| Objection The use of chemical drugs,Immunofluorescence staining,Taq Man QPCR,and Laser scanning confocal microscope to further study of the mechanism of human enterovirus 71 invasion of human neuroblastoma SK-N-SH cells on the basis of previous experiments.Material and Method Human enterovirus 71 and all cell lines derived from The Children's Hospital of Chongqing Medical University Research Institute,which are preserved by the previous experimental study.The first part of experiment was divided into the following three groups : CPZ treatment group(10,20 u M CPZ),NT treatment group(10,20ug/ml NT),and the blank group.Each group contained three parallel holes,and was repeated three times.EV71 infection experiments were performed after SK-N-SH cells were grouped according to different treatment conditions,finally,the Taq Man QPCR and immunofluorescence staining method were used to detect EV71 m RNA and VP1 protein expression levels in the target cells.The second part of the experiment is divided into CPZ treatment group(10,20 u M CPZ)and the blank group(no drug).Each group contained three parallel holes,and was repeated three times.EV71 infection experiments were performed after SK-N-SH cells were grouped according to different treatment conditions,finally,laser scanning confocal microscopy was used to detect the co-location of EV71 and invasion pathway marker molecule.Results 1.Taq Man QPCR results showed that CPZ could significantly inhibit the expression of m RNA EV71(p<0.05),while the effect of NT had no significance(p>0.05).2.Immunofluorescence staining showed that CPZ was able to inhibit the expression of EV71 VP1 protein significantly(p<0.05),while the effect of NT was not significant(p>0.05).3.Confocal laser scanning microscopy showed that EV71 was co-localized with the clathrin.Conclusion EV71 was further proved to be the invasion of SK-N-SH cells by the clathrin dependent endocytosis on the basis of previous studies. |
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