老年性痴呆与TOLL样受体表达的研究

发布时间：2018-07-28 18:14

【摘要】：研究背景阿尔茨海默病(Alzheimer's disease, AD)是一种发生于中枢神经系统的退行性病变,是目前老年人痴呆的最主要类型,至今病因不明。全球范围内约35,000,000人罹患此病。该病特征性表现是进行性记忆力减退和认知功能障碍。AD病理学的特征性改变为β-淀粉样肽(β-amyl oid, Aβ)沉积形成的老年斑,由tau蛋白异常聚集形成的神经原纤维缠结,大脑皮层细胞不同程度的减少,以及星形胶质细胞的增生和肥大。AD原因错综复杂,其中神经炎症机制在阿尔茨海默病的发生和发展进程中发挥了重要作用,Ap沉积激活小胶质细胞引起的炎症反应可能是AD的核心病理机制。TOLL-样受体(Toll-like recepter, TLR)家族成员可通过识别各种病原体相关分子模式(pathogen associated molecular pattern,PAMP)引发机体免疫反应,一些列研究证实Toll样受体2(TLR2)可能在AD的神经变性机制和病情进程中起到重要的作用。而且,TLR2参与细胞对AD相关Aβ的摄取及清除。我们以前的研究已经证实TLR2基因改变与AD的遗传易感风险相关。在人类,TLR2基因均有许多多态性位点,他们所在区域为AD遗传易感相关区域。因此我们首次提出TLR2基因遗传多态性可能为AD的遗传风险因子,而外周血单核细胞TLR2基因和蛋白表达量可能为AD的早期诊断生物标志。方法本研究采用美国神经病学、语言障碍和卒中研究所-老年性痴呆和相关疾病学会(NINCDS—ADRDA)中“很可能AD(probable)"标准,选择LOAD患者60例(年龄≥65岁,民族为汉族,女性33例,平均年龄80.2±8.2岁),并选择同期就诊的60例健康查体者作为对照(女性34例,平均年龄80.6±6.3岁)。所有研究对象均为中国北方汉族人且无亲缘关系。所有入选病人均禁食12小时,抽取清晨空腹血各5.5mLEDTA抗凝,采用实时荧光定量聚合酶链反应(RT-PCR)技术、流式细胞术、单管双向等位基因特异性扩增分别对mRNA、血浆蛋白表达进行检测。使用SPSS11.5统计软件进行统计分析。病例组和对照组各种特征的比较采用t检验或卡方检验。多组之间的比较采用单因素方差分析,组间两两比较采用Bonferroni法。使用Pearson积矩相关来评估mRNA、蛋白与MMSE评分的相关性。P0.05为差异有统计学意义。所有统计学检验均为双侧概率检验,以P0.05为差异有统计学意义的标准。测定研究对象的一般特征,确保两组研究人员在年龄、性别、BMI、血压数值、高密度脂蛋白、低密度脂蛋白、胆固醇、血糖上均无显著差异。结果结果发现和健康对照组相比,病例组中TLR2mRNA的表达均高于对照组(TLR2:0.390±0.204比0.281±0.167,P0.01)。病例组中TLR2的蛋白表达也显著高于健康对照组(TLR2:97.12±1.67%比41.07±18.44%, P0.01)。且无论在病例组还是健康对照组,TLR2mRNA的表达均与TLR2蛋白的表达显著相关(分别为TLR2:r=0.980和0.976,P0.01;)。结论本实验首次对中国北方汉族人群TLR2在LOAD患者外周血中mRNA和血浆蛋白水平的表达进行研究,LOAD组TLR2mRNA及其蛋白水平明显增加,而且TLR2mRNA水平与TLR2的蛋白表达有明显的相关性,证明中国北方汉族人群TLR基因表达与LOAD易感性有关。研究背景 To1l样受体的研究历史虽然仅仅20年,但Toll样受体的发现开创了我们理解先天免疫细胞如何识别外源性(即微生物品种)以及内源(即机体衍生物)致病分子,随后引发炎症反应机制的新纪元。这些研究提供了先天免疫系统复兴的飞跃。对免疫学领域的研究发现,在系统发生上保守的先天性免疫不仅提供了防御微生物入侵的第一道防线,而且还影响组织的损伤反应,控制适应性免疫的活性。阿尔茨海默氏病(AD)的特征表现是脑皮质内形成不溶性β-淀粉样蛋白(Aβ)沉积物。沉积物的形成与小胶质细胞介导的炎性反应有关。近期研究表明Toll-样受体(Toll-like recepter, TLRs)参与该炎性反应。TLR家族成员可通过识别各种病原体相关分子模式(pathogen associated molecular pattern, PAMP)引发机体免疫应,一些列研究证实Toll样受体4(TLR4)可能在AD的神经变性机制和病情进程中起到重要的作用。而且,TLR4参与细胞对AD相关Aβ的摄取及清除我们以前的研究已经证实TLR4基因改变AD的遗传易感风险相关。我们先前的大量研究发现TLR4基因有许多多态性遗传位点,与AD的遗传易感相关。因此我们首次提出TLR4基因遗传多态性可能为AD的遗传风险因子,而外周血单核细胞TLR4基因和蛋白表达量可能为AD的早期诊断生物标志。方法本研究采用美国神经病学、语言障碍和卒中研究所-老年性痴呆和相关疾病学会(NINCDS—ADRDA)中“很可能AD(probable)"标准,选择LOAD患者60例(年龄≥65岁,民族为汉族,女性33例,平均年龄80.2±8.2岁),并选择同期就诊的60例健康查体者作为对照(女性34例,平均年龄80.6±6.3岁)。所有研究对象均为中国北方汉族人且无亲缘关系。所有入选病人均禁食12小时,抽取清晨空腹血各5.5mLEDTA抗凝,采用实时荧光定量聚合酶链反应(RT-PCR)技术、流式细胞术、单管双向等位基因特异性扩增分别对mRNA、血浆蛋白表达及Toll样受体4(TLR4)基因3'UTR11367位点的基因多态性进行检测。使用SPSS11.5统计软件进行统计分析。病例组和对照组各种特征的比较采用t检验或卡方检验。多组之间的比较采用单因素方差分析,组间两两比较采用Bonferroni法,P0.05为差异有统计学意义。所有统计学检验均为双侧概率检验,以P0.05为差异有统计学意义的标准。测定研究对象的一般特征,确保两组研究人员在年龄、性别、BMI、血压数值、高密度脂蛋白、低密度脂蛋白、胆固醇、血糖上均无显著差异。结果结果发现和健康对照组相比,病例组中TLR4mRNA的表达均高于对照组(TLR4:0.503±0.195比0.322±0.183,P0.01)。病例组中TLR4的蛋白表达也显著高于健康对照组(TLR4:66.56±23.74%比14.83±4.31,P0.01)。且无论在病例组还是健康对照组,TLR4mRNA的表达与TLR4蛋白的表达显著相关(TLR4：r=0.938和0.970,P0.01)。在病例组中,TLR4在基因型为CC中的蛋白表达水平显著低于在基因型为GG纯合子和GC杂合子中的表达水平。而在健康对照组中,TLR4的蛋白表达水平在三种基因型中差异无统计学意义。结论本实验首次对中国北方汉族人群TLR4在LOAD患者外周血中mRNA和血浆蛋白水平的表达进行研究,LOAD组TLR4mRNA及其蛋白水平明显增加,而且TLR4mRNA水平与TLR4的蛋白表达有明显的相关性。TLR4基因3’UTR11367基因型CC增加LOAD患者全血TLR4蛋白表达,证明中国北方汉族人群TLR基因表达与LOAD易感性有关。
[Abstract]:Research background
Alzheimer's disease (AD) is a degenerative disease occurring in the central nervous system. It is the most important type of dementia in the elderly, and the cause of the disease is unknown. About 35000000 people worldwide are suffering from this disease. The characteristic features of this disease are progressive memory impairment and the pathological features of cognitive dysfunction.AD. The changes in the formation of beta amyloid peptide (beta -amyl oid, A beta) in the senile plaques were caused by abnormal aggregation of tau protein, the decrease of cortical cells in varying degrees, and the complicated causes of astrocyte proliferation and hypertrophy of.AD, in which the mechanism of neuritis is in the progression and development of Alzheimer's disease. The important role of Ap deposition in the activation of microglia may be the core pathological mechanism of AD, the.TOLL- like receptor (Toll-like recepter, TLR) family members can induce the immune response by identifying various pathogen related molecular models (pathogen associated molecular pattern, PAMP). Some studies confirm Toll Like receptor 2 (TLR2) may play an important role in the neurodegenerative mechanism and progression of AD. Moreover, TLR2 participates in the uptake and removal of AD related A beta. Our previous study has shown that the TLR2 gene changes are associated with the genetic susceptibility to AD.
In humans, there are a number of polymorphic loci in the TLR2 gene, and their region is a genetic susceptibility region of AD. Therefore, we first proposed that the genetic polymorphism of the TLR2 gene may be a genetic risk factor for AD, and the TLR2 gene and protein expression of peripheral blood mononuclear cells may be a biomarker for the early diagnosis of AD.
Method
This study uses the "very likely AD (probable)" standard in the American neurology, language disorder and Stroke Research Institute of Alzheimer's disease and related diseases (NINCDS - ADRDA) to select 60 cases of LOAD patients (age 65 years old, ethnic Han, female 33, average age 80.2 + 8.2 years old), and select 60 healthy subjects as controls at the same time as control. (34 women, with an average age of 80.6 + 6.3 years old). All the subjects were all the Han people in the north of China and had no relationship. All the selected patients were fasted for 12 hours, taking 5.5mLEDTA anticoagulant in the morning empty blood, using real time fluorescence quantitative polymerase chain reaction (RT-PCR), flow cytometry and single tube bidirectional allele specific amplification, respectively. MRNA, plasma protein expression was detected. SPSS11.5 statistical software was used for statistical analysis. The comparison of the characteristics of the case group and the control group was compared with the t test or the chi square test. The comparison among the groups was compared with the single factor analysis of variance, and the 22 of the groups was compared with the Bonferroni method. The evaluation of mRNA, protein and MMSE was evaluated by the correlation of Pearson moment. The correlation.P0.05 was statistically significant. All statistical tests were both bilateral probability tests and P0.05 as a statistically significant standard. The general characteristics of the subjects were measured to ensure that two groups of researchers were not significant in age, sex, BMI, blood pressure, HDL, LDL, cholesterol, and blood sugar. There is a difference.
Result
The results showed that the expression of TLR2mRNA in the case group was higher than that in the control group (TLR2:0.390 + 0.204 / 0.281 + 0.167, P0.01). The protein expression of TLR2 in the case group was significantly higher than that in the healthy control group (TLR2:97.12 1.67% to 41.07 + 18.44%, P0.01), and the expression of TLR2mRNA in both the case group and the healthy control group was all with TLR2. Protein expression was significantly correlated (TLR2:r=0.980 and 0.976, P0.01, respectively).
conclusion
In this experiment, the expression of mRNA and plasma protein levels in peripheral blood of LOAD patients in northern Han population of northern China was studied. The level of TLR2mRNA and its protein in group LOAD was significantly increased, and the level of TLR2mRNA was significantly correlated with the protein expression of TLR2. It was proved that the expression of TLR gene in the Han population in northern China was related to the susceptibility to LOAD.
Research background
The history of the To1l like receptor is only 20 years old, but the discovery of Toll like receptors opens up a new era in understanding how innate immune cells identify exogenous (i.e. microbial species) and endogenous (organism derivatives) pathogenic molecules and subsequently trigger the mechanism of inflammation. These studies provide a leap in the revival of the innate immune system. Studies in the field of immunology have found that conserved innate immunity in the system not only provides the first line of defense against microbial invasion, but also affects the tissue damage response and controls the activity of adaptive immunity.
The characteristics of AD are characterized by the formation of insoluble beta amyloid (A beta) deposits in the cortex. The formation of the sediments is associated with the microglia mediated inflammatory response. Recent studies have shown that the Toll- like receptor (Toll-like recepter, TLRs) participates in the inflammatory response of the.TLR family members by identifying various pathogens. Pathogen associated molecular pattern (PAMP) triggers the immune response to the body. Some column studies have confirmed that Toll like receptor 4 (TLR4) may play an important role in the neurodegenerative mechanism and progression of AD. Moreover, TLR4 participation in the uptake and clearance of AD related A beta has been confirmed by our previous study of TLR4 genes. The risk of genetic susceptibility is related.
A number of previous studies have found that the TLR4 gene has a number of polymorphic loci and is associated with the genetic susceptibility to AD. Therefore, we first proposed that the genetic polymorphism of the TLR4 gene may be a genetic risk factor for AD, and the expression of TLR4 gene and protein in peripheral blood mononuclear cells may be an early diagnostic biomarker of AD.
Method
This study uses the "very likely AD (probable)" standard in the American neurology, language disorder and Stroke Research Institute of Alzheimer's disease and related diseases (NINCDS - ADRDA) to select 60 cases of LOAD patients (age 65 years old, ethnic Han, female 33, average age 80.2 + 8.2 years old), and select 60 healthy subjects as controls at the same time as control. (34 women, with an average age of 80.6 + 6.3 years old). All the subjects were all the Han people in the north of China and had no relationship. All the selected patients were fasted for 12 hours, taking 5.5mLEDTA anticoagulant in the morning empty blood, using real time fluorescence quantitative polymerase chain reaction (RT-PCR), flow cytometry and single tube bidirectional allele specific amplification, respectively. MRNA, plasma protein expression and Toll like receptor 4 (TLR4) gene locus 3'UTR11367 polymorphism were detected. Statistical analysis was performed using SPSS11.5 software. The comparison of the characteristics of the case group and the control group was compared with the t test or chi square test. The comparison between the groups was compared with the single factor analysis of variance, and the 22 of the groups was compared to Bonferro. The Ni method and P0.05 were statistically significant. All statistical tests were both bilateral probability tests, with P0.05 as a statistically significant standard. The general characteristics of the subjects were measured to ensure that two groups of researchers had no significant differences in age, sex, BMI, blood pressure, HDL, LDL, cholesterol, and blood sugar.
Result
The results showed that the expression of TLR4mRNA in the case group was higher than that in the control group (TLR4:0.503 + 0.195 / 0.322 + 0.183, P0.01). The protein expression of TLR4 in the case group was also significantly higher than that in the healthy control group (TLR4:66.56 23.74% to 14.83 + 4.31, P0.01). The expression of TLR4mRNA and TLR4 eggs in the case group and the healthy control group were also significantly higher than that in the healthy control group. The expression of white was significantly correlated (TLR4:r=0.938 and 0.970, P0.01). In the case group, the expression level of TLR4 in the genotype CC was significantly lower than that in the genotype GG homozygote and GC heterozygote. In the healthy control group, the protein expression level of TLR4 was not statistically significant in the three genotypes.
conclusion
This experiment was the first time to study the expression of mRNA and plasma protein in peripheral blood of patients with LOAD in the Han population of northern China. The level of TLR4mRNA and its protein in group LOAD increased significantly, and the level of TLR4mRNA was significantly correlated with the protein expression of TLR4..TLR4 gene 3 'UTR11367 genotype increased the expression of whole blood TLR4 protein in LOAD patients. It is proved that the expression of TLR gene is related to LOAD susceptibility in Han population of northern China.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16

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