Effects Of Heterozygous HTRA1 Gene Missense Mutation On Oxidative Stress Response In Mouse Brain-derived Endothelial Cells.3

Objective: CADASIL type 2 is associated with missense mutation of HTRA1 gene.To explore the possible molecular mechanism of HTRA1 missense mutation（NM₀02775.4,Exon4,c.905G>A,p.Arg302Gln）,this study constructs a mouse microvascular endothelial cell model infected by lentivirus vector,and reveals the influence of heterozygous HTRA1 missense mutation on oxidative stress response in rat brain-derived Endothelial cells.3.Methods: We cultured bEnd.3 cells and constructed the overexpressing lentiviral vectors of HTRA1,then infected the cells.The experiment was conducted in five groups,they are blank control（NULL group）,negative control（NC group）,wild overexpression（WT group）,heterozygous mutation overexpression（Het group）and homozygous mutation overexpression.The five groups of stable strains were obtained suffering screening Puromycin.The expression of ROS in each groups was tested by DCFH-DA;The NOX4 mRNA expression was detected by fluorescence quantitative PCR and the expression of NOX4 protein was determined by Western Blot.Results: Five groups of cell stable strains are successfully constructed.The level of intracellular oxidative stress is measured by DCFH-DA,there is no significant difference in intracellular fluorescence expression between NULL group and NC group（P>0.05）;intracellular fluorescence expression in Het group and Hom group increases by 2 times（P<0.05）and 2.6 times（P<0.05）than NC group;compared with WT group,the intracellular fluorescence expression in Het group and Hom group increases by 1.75 times（P<0.05）and 2.3 times（P<0.05）;there is no difference between Het group and Hom group.According to fluorescence quantitative PCR,the expression of NOX4 mRNA in cells is detected.There is no significant difference in intracellular NOX4 mRNA expression between NULL group and NC group（P>0.05）;intracellular NOX4 mRNA expression in WT group,Het group and Hom group increases by 0.11 times（P>0.05）,1.03 times（P>0.05）and 2.37 times（P>0.05）than NC group;intracellular NOX4 mRNA expression is increased by 0.83 times（P<0.05）and 2.03 times（P<0.05）in Het group and Hom group compared with WT group;intracellular NOX4 mRNA expression in Hom group is 0.66 times higher than that in Het group（P<0.05）.The expression of NOX4 protein in cells is determined by Western Blot.There is no difference in intracellular NOX4 protein expression between NULL group and NC group（P>0.05）;compared with NC group,intracellular NOX4 protein expression in WT group,Het group and Hom group is increased by 0.02 times（P>0.05）,0.17 times（P>0.05）and 0.40 times（P<0.05）;intracellular NOX4 protein expression is up-regulated by 0.15 times（P>0.05）and 0.37 times（P<0.05）in Het group and Hom group compared with WT group;intracellular NOX4 protein expression in Hom group is 0.20 times higher than in Het group（P<0.05）.Conclusion In this study,the experimental cell stable strain model was successfully constructed.Heterozygous HTRA1 gene missense mutation resulted in increased intracellular reactive oxygen species,NOX4 mRNA and NOX4 protein expression levels of bEnd.3,which initially reveale the effect of Heterozygous HTRA1 gene missense mutation on intracellular oxidative stress response of bEnd.3;In bend.3 cells,NOX4 expression was decreased at the transcription and translation levels between missense mutation of HTRA1 gene and homozygous mutation of HTRA1 gene,which will enrich the possible mechanism of the difference from the perspective of oxidative stress response.