PERV的整合对HEK293细胞中HERV-W mRNA表达的影响

Effects of the integration of PERV on HERV-W mRNA expression in HEK293 cells

目的明确PERV的整合是否影响HEK293细胞中HERV-W各基因表达水平。方法根据GenBank中登录的基因序列,分别设计HERV-W gag、HERV-W pol、HERV-Wenv、syncytin-1和humanβ-actin引物,并分别建立SYBR Green Ⅰ荧光定量PCR检测方法,用于检测HEK293、HEK293-PERV中各基因mRNA的表达。结果本研究建立的检测方法均具有良好的特异性、敏感性和稳定性,标准曲线的相关系数大于0.99,扩增效率介于95%~110%,可用于检测。结论检测结果经2^(-△△Ct)分析后,发现与对照相比,PERV整合后的HEK293-PERV中HERV-Wgag、HERV-Wpol、HERV-Wenv和syncytin-1 mRNA相对表达量分别升高37.08倍、42.56倍、2.49倍和13.17倍,为进一步明确PERV在异种移植中的安全性提供参考。

To find out whether the integration of PERV in HEK293 cells influence the expression profile of HERV-W,based on the gene sequences of HERV-W in GenBank,the primers of HERV-Wgag,pol,env,envfrom locus 7q21.2 genes as well as humanβ-actin were synthesized respectively and were used to develop the means of SYBR Green Ⅰ real-time reverse transcription-quantitative polymerase chain reaction（RT-qPCR）to detect the mRNA expression of these genes in HEK293 cells and PERV-infected HEK293 cells.Experiments showed that these RT-qPCR methods were of good specificity,sensitivity and stability：the standard curves could ensure the correlation coefficients to be all above 0.99 and the amplification efficiency were between 95% and 110%,which verified that these methods could be used to detect the mRNA of HERV-W and humanβ-actinin culture cells.Through the detection and analysis of relative gene expression data using the 2^（-△△Ct） method,we found that the mRNA level of HERV-Wgag,pol,env,envfrom locus 7q21.2 genes as well as humanβ-actinin PERV-infected HEK293 cells increased,after integration,by 37.08,42.56,2.49 and 13.17 times than in non-infected HEK293 cells,respectively.Results provide a reference to further evaluate the safety of PERV pathogen in xenotransplantation.