脂筏在柯萨奇病毒A组16型感染RD细胞中的作用

The role of lipid rafts in CA16 infecting RD cells

目的探究脂筏在柯萨奇病毒A组16型(CA16)感染RD细胞中的作用。方法使用胆固醇抑制剂甲基-β-环糊精(MβCD)驱散细胞膜胆固醇并破坏脂筏,分别采用RT-PCR和Western blot技术,在mRNA以及蛋白质水平检测MβCD处理对于CA16病毒感染RD细胞的影响。在RD细胞感染不同时间使用MβCD处理,并检测其对CA16病毒感染的影响。MβCD处理后,使用添加外源胆固醇的方式对细胞膜胆固醇进行补充,观察CA16病毒感染的变化。通过Western blot检测CA16感染后宿主细胞Akt磷酸化水平的变化,以及MβCD处理对宿主细胞Akt磷酸化的影响。结果 MβCD以剂量依赖方式抑制CA16病毒对RD细胞的感染,MβCD处理浓度为2.5、5、7.5、10 mmol/L时CA16 VP1的mRNA水平和蛋白质水平表达均出现降低,差异均具有统计学意义(P<0.05)。在感染前1 h使用MβCD处理CA16 VP1 mRNA和蛋白质水平的表达分别降低了(84.23±3.70)%和(81.80±5.09)%,与未做处理的对照组相比具有统计学意义(P<0.05)。在MβCD处理后添加200、300、400μg/ml的外源胆固醇,VP1 mRNA水平的表达恢复了(26.63±7.69)%、(77.06±6.91)%、(91.36±12.09)%,VP1蛋白质水平的表达恢复了(28.71±8.27)%、(51.14±5.82)%、(58.14±7.35)%,且VP1表达与MβCD单独处理相比差异均具有统计学意义(P<0.05)。CA16感染30 min时pAkt表达上升了(91.86±27.14)%,差异具有统计学意义(P<0.05)。CA16感染MβCD预处理过的细胞pAkt未发生显著变化(P>0.05)。结论脂筏在CA16病毒感染RD细胞与激活PI3K/Akt通路中起重要作用。

Objective To investigate the role of lipid rafts in CA16 infecting RD cells. Methods MβCD, an inhibitor of cholesterol, was used to remove cytomembrane cholesterol and destroy lipid rafts. The effects of MβCD treatment on CA16 infection were determined using RT-PCR and Western blotting to measure levels of mRNA and protein. Cells were treated with MβCD at different times during infection, and its effect on CA16 infection was observed. Cholesterol was supplemented after MβCD treatment, and then the effect of cholesterol supplementation on CA16 infection was determined. The level of phosphorylation of Akt in host cells after CA16 infection and the effect of MβCD treatment on the phosphorylated Akt were determined using Western blotting. Results MβCD inhibited CA16 from infecting RD cells in a dose-dependent manner. The levels of CA16 VP1 mRNA and protein decreased after treatment with 2.5, 5,7.5, or 10 mmol/L of MβCD. Differences in those levels were significant(P<0.05). The levels of CA16 VP1 mRNA and protein decreased significantly(84.23±3.70% and 81.80±5.09%) with MβCD treatment 1 h prior to CA16 infection compared to levels in untreated cells(P<0.05). The level of mRNA increased 26.63±7.69% with 200 μg of cholesterol supplementation after MβCD treatment, 77.06±6.91% with 300 μg of cholesterol supplementation, and 91.36±12.09% with 400 μg of cholesterol supplementation. The level of protein increased 28.17±8.27% with 200 μg of cholesterol supplementation, 51.14±5.82% with 300 μg of cholesterol supplementation, and 58.14±7.35% with 400 μg of cholesterol supplementation. The increase in the levels of VP1 mRNA and protein was significant compared to levels in cells treated with MβCD(P<0.05). The expression of phosphorylated Akt increased significantly(91.86±27.14%) 30 min after infection(P<0.05). The level of phosphorylated Akt did not change significantly after MβCD treatment(P>0.05). Conclusion Lipid rafts play an important role in CA16 infection of RD cells and the activation of the PI3 K/Akt pathway.