去氢骆驼蓬碱致细粒棘球蚴DNA损伤修复相关基因表达量的检测

Detection of Echinococcus granulosus genes related to repair of DNA damage caused by harmine

目的观察去氢骆驼蓬碱(HM)对细粒棘球蚴不同类型DNA损伤修复基因表达量的影响。方法以细粒棘球蚴为实验材料,以HM为药物,以虫体不同类型DNA损伤修复方式(同源重组修复、非同源末端连接修复、碱基切除修复)及核苷酸切除修复通路的关键修复基因为目的基因设计引物,筛选确定qRT-PCR反应体系,采用该体系检测基因的差异表达水平,分析HM对细粒棘球蚴不同类型DNA损伤修复相关基因的影响。结果筛选的细粒棘球蚴DNA损伤修复类型代表基因BRCA1引物F:5’-TGATTGCGACCTAAGAGAC-3’,R:5’-TTCCATACACAAGCCATCC-3’);KU70引物F:5’-TGATTGCGACCTAAGA GAC-3’,R:5’-GTCGCCATCTCAACTTCA-3’);OGG1引物5’-CGCTATGGTAAGAAGATGTG-3’,R:5’-CGTGAAGATGGATGGAGAA-3’);ERCC1引物F:5’-TCGTGCTCATTGTGTTAGT-3’,R:5’-CTCCTCTGGTGGCTTATTC-3’)。各基因扩增曲线Ct值可用,溶解曲线特异性良好。qRT-PCR检测各样品组BRCA1,KU70,OGG1基因表达均有不同程度上调,DOX组分别为26.05倍,323.44倍,29.63倍,HM组分别为7.24倍,55.65倍,3.22倍,以KU70和BRCA1基因上调尤为明显。ERCC1基因表达量DOX组显著上调(上调倍数25.24),HM组无显著变化。结论 HM对DNA双链断裂修复类型代表基因BRCA1,KU70,OGG1表达量有较大影响,而对ERCC1基因表达无显著影响。

Objective To observe the effects of harmine(HM) on the expression of different genes related to the repair of DNA damage. Methods E. granulosus was treated with HM Primers were designed for the key genes that repair DNA damage in different ways(homologous recombination repair, non-homologous end joining repair, and base excision repair) and for target genes in the nucleotide excision repair pathway. A qRT-PCR system was prepared via primer design and screening. The system was then used to detect the differential expression of genes and to analyze the effects of HM on different E. granulosus genes related to the repair of DNA damage. Results The primer for BRCA1, a typical E. granulosus gene related to repair of DNA damage:(F: 5’-TGATTGCGACCTAAGAGAC-3’, R: 5’-TTCCATACACAAGCCATCC-3’), the primer for KU70:(F: 5’-TGATTGCGACCT AAGAGAC-3’, R: 5’-GTCGCCATCTCAACTTCA-3’), the primer for OGG1:(F: 5’-CGCTATG GTAAGAAGATGTG-3’, R: 5’-CGTGAAGATGGATGGAGAA-3’), and the primer for ERCC1:(F: 5 ’-TCGTGCTCATTGTGTTAGT-3’, R: 5’-CTCCTCTGGTGGCTTAT TC-3). Ct was determined for each gene amplification curve, and the melting curve was specific. The level of expression of BRCA1, KU70, and OGG1 in each sample group was adjusted to different degrees with qRT-PCR. Expression of BRCA1, KU70, and OGG1 was respectively 26.05-fold, 323.440-fold, and 29.63-fold in a DOX group and 7.24-fold, 55.65-fold, and 3.22-fold in an HM group. Up-regulation of the KU70 and BRCA1 genes was particularly marked. The level of expression of the ERCC1 gene was significantly up-regulated in the DOX group(upregulated 25.24-fold), and there were no significant changes in the HM group. Conclusion HM had a significant effect on the expression of BRCA1, KU70, and OGG1, i.e. genes related to the repair of DNA double-strand breaks, but it had no marked effect on expression of the ERCC1 gene.