基于CRISPR/Cas9系统的猪内皮细胞UHRF1定向编辑及其对单核细胞趋化蛋白-1影响的研究

The targeted editing of UHRF1 based on CRISPR/Cas9 system and its effects on the expression of MCP-1

目的对基于CRISPR/Cas9系统的猪内皮细胞泛素样含PHD和环指域1(UHRF1)定向编辑进行研究,并分析其对单核细胞趋化蛋白-1表达的影响。方法靶向设计并构建UHRF1基因外显子区域gRNA序列载体,并将其与h Cas9载体共转染猪内皮细胞制备单克隆抗体,并分析单核细胞趋化蛋白-1表达变化。结果本研究共获重组质粒5个,转染后获得细胞株a、细胞株b和细胞株c三株UHRF1蛋白表达极低细胞株;UHRF1蛋白敲除后单核细胞趋化蛋白-1和MMP9、IL1、IL2及Bax蛋白表达分析水平明显降低而Bcl2表达明显升高,且与野生型细胞株相比差异有显著的统计学意义(P<0.05)。结论成功获得基于CRISPR/Cas9系统的猪内皮细胞UHRF1定向编辑,且获得细胞株内单核细胞趋化蛋白-1表达明显降低。

Objective The present research aimed to explore the targeted editing of UHRF1 based on CRISPR/ Cas9 system and its effects on the expression of MCP - 1. Methods gRNA were designed for targeting on UHRF1 gene. The gRNA vectors combined with hCas9 were co -transfected to endothelial cells. The expression of MCP - 1,apoptosis related proteins and MMP9 as well as interleukins were assayed. Results 5 strains of co - transfected plasmids were harvested. At the meaning time,3 strains that low UHRF1 protein expression were harvested. The re-sults also showed that the expression of MCP - 1,MMP9,Bax,IL1,IL2 were decreased greatly while the expression of Bcl2 protein was increased significantly when compared with wild type cell strains. Conclusion The present research successfully targeted editing the UHRF1 in endothelial cells based on CRISPR/ Cas9 system and the expression of MCP - 1 protein was decreased greatly in UHRF1 knock out cell lines.