微管相关蛋白tau转基因细胞和动物模型的建立及tau蛋白病变表征  

Establishment and characterization of microtubule-associated protein tau overexpression cell and transgenic animal models

作　　者：马登磊 张旭[2,4] 罗艺 黄蕊[1,2,3] 李雅莉[1,2,3] 李林 张兰[1,2,3] Ma Denglei;Zhang Xu;Luo Yi;Huang Rui;Li Yali;Li Lin;Zhang Lan(Department of Pharmacy,Xuanwu Hospital,Capital Medical University,Beijing 100053,China;Key Laboratory for Neurodegenerative Diseases of Ministry of Education,Beijing 100053,China;Beijing Engineering Research Center for Nerve System Drugs,Beijing 100053,China;Central Laboratory,Xuanwu Hospital,Capital Medical University,Beijing 100053,China)

机构地区：[1]首都医科大学宣武医院药学部,北京100053 [2]神经变性病教育部重点实验室,北京100053 [3]北京市神经药物工程研究中心,北京100053 [4]首都医科大学宣武医院中心实验室,北京100053

出　　处：《首都医科大学学报》2019年第4期582-587,共6页Journal of Capital Medical University

基　　金：国家自然科学基金(81473373,81874351,81673406);国家重大新药创制专项(2015ZX09101-016);北京市高层次卫生技术人才(2011-1-7,2014-2-014)~~

摘　　要：目的构建并表征tau及突变tau蛋白细胞和动物模型,检测模型tau蛋白及磷酸化tau蛋白表达情况及微管的形态,为tau蛋白及相关疾病的研究提供疾病模型。方法将不同的质粒分别转染到HEK293细胞中,构建过表达野生型tau以及P301L/P301S突变tau的细胞模型。引进并繁育rTg4510小鼠(P301L突变tau转基因小鼠)以及PS19小鼠(P301S突变tau转基因小鼠)。通过Western blotting法检测总tau及磷酸化tau蛋白的表达,应用免疫组织化学的方法检测转基因小鼠脑内磷酸化tau蛋白的表达情况,应用免疫荧光的方法检测HEK293细胞微管形态的变化。结果本研究成功构建过表达tau蛋白的细胞模型,繁育了两种tau转基因小鼠,在这些模型中tau蛋白表达及tau蛋白的磷酸化水平显著高于对照组。在不同突变类型的细胞和动物模型中,tau蛋白不同位点的磷酸化水平变化以及微管形态变化略有差异。结论本研究成功构建了P301L/P301S突变tau蛋白的细胞和动物模型,这些模型均表现了显著的tau蛋白过度磷酸化的病变,并引起了细胞微管形态或tau蛋白病理的变化,进而为包括AD在内的tau蛋白病变提供了实验模型。Objective To construct and characterize tau and mutant tau protein cell and animal models.Methods Different plasmids were transfected into HEK293 cells,and cell models overexpressing wild-type tau or P301L/P301S mutant tau were constructed.rT g4510 mice(P301L mutant tau transgenic mice) and PS19 mice(P301S mutant tau transgenic mice) were introduced and bred.The expression of total tau and phosphorylated tau protein was detected with Western blotting.The expression of phosphorylated tau protein in the brain of transgenic mice was detected with immunohistochemistry.The morphology of HEK293 cells was observed with immunofluorescence.Results In this study,cell and animal models overexpressing tau were successfully constructed.The tau protein expression in these models were significantly increased,and the phosphorylation levels of tau protein were significantly higher than that of the control group.In the cell and animal models of different mutation types,changes in phosphorylation levels and microtubule morphology at different sites showed slight differences.Conclusion We successfully constructed P301L/P301S mutant tau cell and animal models with significant tau hyperphosphorylation and changes in cell microtubule morphology.These models might provide alternative for proper disease model in the studies of tau and tauopathies,such as Alzheimer’s disease.

关 键 词：微管相关蛋白tau 细胞转染模型 转基因动物模型 微管 

分 类 号：R592[医药卫生—老年医学]