大鼠脑组织中甲状腺素受体THRα1的表达与阿尔茨海默病发病机制的实验研究
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摘要
目的：复制阿尔茨海默病动物模型,观察其大脑皮层和海马区脑组织中甲状腺素受体THR α1的表达与阿尔茨海默病发生的相关性,并从THRα1表达增加对神经细胞tau蛋白磷酸化影响的角度对其机制进行初步探讨。
方法：SD大鼠腹腔注射D-半乳糖50mg·kg-1·d-1,复制阿尔茨海默病动物模型,另设正常对照组,腹腔注射等量生理盐水。连续给药6w后观察观察动物一般情况如精神状态、活动情况、皮毛等；经二次固定法固定后取全脑组织制作病理切片,进行HE染色和改良Bicschowsky染色,用以观察大脑皮层及海马组织中老年斑,神经元纤维缠结及神经元数量、体积、排列状况等形态学变化；分别用实时荧光定量PCR (Real time fluorescence quantitation PCR, FQ-PCR)法和Western blot法检测阿尔茨海默病动物与正常大鼠脑组织中THRα1、CDK-5及p35mRNA的差异性表达和pser404-tau蛋白的表达水平。
结果：与正常对照组相比,阿尔茨海默病模型组动物神情呆滞，反应迟钝,撮毛等；镜下见大脑皮层及海马区神经元排列紊乱、层次减少,核呈固缩、深染状,神经元轴突染色较深呈拖尾状,形成神经元纤维缠结,尤以海马区表现更为显著。与对照组相比,模型组皮层区THRα1、CDK-5和p35mRNA表达量增加,分别为1.20±0.30、4.71±0.54、2.11±0.40,差异有统计学意义(P<0.05)；模型组海马区三者的表达量分别为2.53±0.65、18.51±2.77、5.96±0.49,与对照组相比亦有统计学差异(P<0.05)模型组pser404-tau蛋白呈高表达状态,与正常对照组相比P<0.05。
结论：阿尔茨海默病动物大脑皮层及海马区神经细胞中THRα1mRNA的表达量显著高于正常对照组大鼠,过高THRα1可提高tau蛋白磷酸化相关激酶CDK-5mRNA及其调节因子p35mRNA的表达水平,进而加剧神经细胞tau蛋白磷酸化,导致动物大脑皮层及海马区出现神经元丢失、神经纤维缠结等典型的AD形态学变化,这可能是THRα1过表达导致阿尔茨海默病发生的主要机制之一。
Objective:To study the correlation between Alzheimer's disease (AD) and the expression of thyroid receptor al (THR al) in brain tissues by establishing a rat model of AD, and therefore to investigate its mechanism primarily from the angle of phosphorylation of protein tau aggravated by the over-expression of THR al.
Methods:D-galactose (50mg·kg-1·d-1) was administered by intraperitoneal injection into SD rats from AD group, while equal amount of saline into that from control group. After six weeks' administration,these rats were observed mental state, activities, etc. Tissues from the whole brain were made into pathological sections by the secondary fixed, whose morphological changes were then observed under optical microscope. The differential expression of mRNA encoding THR al, CDK-5, p35and that of protein pser404-tau were determined using real time fluorescence quantitative PCR and Western-blot respectively.
Results:Compared with control group, AD rats were dull appearance and slow reaction., the neurons of cerebral cortex and hippocampus in particular from AD group were disorganized, with their nuclei hyperchromatic、pyknosis, their axons hyperchromatic, trailing, and formed the neurons fiber tangles.The amount of expression of THRα1、CDK-5and p35mRNA increased, were1.20±0.30、4.71±0.54、2.11±0.40with statistical significance(P<0.05); while in the same time, in hippocampal, the amount of expression of them were2.53±0.65、18.51±2.77、5.96±0.49respectively with statistical significance (P<0.05)。And the amount of pser404-tau protein is significantly higher than that of the rats in the control group(P<0.05).
Conclusion:The expression of mRNA encoding THR al in neurons of cerebral cortex and hippocampus from AD group is significantly higher than control group. Over-expression of THR al can increase the expression of mRNA encoding protein phosphorylation related kinase, namely CDK-5. And it in turn aggravates the phosphorylation of protein tau in neurons, which may participate or contribute to the pathological process of AD.
方法：SD大鼠腹腔注射D-半乳糖50mg·kg-1·d-1,复制阿尔茨海默病动物模型,另设正常对照组,腹腔注射等量生理盐水。连续给药6w后观察观察动物一般情况如精神状态、活动情况、皮毛等；经二次固定法固定后取全脑组织制作病理切片,进行HE染色和改良Bicschowsky染色,用以观察大脑皮层及海马组织中老年斑,神经元纤维缠结及神经元数量、体积、排列状况等形态学变化；分别用实时荧光定量PCR (Real time fluorescence quantitation PCR, FQ-PCR)法和Western blot法检测阿尔茨海默病动物与正常大鼠脑组织中THRα1、CDK-5及p35mRNA的差异性表达和pser404-tau蛋白的表达水平。
结果：与正常对照组相比,阿尔茨海默病模型组动物神情呆滞，反应迟钝,撮毛等；镜下见大脑皮层及海马区神经元排列紊乱、层次减少,核呈固缩、深染状,神经元轴突染色较深呈拖尾状,形成神经元纤维缠结,尤以海马区表现更为显著。与对照组相比,模型组皮层区THRα1、CDK-5和p35mRNA表达量增加,分别为1.20±0.30、4.71±0.54、2.11±0.40,差异有统计学意义(P<0.05)；模型组海马区三者的表达量分别为2.53±0.65、18.51±2.77、5.96±0.49,与对照组相比亦有统计学差异(P<0.05)模型组pser404-tau蛋白呈高表达状态,与正常对照组相比P<0.05。
结论：阿尔茨海默病动物大脑皮层及海马区神经细胞中THRα1mRNA的表达量显著高于正常对照组大鼠,过高THRα1可提高tau蛋白磷酸化相关激酶CDK-5mRNA及其调节因子p35mRNA的表达水平,进而加剧神经细胞tau蛋白磷酸化,导致动物大脑皮层及海马区出现神经元丢失、神经纤维缠结等典型的AD形态学变化,这可能是THRα1过表达导致阿尔茨海默病发生的主要机制之一。
Objective:To study the correlation between Alzheimer's disease (AD) and the expression of thyroid receptor al (THR al) in brain tissues by establishing a rat model of AD, and therefore to investigate its mechanism primarily from the angle of phosphorylation of protein tau aggravated by the over-expression of THR al.
Methods:D-galactose (50mg·kg-1·d-1) was administered by intraperitoneal injection into SD rats from AD group, while equal amount of saline into that from control group. After six weeks' administration,these rats were observed mental state, activities, etc. Tissues from the whole brain were made into pathological sections by the secondary fixed, whose morphological changes were then observed under optical microscope. The differential expression of mRNA encoding THR al, CDK-5, p35and that of protein pser404-tau were determined using real time fluorescence quantitative PCR and Western-blot respectively.
Results:Compared with control group, AD rats were dull appearance and slow reaction., the neurons of cerebral cortex and hippocampus in particular from AD group were disorganized, with their nuclei hyperchromatic、pyknosis, their axons hyperchromatic, trailing, and formed the neurons fiber tangles.The amount of expression of THRα1、CDK-5and p35mRNA increased, were1.20±0.30、4.71±0.54、2.11±0.40with statistical significance(P<0.05); while in the same time, in hippocampal, the amount of expression of them were2.53±0.65、18.51±2.77、5.96±0.49respectively with statistical significance (P<0.05)。And the amount of pser404-tau protein is significantly higher than that of the rats in the control group(P<0.05).
Conclusion:The expression of mRNA encoding THR al in neurons of cerebral cortex and hippocampus from AD group is significantly higher than control group. Over-expression of THR al can increase the expression of mRNA encoding protein phosphorylation related kinase, namely CDK-5. And it in turn aggravates the phosphorylation of protein tau in neurons, which may participate or contribute to the pathological process of AD.
引文
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[2]Selina Wrayl,Wendy Noble2.Linking Amyloid and Tau Pathology in Alzheimer's Disease:The Role of Membrane Cholesterol in A(3-Mediated Tau Toxicity[J]. The Journal of Neuroscience, 2009,29(31):9665-9667.
[3]Selkoe DJ.Alzheimer's disease:genes, proteins, and therapy [J]. Physiol Rev. 2001,81(2):741-66.
[4]Vina J, I loret A, Orti R. Molecular bases of the treat-ment of Alzheimer's disease with antioxidants:preven-tion of oxidative stressl [J]. Molecular Aspects of Med,2004(25): 117-123.
[5]Mecocci P. Oxidative stress in mild cognitive impair-ment and Alzheimer's disease:a continuum [J]. Alzheimer's Disease,2004(6):159-163.
[6]Jorm AF. Cross—national comparisons of the occurrence of Alzheimer'S and vascular dementias [J]. EurArchPsychiatryClinNeurosci,1991(4):218-222.
[7]Heben LE, Scherr PA, Bienias JL, et al. Alzheimer disease in the US population:prevalence estimates using the 2000 Census [J]. Arch Neurol.2003.60:1119-1122.
[8]张朝峰、杜会枝、杨频.阿尔茨海默病分子机理的离子通道假说.[J]化学进展2006(18)1194-1199.
[9]Saido TC. Towards presy ptommatic diagnosis, prevention and treament of Alzheimer'S disease [J]. Rinshoshinkeigaku,2004(11):824—826。
[10]魏群、于大禹、吴和珍.阿尔茨海默病与tau蛋白磷酸化及去磷酸化.[J]中国航天医药杂志2004(6)：1-5.
[11]许飞、程灶火、杨敏.Tau蛋白、Aβ蛋白与阿尔茨海默病的关系及其作用.[J]实用临床医药杂志2008(12)：118-120.
[12]龚成新、王建枝.tau蛋白在老年性痴呆症神经原纤维退行性变中的作用.[J]医学分子生物学杂志,2006(3)：163-169.
[13]吴琪Alzheime病神经原纤维缠结Tau蛋白研究[J].中国神经精神疾病杂志,2000,26(1)：63-64.
[14]Berriman J, SevpellLC, ObergKA, et al. Tau filamentsfromhuman Brain and from in vitro assembly of recombinantprotein show cro68 bet structure[J]. Proc Nat Acad SciUSA,2003(15): 9034-9038
[15]曾晖,潘希锋.雌激素、Tau蛋白及海马体积与阿尔茨海默病的相关性研究进展[J].神经损伤与功能重建,2008(1)：59-60.
[16]Du J T, Yu C H, Zhou L X, et al. Phosphorylation modulates the local conformation and self-aggregation ability of a peptide from the fourth tau microtubule-binding repeat [J].FEBS J, 2007,274 (19):5012.
[17]刘英华、尹君、王建枝.腔注射脂多糖对大鼠海马tau蛋白磷酸化的影响[J].中国病理生理杂志,2006(22)：2086-2089.
[18]HAASE C, STIELER J, ARENDT T, et al。Pseudophosphorylation of tau protein alters its ability for self-aggregation.[J].J Neurochem,2004,88:1509-1520.
[19]王英鹏、宋俊峰、饶志仁.CDK-5与神经退行性疾病[J]生理科学进展,2004(35)：48-45.
[20]李红丽、杨忠、李泽桂.小鼠脑发育中CDK-5 mRNA表达变化的研究[J].第三军医大学学报2004(26)：2223-2226.
[21]张昭军、齐众、乔明强.Cdk5/p35激酶与肌动蛋白细胞骨架结合关系的鉴定[J].细胞生物学杂志,2008(30)：489—493.
[22]Patrick GN, Zukerberg I, Mikolic M el al. Conversion of p35 to p25 deregulates cdk5 activity and promotes neurodegeneration[J]. Nalure,1999,402(6 762):615-622.
[23]Evans D B,Rant K B,Bhattacharya K, et al.Tau phosphorylation at serine396 and serine404 by human recombinant tau protein kinase inhibits.Tau's ability to promote microtubule assembly. [J]J Biol chem,2000,275:24877-83.
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