EV71入侵人神经母细胞瘤SK-N-SH细胞机制的初步研究

Mechanism of enterovirus 71 entering into human neuroblastoma cells

摘要:

目的：初步研究肠道病毒71型（enterovirus type 71，EV71）入侵人神经母细胞瘤SK-N-SH细胞的机制。方法：将临床EV71分离株接种于人横纹肌肉瘤（rhabdomyosarcoma，RD）细胞，扩增和纯化病毒；MTT法检测不同病毒胞吞途径阻断剂对SK-N-SH细胞生长抑制作用；用特异性的化学阻断剂预处理靶细胞后，TaqMan real-time PCR验证其对EV71 mRNA表达的影响。结果：RD细胞能够成功扩增出EV71病毒，病毒滴度为1×105 TCID50。随着药物浓度梯度的增加，SK-N-SH细胞的生长增殖受到抑制。TaqMan荧光定量RT-PCR结果显示氯丙嗪（chlorpromazine，CPZ）能够抑制EV71 mRNA的表达（Ρ＜0.05），制霉菌素（nystatin，NT）对其影响不大（Ρ ＞0.05）。结论：初步推测EV71入侵SK-N-SH细胞是通过网格蛋白依赖性的内吞作用入胞。

Abstract:

Objective：To research the mechanism of enterovirus type 71（EV71） entering into human neuroblastoma SK-N-SH cells. Methods：The clinical EV71 isolates were inoculated from hu－man rhabdomyosarcoma（RD） cells for amplifying and purify－ing virus. MTT assay was conducted to detect the effect of dif－ferent virus endocytosis blockers on inhibition of SK-N-SH cells. The impact of target cells SK-N-SH on EV71 mRNA expression was validated by TaqMan real-time PCR after treating by specific chemical blockers. Results：EV71 viruses were success－fully amplified in RD cells and the virus titer was 1×105 TCID50. With the increase of drug concentration gradient，the growth rate of SK-N-SH cell was inhibited. TaqMan real-time PCR showed that the expression of EV71 mRNA was inhibited by pretreatment of chlorpromazine（CPZ）（Ρ<0.05） and nystatin（NT） exerted little influence（Ρ>0.05）. Conclusion：EV71 invading into SK-N-SH cells by clathrin-dependent endocytosis is initially speculated.