甲钴胺对VZV病毒感染施万细胞中朊蛋白和TNF-α转换酶表达的影响  

The effects of methylcobalamin on the expression of cellular prion protein and TNF-αconverting enzyme in VZV-infected Schwann cells

作　　者：许纲[1] 周朝生[1] 唐维桢[1] 张雨 徐刚[1] 程超[1] 许洁[1] 王晓梅[1] Xu Gang;Zhou Chaosheng;Tang Weizhen;Zhang Yu;Xu Gang;Cheng Chao;Xu Jie;Wang Xiaomei(Zoster-associated Pain Center,Shanghai Tenth People’s Hospital,Affiliated Tenth People’s Hospital of Tongji University,Shanghai 200072,China)

机构地区：[1]同济大学附属第十人民医院上海市第十人民医院带状疱疹神经痛诊疗中心,上海200072

出　　处：《神经解剖学杂志》2022年第2期171-178,共8页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(81771209)。

摘　　要：目的:探究感染水痘-带状疱疹病毒(VZV)对人施万细胞(hSC)朊蛋白(PrP^(C))、肿瘤坏死因子-α转换酶(TACE)和肿瘤坏死因子-α受体1(TNFR1)表达的影响,及甲钴胺(MCbl)的调节作用。方法:将传代培养的hSC分为:空白对照组、VZV感染组、VZV感染加MCbl干预组、VZV感染PrP^(C)基因(Prnp)siRNA转染细胞组、VZV感染Prnp-siRNA转染细胞加MCbl干预组。设计合成Prnp的siRNA序列转染hSC,构建PrP^(C)表达下调的hSC模型。以感染复数为1.0的VZV分别感染后4组细胞3 d后,加药组分别加入250μg/ml的MCbl干预。感染7 d后采用CCK-8法评估细胞活力,real time RT-PCR检测Prnp mRNA表达,Western Blot分析PrP^(C)糖基化的变化,免疫荧光双染检测TACE和TNFR1的表达,TACE试剂盒检测其酶活性的变化,酶联免疫吸附试验(ELISA)测定细胞上清液中可溶性TNFR1(sTNFR1)的浓度。结果:与对照组比较,感染组细胞活力明显下降(P<0.05)。感染组细胞Prnp的mRNA表达与对照组相比上调了2.7倍,PrP^(C)的单糖基化条带密度明显增加。TACE染色颗粒明显变小,TACE活性为对照组的36%,TNFR1表达与对照组比较明显增多,sTNFR1含量为(16.77±4.57)pg/ml。感染加药组Prnp mRNA表达上调3.7倍,显著高于感染组,PrP^(C)向单糖基化迁移的比例较感染组明显减少。TACE染色颗粒变大,且TACE的活性为对照组的76%,TNFR1表达较感染组减少,sTNFR1水平达到(231.23±41.04)pg/ml,较感染组明显增加。Prnp-siRNA转染细胞感染VZV后,TACE和TNFR1等表达及活性与VZV感染组比较无明显差异,sTNFR1含量与感染组比较无明显差异。Prnp-siRNA转染细胞感染VZV加药组对TACE和TNFR1的表达及活性调节作用不明显,sTNFR1含量与感染组比较无明显差异。结论:VZV可诱导hSC中PrP^(C)稳定性下降,TACE活性降低。而MCbl可稳定PrP^(C),调节TACE活性,促进TNFR1的脱落,从而增强hSC的抗TNF-α神经毒性能力。Objective:To explore the effects of varicella-zoster virus(VZV)on the glycosylation characteristics of cellular prion protein(PrP^(C)),tumor necrosis factor-α(TNF-α)converting enzyme(TACE)and TNF-αreceptor 1(TNFR1)expressions in human Schwann cells(hSC),and the regulation of methylcobalamin(MCbl).Methods:The subculture cells were divided into 5 groups,the control group,VZV infection group,VZV infection added with MCbl group,VZV infected with Prnp-siRNA hSC group,VZV infected with Prnp-siRNA hSC added with MCbl group.The siRNA for Prnp was designed,synthesis and transfected into the hSC,and cells of the specific downregulated PrP^(C) expression were constructed.The hSC of the latter 4 groups were inoculated with VZV at 1.0 multiplicity of infection,then 250μg/ml of MCbl were added to the culture medium at 3 days post-infection(DPI)in the treatment groups.The samples of each group were collected at 7 DPI,the cell viability was examined using Cell Count-kit 8(CCK-8),expression of Prnp mRNA was quantified by real time RT-PCR,and the glycosylation changes of PrP^(C) were evaluated using Western Blot.In addition,the samples were used to measure the expression of TACE and TNFR1 by double-labeling immunofluorescence staining,and then the sheddase activity was measured by using TACE activity assay kit,the concentration of soluble TNFR1(sTNFR1)in the culture supernatant was detected by enzyme-linked immunosorbent assay(ELISA).Results:Compared with the control group,VZV significantly reduced the cell viability of hSC in the VZV infection group(P<0.05).The relative level of Prnp mRNA was up-regulated by 2.7 times(P<0.05)and the density of monoglycosylated bands of PrP^(C) was increased compared with that in the control group(P<0.05).TACE staining particles were significantly smaller,the TACE activity decreased to 36%to the control group(P<0.001),the expression of TNFR1 was significantly increased compared to the control group,the content of sTNFR1 in the culture supernatant was(16.77±4.57)pg/ml.When the VZV-infected cel

关 键 词：水痘-带状疱疹病毒 细胞型朊蛋白 肿瘤坏死因子-α转换酶 肿瘤坏死因子-α受体1 甲钴胺 施万细胞 

分 类 号：R752.12[医药卫生—皮肤病学与性病学]