【作者】 吴丹；

【导师】 王光辉；

【作者基本信息】 苏州大学 ， 药理学， 2018， 硕士

【摘要】 目的:自噬在神经退行性疾病中扮演着重要的角色。本研究考察囊泡相关膜蛋白相关蛋白B(VAPB)是否参与自噬调控及其机制研究。方法:我们分别在HEK293T、HeLa和SH-SY5Y三种细胞系中,转染了两种VAPB干扰小RNA来沉默VAPB的表达,利用蛋白免疫印迹法(Western blot)检测细胞中LC3和Beclin 1的蛋白水平。在HEK293T细胞中过表达FLAG-VAPB,检测细胞中的LC3和Beclin 1的蛋白水平。在HEK293中敲减VAPB,同时过表达GFP-LC3和FLAG-P62,通过免疫荧光的方法,检测GFP-LC3和FLAG-P62的共定位情况。在HEK293T细胞中沉默VAPB的表达,通过实时定量PCR(Real-time q PCR)检测Beclin1 m RNA水平的变化。在HEK293T细胞中同时沉默VAPB和Beclin 1的表达,检测细胞中的LC3蛋白水平。在m Cherry-LC3稳定转染的HEK293细胞中,同时沉默VAPB和Beclin 1的表达,检测细胞中m Cherry-LC3点状聚集的数量。在HeLa细胞中,敲减VAPB后过表达m Cherry-EGFP-LC3B,另一组加入自噬抑制剂Bafilomycin A1(Baf A1),最后在荧光显微镜下检测Cherry和GFP的点状聚集。在HEK293细胞中过表达GFP-HTT60、m Cherry-LC3和FLAG-P62,或者RFP-SOD1-G93A、GFP-LC3和FLAG-P62,通过免疫荧光的方法,检测三种蛋白的共定位情况。在HEK293细胞中敲减VAPB,然后过表达GFP-HTT60或RFP-SOD1-G93A,并加入蛋白酶体抑制剂MG132或自噬抑制剂Baf A1,在荧光显微镜下检测这些的点状聚集数量。结果:在HEK293T、HeLa和SH-SY5Y三种细胞系中,敲减VAPB显著地上调了LC3Ⅱ和Beclin 1的蛋白水平,而在HEK293T中过表达FLAG-VAPB则导致了LC3Ⅱ和Beclin 1的蛋白水平的下降。在HEK293中敲减VAPB后,GFP-LC3和FLAG-P62的共定位增多。敲减VAPB后,Beclin 1的m RNA水平增加。在敲减Beclin 1后,敲减VAPB则LC3Ⅱ水平和m Cherry-LC3的点状聚集不再增加。在HeLa细胞中敲减VAPB后转染m Cherry-EGFP-LC3B,我们发现红色荧光比黄色荧光的比值有所增加,这说明自噬流增强;敲减VAPB后,加入100 n M Baf A1处理4小时后,黄色荧光点的数量增多,这再次表明敲减VAPB起始自噬。在HEK293细胞中过表达GFP-HTT60、m Cherry-LC3和FLAG-P62,或者RFP-SOD1-G93A、GFP-LC3和FLAG-P62,这三个蛋白有共定位,说明GFP-HTT60或RFP-SOD1-G93A均与自噬体共定位。在HEK293细胞中敲减VAPB后,GFP-HTT60和RFP-SOD1-G93A的聚集在转染细胞中的比例下降,这说明RFP-SOD1-G93A或者GFP-HTT60的降解增加。在用MG132处理细胞后,这种蛋白聚集下降的现象仍然可见,而在用Baf A1处理细胞后,该现象消失,这说明GFP-HTT60和RFP-SOD1-G93A聚集体的降解与VAPB敲减所诱导的自噬有关。结论:敲减VAPB可以上调Beclin 1的转录表达,进而诱导自噬。VAPB缺失所诱导的自噬是依赖于Beclin 1的。VAPB敲减增强自噬流,并促进易聚集蛋白GFP-HTT60和RFP-SOD1-G93A的降解。更多还原

【Abstract】 Aim:Autophagy plays an important role in neurodegenerative diseases.This study examines whether vesicle-associated membrane protein-associated protein B(VAPB)plays a role in autophagy.Methods:VAPB was silenced by transfecting with two sets of si-RNAs in three cell lines including HEK293 T,He La and SH-SY5 Y.The protein levels of LC3 and Beclin 1 were detected with immunoblot analysis.FLAG-VAPB was transfected into HEK293 T cells,and then the levels of LC3 and Beclin 1 were detected.VAPB was knocked down in HEK293,and then GFP-LC3 and FLAG-P62 were transfected into HEK293 cells.The co-localization of GFP-LC3 and FLAG-P62 was detected by immunofluorescence assays.The expression of VAPB was silenced in HEK293 T cells,and the levels of Beclin 1 m RNA were detected by real-time quantitative PCR(RT-q PCR).Futhermore,Beclin 1 was silenced in VAPB knockdown HEK293 T cells,and then the levels of LC3 protein were detected.In m Cherry-LC3 stable transfected HEK293 cells,Beclin 1 was further silenced in VAPB knockdown cells,and then the number of m Cherry-LC3 puncta was detected.Finally,the level of autophagy flux was detected in VAPB deficiency and m Cherry-EGFP-LC3 B overexpressed He La cells.Dots of Cherry and GFP were detected using fluorescence microscopy.GFP-HTT60,m Cherry-LC3 and FLAG-P62,or RFP-SOD1-G93 A,GFP-LC3 and FLAG-P62 were transfected in HEK293 cells,and then the co-localization of these proteins was detected with immunofluorescence assays.In VAPB knockdown HEK293 cells,GFP-HTT60 or RFP-SOD1-G93 A was transfected and the proteasome inhibitor MG132 or autophagy inhibitor Baf A1 was administrated.The aggregation of these proteins was detected using fluorescence microscopy.Results:The loss of VAPB significantly increased the protein levels of LC3Ⅱ and Beclin 1 in HEK293 T,He La and SHSY-5Y,while the overexpression of FLAG-VAPB decreased the protein levels of LC3Ⅱ and Beclin 1 in HEK293 T.In HEK293 cells that VAPB was knocked down,the co-localization of GFP-LC3 and FLAG-P62 was increased,accompanied by an increase of the m RNA levels of Beclin 1.The increase of LC3Ⅱ resulting from the loss of VAPB was blocked by the knockdown of Beclin 1.In He La cells that VAPB was knocked down and m Cherry-EGFP-LC3 B was transfected,the ratio of red fluorescence to yellow fluorescence increased,which indicates increases of the autophagic flow.Moreover,in above cells after 4 hours of 100 n M Baf A1 treatment,the number of yellow fluorescence dots increased compared with the control,which was consistent with the result that loss of VAPB initiated the autophagy.We found that GFP-HTT60,m Cherry-LC3 and FLAG-P62,or RFP-SOD1-G93 A,GFP-LC3 and FLAG-P62 were co-localizated in HEK293 cells,indicating that the aggregates of GFP-HTT60 or RFP-SOD1-G93 A were co-localizated with autophagosomes.In HEK293 cells that VAPB was knocked down,the number of aggregates of GFP-HTT60 and RFP-SOD1-G93 A was decreased in the transfected cells,indicating an increased degradation of GFP-HTT60 or RFP-SOD1-G93 A.The aggregation degradation caused by VAPB deficiency was induced in the MG132 treated cells,but blocked in the Baf A1 treated cells,which indicated that the degradation of GFP-HTT60 or RFP-SOD1-G93 aggregation was related to the autophagy induced by VAPB deficiency.Conclusion:Knockdown of VAPB up-regulates Beclin 1 at a transcriptional level,then induces autophagy.And the autophagy induced by loss of VAPB is dependent on Beclin 1.VAPB deficiency enhances autophagy flow and promotes the degradation of GFP-HTT60 and RFP-SOD1-G93 A protein.更多还原