摘要
目的构建阿尔茨海默病(Alzheimer′s disease,AD)Tau蛋白异常磷酸化体外模型,探究磷酸化TauS214对细胞焦亡及炎症的影响。方法以NCBI中人源Tau40(h-Tau40)基因序列为模板设计突变引物,采用聚合酶链反应(polymerase chain reaction,PCR)扩增目的基因,经限制性内切酶(restriction endonuclease,RE)BglⅡ和EcoR I双酶切后定向克隆到载体pEGFP-C1中,构建Tau突变体(TauS214E、TauS214A、TauP301L),测序鉴定正确后将质粒瞬时转染至HEK293T和HT22细胞,免疫荧光法检测重组Tau蛋白定位情况,蛋白质印迹法检测pTauS214、pTauS404和Tau5的表达,检测NLR家族Pyrin域蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白酶1(Caspase-1)、Gasdermin D N端片段(GSDMD-N)、白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)、白细胞介素-6(IL-6)的表达水平。结果成功构建Tau蛋白重组质粒并在两种细胞中稳定表达;Tau40和TauP301L组中pS214表达显著降低,pS404表达升高(P<0.05),NLRP3、Caspase-1、GSDMD-N、IL-1β、IL-18、IL-6表达显著增加(P<0.05);TauS214E与Tau40相比,pS214表达显著升高,pS404表达显著降低(P<0.05),NLRP3、Caspase-1、GSDMD-N、IL-1β、IL-18、IL-6表达显著降低(P<0.05)。结论Tau40和TauP301L通过调节Tau蛋白磷酸化水平显著促进细胞焦亡和炎症反应,而TauS214E位点抑制炎性小体激活或阻断细胞焦亡。
Objective To establish an in vitro model of abnormal Tau protein phosphorylation in Alzheimer's disease(AD)and investigate the effect of phosphorylated TauS214 on cellular pyroptosis and inflammatory responses.Methods Designed mutagenic primers based on the human Tau40(h-Tau40)gene sequence from NCBI.Amplified the target gene by polymerase chain reaction(PCR).Performed double digestion with restriction endonucleases(RE)BglⅡand EcoRⅠ,followed by directional cloning into the pEGFP-C1 vector.Constructed the Tau mutants(TauS214E,TauS214A,TauP301L)and validated by sequencing.The plasmid was transiently transfected into HEK293T and HT22 cells after sequencing identification.Immunofluorescence(IF)was used to detect the localization of recombinant Tau protein,and Western blot(WB)was used to detect the expression levels of pTauS214,pTauS404,and Tau5,as well as NLR family Pyrin domain protein 3(NLRP3),Cysteinyl aspartate-specific proteinase-1(Caspase-1),Gasdermin D-N-terminal fragment(GSDMD-N),interleukin 1β(IL-1β),interleukin 18(IL-18),and interleukin 6(IL-6).Results The recombinant Tau protein plasmid was successfully constructed and stably expressed in two types of cells.In the Tau40 and TauP301L groups,pS214 expression was significantly reduced,while pS404 expression was increased(P<0.05).Additionally,the expression levels of NLRP3,Caspase-1,GSDMD-N,IL-1β,IL-18,and IL-6 were significantly elevated(P<0.05).Compared with the Tau40 group,the TauS214E group exhibited significantly higher pS214 expression but lower pS404 expression(P<0.05),along with significantly reduced expression of NLRP3,Caspase-1,GSDMD-N,IL-1β,IL-18,and IL-6(P<0.05).Conclusion Tau40 and TauP301L significantly promote pyroptosis and inflammatory responses by modulating Tau protein phosphorylation levels,whereas the TauS214E site inhibits inflammasome activation or blocks pyroptosis.
作者
陈慧琳
操蓉
文亿
王能琴
齐晓岚
唐智
CHEN Huilin;CAO Rong;WEN Yi;WANG Nengqin;QI Xiaolan;TANG Zhi(Key Laboratory of Endemic and Ethnic Diseases,Ministry of Education&Key Laboratory of Medical Molecular Biology,Guizhou Medical University,Guiyang 550004,Guizhou,China)
出处
《贵州医科大学学报》
2025年第8期1093-1105,1119,共14页
Journal of Guizhou Medical University
基金
国家自然科学基金项目(82360281)。
