朊病毒感染与VCP关系初探

A preliminary study on the relationship between prion infection and VCP

为了探究感染朊病毒后含缬酪肽蛋白(valosin-containing protein,VCP)的变化情况及其对内质网相关蛋白降解(endoplasmic reticulum-associated degradation,ERAD)信号通路的影响,试验采用细胞免疫荧光和实时荧光定量PCR等方法分析朊病毒感染的细胞系中VCP及其基因表达情况;Western-blot方法检测朊病毒感染的细胞、小鼠脑组织中VCP含量变化,白藜芦醇动态清除朊蛋白(prion protein,PrP)的SMB-S15细胞、PrP重组质粒pcDNA3.1(+)-Cyto-PrP瞬时转染的293T细胞、蛋白酶体抑制剂MG-132处理的SMB-S15细胞中VCP的表达情况,以及朊病毒感染的细胞系中ERAD信号通路关键因子的表达水平。结果表明:与对照组相比,在朊病毒感染的细胞中VCP及基因表达水平差异不显著(P>0.05);在终末期小鼠脑组织中VCP表达水平差异不显著(P>0.05);经动态清除PrP、细胞内聚集PrP以及MG-132处理后,VCP表达水平均差异不显著(P>0.05);在朊病毒感染细胞中ERAD信号通路关键因子calnexin表达水平极显著升高(P<0.01),OS-9表达水平显著高于对照SMB-PS细胞(P<0.05)。说明感染朊病毒后VCP变化不显著,calnexin和OS-9发生明显变化,提示ERAD信号通路可能会参与朊病毒的复制过程。

To explore the changes of Valosin-containing protein(VCP) after prion infection and its influence on the endoplasmic reticulum-associated degradation(ERAD) signaling pathway, the expression of VCP in prion infected cells was analyzed by cell immunofluorescence and real time fluorescence quantitative PCR;the content of VCP protein in prion-infected cells and mouse brain tissue was detected by Western-blot;The expression of VCP in the SMB-S15 cells was treated with Resveratrol removal prion protein(PrP) and 293 T cells transiently transfected with PrP plasmid and the SMB-S15 cells were treated with proteasome inhibitor MG-132, and then the expression of VCP protein was detected. The protein expression levels of key factors of ERAD signaling pathway in prion-infected cell lines were detected. Results showed that compared with the control group, there was no significant change in VCP when prion infected cells(P>0.05). There was no significant change in VCP when prion infected brain tissue of end-stage animals(P>0.05). There was no significant change in VCP when dynamic scavenging of PrP and intracellular aggregation of PrP after transfected and after treatment with MG-132(P>0.05). The key factors of ERAD signaling pathway in prion infected cells Calnexin was significantly increased(P<0.01) and OS-9 was increased(P<0.05). It indicated that VCP was no significant change after prion infection, and the protein level of Calnexin and OS-9 was significantly increased than SMB-PS cell, suggesting that the ERAD signaling pathway may participate in the prion replication process.