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Ankfy1与Sacsin相互作用参与常染色体隐性痉挛性共济失调(ARSA)致病机制的研究
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作者 付容 丁曼 卢祖能 《卒中与神经疾病》 2023年第2期111-117,共7页
目的探讨体外研究Ankfy1(Ankyrin repeat and FYVE domain containing 1)基因敲低后细胞内Sacsin molecular chaperone(SACS)基因的表达水平变化及体内研究条件性敲除小鼠小脑浦肯野细胞内Ankfy基因后SACS基因表达水平变化,确定Ankfy1与... 目的探讨体外研究Ankfy1(Ankyrin repeat and FYVE domain containing 1)基因敲低后细胞内Sacsin molecular chaperone(SACS)基因的表达水平变化及体内研究条件性敲除小鼠小脑浦肯野细胞内Ankfy基因后SACS基因表达水平变化,确定Ankfy1与Sacsin蛋白相互作用关系在常染色体隐性痉挛性共济失调疾病中的发病机制。方法体外培养人胶质母细胞瘤细胞A172细胞和人胚肾细胞293T细胞,随机分为对照组和短发夹RNA(Short hairpin RNA,shRNA)干扰Ankfy1组,即shRNA-Ankfy1组;对照组加入空载慢病毒,shRNA-Ankfy1组加入敲低Ankfy1的慢病毒,使用实时定量聚合酶链反应(Real-time quantitative polymerase chain reaction,RT-qPCR)法检测细胞内SACS的信使核糖核酸(Messenger RNA,mRNA)水平,使用免疫荧光检测Sacsin在细胞内的分布和荧光密度;使Cre重组酶(Cre recombinase)切除Flox(Locus of X-over P1)位点间的靶向基因的技术(Cre-Flox系统)条件性敲除C57BL/6J小鼠的小脑浦肯野细胞内Ankfy1,将此小鼠作为条件性敲除CKO(Conditional gene knoctout)组,同窝未敲除Ankfy1小鼠作为对照组,使用RT-qPCR法检测小鼠小脑内SACS的mRNA水平,使用免疫荧光检测小脑内Sacsin的荧光密度。结果体外细胞实验发现,与对照组比较,shRNA-Ankfy1组内SACS的mRNA表达水平增高(P<0.05),Sacsin蛋白平均荧光强度增加(P<0.05),且在细胞内分布不均,出现聚集;体内小鼠实验得出,与对照组小鼠比较,CKO组小鼠小脑SACS的mRNA表达水平增高(P<0.05),Sacsin在小脑颗粒层和浦肯野细胞层荧光强度增加,在分子层内出现聚集。结论Ankfy1下调可能导致Sacsin运输障碍,进而导致其在细胞内出现聚集和代偿性增加,推测Ankfy1引起小脑性共济失调的可能机制是阻断Sacsin的运输,同时也表明常染色体隐性遗传性共济失调的异质性,各致病基因之间可能具有复杂的网络关系。 展开更多
关键词 ankfy1 SACS Sacsin 常染色体隐性痉挛性共济失调 遗传异质性
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Ankfy1参与遗传性共济失调的发病机制及其对自噬的影响
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作者 唐洁 丁曼 卢祖能 《卒中与神经疾病》 2021年第3期263-267,共5页
目的探讨Ankfy1(Ankyrin repeat and FYVE domain containing 1,Ankfy1)基因敲低或过表达后对细胞自噬影响,体外实验研究Ankfy1基因突变参与遗传性共济失调的发病机制。方法培养人恶性脑胶质瘤细胞系(U87)细胞,建立Ankfy1基因敲低和过... 目的探讨Ankfy1(Ankyrin repeat and FYVE domain containing 1,Ankfy1)基因敲低或过表达后对细胞自噬影响,体外实验研究Ankfy1基因突变参与遗传性共济失调的发病机制。方法培养人恶性脑胶质瘤细胞系(U87)细胞,建立Ankfy1基因敲低和过表达的细胞模型,运用电镜扫描观察细胞内的自噬小体;运用Western blot方法检测自噬相关蛋白丝氨酸/苏氨酸蛋白激酶(Serine/threonine-protein kinase,ULK1)、磷酸化丝氨酸/苏氨酸蛋白激酶(Phosphorylation serine/threonine-protein kinase,pULK1)、泛素结合蛋白p62(Sequestosome 1,p62)、微管相关蛋白LC3(Microtubule-associated protein 1 light chain 3 alpha,LC3)、溶酶体相关膜蛋白1(Lysosomal associated membrane protein 1,Lamp1)和溶酶体相关膜蛋白2(Lysosomal associated membrane protein 2,Lamp2)的水平。结果扫描电镜观察发现Ankfy1敲低组和Ankfy1过表达组未见完整的自噬小体,但可见形态不规则的自噬溶酶体;对照组可见丰富的自噬小体。敲低组和过表达组的自噬相关蛋白的ULK1,pULK1,LC3,Lamp1和Lamp2的表达水平均较对照组高,p62的表达水平均较对照组低。当Ankfy1基因上调或者下调时会对细胞环境造成应激状态,诱导自噬发生,引起自噬通量的增加,但自噬溶酶体形态异常、自噬溶酶体通路受损。结论Ankfy1基因的上调或者下调会导致细胞自噬溶酶体通路异常,这也许是遗传性共济失调发病的机制之一。 展开更多
关键词 ankfy1 共济失调 发病机制 自噬
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Identification of novel genes associated with duck OASL in response to influenza A virus
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作者 WANG Xiao-xue LU Chang +6 位作者 RONG En-guang HU Jia-xiang XING Yan-ling LIU Zheng-yu GAO Chu-ze LIU Jin-hua HUANG Yin-hua 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2019年第7期1451-1459,共9页
2′-5′-Oligoadenylate synthetase like protein(OASL) plays a key role in response to viral infections through selectively activating the OAS/RNase L or OASL/RIG-I signaling pathway.Although classic pathway of OASL is ... 2′-5′-Oligoadenylate synthetase like protein(OASL) plays a key role in response to viral infections through selectively activating the OAS/RNase L or OASL/RIG-I signaling pathway.Although classic pathway of OASL is well-known,its regulated genes or co-actors are largely unknown.To study the possible molecular mechanism of duck OASL(dOASL),we performed RNA-sequencing(RNA-seq) and immunoprecipitation and mass spectrometry(IP-MS) at the level of mRNA and protein,respectively.For RNA-seq,we used DF1 cell lines(DF1 dO ASL+/+,DF1 cO ASL–/–,and DF1) with or without the CK/0513 H5 N1 virus(A/chicken/huabei/0513/2007) infection.1 737 differentially expressed genes(DEGs) were identified as candidate target genes regulated by dOASL.Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis and Weighted Correlation Network Analysis(WGCNA) were performed.We identified one important yellow co-expression module correlated with antiviral immune response.In this module,Ankyrin repeat and FYVE domain containing 1(ANKFY1),harboring a BTB domain similar to the methyl CpG-binding protein 1(MBD1) which bound to OASL in human,was regulated by dOASL.At protein level,133 host proteins were detected.Interestingly,ANKFY1 was one of them binding to dOASL protein.Further phylogenomic and chromosomal syntenic analysis demonstrated MBD1 was absent in birds,while mammals retained.It is suggested that OASL-ANKFY1 interaction might act as a compensatory mechanism to regulate gene expression in birds.Our findings will provide a useful resource for the molecular mechanism research of dOASL. 展开更多
关键词 DUCK OASL ankfy1 COMPENSATORY MECHANISM INFLUENZA A VIRUS
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