而GAPDH突变转染与rot联用可增强凋亡作用。流式细胞仪中观察到GAPDH突变转染后的细胞凋亡增加,加用鱼藤酮后,凋亡作用更强。DCFH-DA处理后的细胞可见GAPDH突变转染的细胞中ROS水平增高,鱼藤酮可增强这一作用。GAPDH基因5’非编码区突变转染后的细胞内SOD水平下降,GAPDH基因5’非编码区突变转染后细胞内脂质过氧化水平增高,而鱼藤酮使SOD水平下降及脂质过氧化增高得更明显。结论：GAPDH基因5’非编码区突变,可引起mRNA表达异常,蛋白折叠障碍,异常GAPDH聚集,最终引起细胞死亡,加入鱼藤酮后,细胞内凋亡各项指标发生改变。第三节GAPDH过表达对细胞功能的影响目的：构建GAPDH过表达载体,探讨GAPDH基因过表达与PD的关系。GAPDH基因过表达与GAPDH突变共同在PD中的关系研究。方法：进行SH-SY5Y细胞培养,构建GAPDH过表达载体。根据分组采用鱼滕湒或载体单个或同时进行干预细胞。摸索载体转染细胞的合适浓度及转染时间以及检验时间。使用DCFH-DA处理细胞后在流式细胞仪中检测,对各组细胞中神经元中氧化应激水平作出比较。用SOD Assay kit检测细胞内SOD活性水平；采用TBARS Assay kit检测细胞内脂质过氧化水平。使用western blot观察各组细胞中GAPDH表达的情况及观察各组中凋亡情况。使用流式细胞仪对各组细胞中神经元凋亡进行检测,比较各组细胞经干预后细胞凋亡的情况。使用非变性PAGE胶印迹GAPDH基因5’非编码区突变及GAPDH过表达以及鱼藤酮处理细胞后细胞内GAPDH多聚体表达情况。结果：western blot示过表达载体转染的细胞内GAPDH表达增高,流式细胞仪观察细胞凋亡增多。DCFH-DA示过表达载体转染后的细胞中ROS水平增高。而当GAPDH突变与过表达同时存在时这一作用被增强。非变性PAGE胶印迹示GAPDH基因5’非编码区突变及GAPDH过表达以及鱼藤酮处理细胞后细胞内异常GAPDH表达增多。结论：GAPDH过表达时细胞内氧化水平增高,促进细胞凋亡,当过表达与突变同时存在时,细胞凋亡作用更强。
[Abstract]:Study on the polymorphism of the first part of parkinson's disease Section 1. GAPDH gene polymorphism and PD association analysis: a case-control study was used to make a genetic analysis of the GAPDH gene in the PD patients and the normal control group, and to explore the risk of GAPDH gene and PD and the correlation. Methods:265 patients with primary PD and 269 normal subjects with sex and age were collected, and the peripheral blood DNA extraction kit was used to extract the genome. DNA. The target gene was detected by the method of SNaPshot and divided Results: The gene polymorphism of rs1136666 in the 5 'non-coding region of GAPDH gene and P D. The G is the protective base, and there is a significant difference in the male case-control study of this point, and there is no female study. Statistical significance. This point was statistically significant in the study of the frequency of the allele in the early-onset case-control study, but the genotypic study did not Statistical significance. The frequency and genotype of the allele in the late-onset group were all Conclusion: The polymorphism of rs1136666 in the 5 'non-coding region of GAPDH gene is related to the occurrence of PD, and its correlation is related to that of PD. Sex, age-related. Male, old is the gene related to PD Risk factors for morbidity. Section II NCAPD2, GAPDH family, PKP2P1, PPM1H, CD9, ETV6 gene Objective: To study the genetic analysis of the other enrolled genes in the genome of PD patients and the normal control group by case-control study, and to explore these genes. Methods:265 patients with primary PD and 269 normal subjects with sex and age were collected, and the peripheral blood was extracted from peripheral blood. taking the kit, extracting the genomic DNA, using the SNaPshot, Results: The genetic polymorphism of rs7311174 and rs2072374 in the NCAPD2 gene in the upstream of the GAPDH gene was of statistical significance in the control study of PD. There was no statistical significance in the control of PD cases. rs2029721, s4806173, rs12984928 in the GAPDH family site were polymorphic at P There was no statistical significance in the D case control study. rs10492243 was There is a strong association with PD in this study. rs2008134, rs342169, rs740839, rs732868 are here There was no statistical significance in the secondary case control study. On the relationship between the NCAPD2 gene and the PD, the site of the GAPDH family is included. The three points of this study were not found to be associated with PD. The first part of the study on the relationship between the gene mutation of the second part of GAPDH and Study on the level of oxidation of SH-SY5Y cells treated with rotenone The intracellular oxygen of SH-SY5Y cells after the intervention of ketone Methods: SH-SY5Y cell culture was performed. culturing; grouping the cells according to a concentration gradient, and the MTT method The changes of cell morphology were observed under light microscope. The level of intracellular oxidation was observed by flow cytometry using DCFH-DA, and the level of SOD activity in cells was detected by using the SOD Assay kit. Detection of the level of lipid peroxidation in the cells by the TBARS Assay kit. The pre-concentration was 500 nnM, and the cell viability and the concentration of the intervention were in a concentration-dependent manner. The results showed that the level of ROS in the cells was increased by using DCFH-DA, and the level of SOD activity in the cells was decreased. The increase of lipid peroxidation in the cells was detected by the TBARS Asay kit. After the cell, the level of internal oxidation of the cell is increased, the level of SOD is reduced, and the lipid peroxidation is reduced. The effect of the mutation of the second GAPDH gene 5 and the non-coding region on the function of the cell: the structure The mutation vector of rs1136666 was constructed to study the relationship between GAPDH gene mutation and PD. Method: SH-SY5Y cell culture, clone point rs1 The segment where the 136666 point is located. The fish-tented or carrier sheet is used according to the grouping. One or the same time for the intervention of the cells. The appropriate concentration of the carrier-transfected cells and the time of transfection A real-time fluorescence quantitative PCR was used to observe the expression of GAPDH gene in each group. The expression of GAPDH in each group was observed by ern-blot and the apoptosis in each group was observed. The level of SOD activity was detected. The level of lipid peroxidation in the cells was detected by the TBARS Assay kit. The apoptosis of the cells in each group was detected by flow cytometry. Results: The 5 'non-coding region mutation of GAPDH gene was reduced by real-time fluorescence quantitative PCR, and 5' non-coding of GAPDH gene was shown by western blot. The mutation of the region can cause the expression of the apoptosis protein in the cell to be increased, and the anti-apoptotic protein table The increase of GAPDH expression and the increase of GAPDH expression and the combination of GAPDH mutation and rot can enhance the effect of apoptosis. The cell apoptosis increased after the GAPDH mutation was observed in the cytometer, and the apoptosis was stronger after the addition of the rotenone. The DCFH-DA treatment The level of ROS in the cells transfected with the GAPDH mutation was increased in the cells of the GAPDH gene. The level of SOD in the cells of the 5 'non-coding region of the GAPDH gene was decreased, and the non-coding region of the GAPDH gene was mutated. Conclusion: The 5 'non-coding region of GAPDH gene can cause abnormal expression of mRNA and protein folding. Abnormal GAPDH aggregation, resulting in cell death, After the addition of the rotenone, the apoptosis indexes of the cells were changed. The purpose of cellular function: to construct a GAPDH overexpression vector and to explore the GAP The relationship between the overexpression of DH gene and PD and the overexpression of GAPDH gene and GAPDH A study of the relationship between mutations in PD. Method: SH- SY5Y cell culture to construct a GAPDH overexpression vector. The appropriate concentration of the transfected cells and the time of transfection and the time of the test were found in the group. The level of oxidative stress in the neurons of each group was compared by using DCFH-DA to process the cells, and the levels of oxidative stress in the cells of each group were compared. Assay of the level of SOD activity in the cells by the ay kit; the detection of intracellular lipid with the TBARS Assay kit The expression of GAPDH in each group was observed by western blot and the expression of GAPDH in each group was observed by western blot. Apoptosis was detected by flow cytometry. The apoptosis of the cells in each group was compared by flow cytometry. The non-modified PAGE gel was used to print the GAPD. H gene 5 'non-coding region mutation and GAPDH overexpression and the expression of GAPDH multimer in cells after the treatment of cells with rotenone. : Western blot showed the intracellular GAPD in the cells transfected with the expression vector H-expression, flow cytometry to observe the increase of apoptosis, DCFH-D A showed an increase in the level of ROS in the cells transfected with the expression vector. This action was enhanced when the GAPDH mutation was present at the same time as the overexpression. APDH gene 5 'non-coding region mutation and GAPDH overexpression and abnormal GAPDH expression in the cells after the treatment of the cells with the rotenone.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.5

【共引文献】