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VZV对Schwann细胞朊蛋白糖基化的影响及甲钴胺的调节作用

Effect of VZV on the cellular prion protein glycosylation of Schwann cells and the regulation of methylcobalamin

摘要目的:探究水痘-带状疱疹病毒(varicella-zoster virus, VZV)对人Schwann细胞(human Schwann cells, hSC)朊蛋白(cellular prion protein, PrP C)糖基化特征的影响,及甲钴胺(methylcobalamin, MeB 12)的调节作用。 方法:以感染复数1.0的VZV感染细胞48 h,加入250 μg/ml的MeB 12培养48 h,用抗体3F4分别包被上清和沉淀中PrP C,凝集素-ELISA法筛查PrP C的糖基化特征,并测定上清中超氧化物歧化酶(superoxide dismutase, SOD)活性与丙二醛(malondialdehyde, MDA)含量。 结果:VZV感染组细胞上清与沉淀中的PrP C聚糖比例与未感染组比较有明显变化,总聚糖比分别为1∶1.5和1∶2.6(F=24.18, P<0.001, LSD-t=8.27, P<0.001),提示VZV感染后PrP C稳定性下降,相应的SOD活性(4.43±2.05 U/mg)与未感染组(14.23±1.27 U/mg)比较明显下降(F=18.19, P=0.001, LSD-t=6.54, P<0.001),MDA水平(11.17±1.89 nmol/mg)与未感染组(3.73±0.35 nmol/mg)比较明显升高(F=30.70, P<0.001, LSD-t=8.25, P<0.001),差异均有统计学意义。加入MeB 12后,VZV感染细胞沉淀中的聚糖较VZV感染未加药组有明显增加,总聚糖比为1∶2.4,提示MeB 12增强了PrP C的稳定性,相应地SOD活性明显增高(11.07±2.07 U/mg, LSD-t=4.42, P =0.002),MDA水平明显下降(5.23±0.96 nmol/mg, LSD-t=6.58, P<0.001),与感染未加MeB 12组比较差异有统计学意义。 结论:VZV可改变hSC中PrP C的糖基化特征,而MeB 12可调节PrP C的糖基化特征,增强PrP C的稳定性,从而提高hSC的抗氧化能力。

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abstractsObjective:To explore the effects of varicella-zoster virus (VZV) on the glycosylation characteristics of cellular prion protein (PrP C) in human Schwann cells (hSC) and the regulation of methylcobalamin (MeB 12). Methods:The hSC were inoculated with VZV at 1.0 multiplicity of infection for 48 hours, then 250 μg/ml of MeB 12 were added and cultured for 48 hours. PrP C from the supernatant and sediment were coated with anti-PrPC antibody (3F4) respectively and subjected to screening for glycans by sandwich lectin-ELISA. Meanwhile, the superoxide dismutase (SOD) and malondialdehyde (MDA) from the supernatant were detected by diagnostic reagent kit. Results:The ratio of PrP C glycans in the supernatant to sediment of VZV-infected cells was found to be significantly different compared with those in the VZV-non-infected cells. The overall glycans ratios of the supernatant to the sediment was 1∶2.6 in the uninfected cells, while the ratio was 1∶1.5 in the VZV-infected cells (F=24.18, P<0.001, LSD-t=8.27, P<0.001), suggesting that stability of PrP C decreased after VZV infection, and correspondingly the activity of SOD (4.43±2.05 U/mg) was significantly reduced in the VZV-infected hSCs compared with those(14.23±1.27 U/mg) in the uninfected cells (F=18.19, P=0.001, LSD-t=6.54, P<0.001), the level of MDA (11.17±1.89 nmol/mg) was significantly elevated in the VZV-infected hSCs compared with those (3.73±0.35nmol/mg) in the uninfected cells (F=30.70, P<0.001, LSD-t=8.25, P<0.001). When the VZV-infected cells were added with 250 μg/ml MeB 12, glycans in the sediment of infected cells significantly increased compared with those in the VZV-infected cells without MeB 12, the overall glycans ratio of the supernatant to the sediment was 1∶2.4, suggesting that MeB 12 improved the stability of PrP C. Moreover, SOD activity (11.07±2.07 U/mg) was significantly increased (LSD-t=4.42, P=0.002), MDA level (5.23±0.96 nmol/mg) was significantly decreased (LSD-t=6.58, p<0.001) in the VZV-infected cells added with MeB 12 compared with those in the VZV-infected cells without MeB 12. Conclusions:The glycosylation characteristics of PrP C in hSC could be changed by VZV, while MeB 12 could regulate the glycosylation characteristics to improve the stability of PrP C, thereby increase the antioxidant capacity of hSC.

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