PERV的整合对HEK293细胞中HERV-W mRNA表达的影响

摘要

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-W env、syncytin-1和humanβ-actin引物,并分别建立SYBRGreen 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-△△o分析后,发现与对照相比,PERV整合后的HEK293-PERV中HERV-W gag、HERV-W pol、HERV-W env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 H ERV-W in GenBank,the primers of H ERV-W gag,pol,env,env from 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-actin in 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-W gag,pol,env,env from locus 7q21.2 genes as well as humanβ-actin in 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.