斑马鱼cetp基因敲除模型的建立及其肝脏转录组学分析

Establishment of cetp^-/- model and liver transcriptomics study in zebrafish

目的建立斑马鱼cetp基因敲除模型,探讨cetp基因敲除后在动脉粥样硬化中的转录组学变化。方法采用CRISPR/Cas9基因编辑技术敲除斑马鱼cetp基因,构建cetp^-/-斑马鱼模型。采用转录组测序观察cetp基因敲除后基因表达谱变化,通过京都基因与基因组百科全书(KEGG)数据库对差异表达基因进行功能富集分析。结果采用CRISPR/Cas9技术敲除斑马鱼cetp基因,斑马鱼产生增加5个碱基的移码突变。转录组测序发现,cetp^-/-斑马鱼肝脏基因表达谱发生显著变化,共筛选到3808个发生显著变化的基因(P<0.05),其中显著上调基因有1918个,显著下调基因1890个。脂质代谢相关基因如abca1、abcg2a基因显著上调,lipg显著下调;与自噬相关信号通路基因如atg13显著上调;下调基因富集到DNA及RNA合成、三羧酸循环等相关通路。结论建立了斑马鱼cetp^-/-模型,可用于胆固醇酯转运蛋白(CETP)在动脉粥样硬化疾病中作用机制的研究。尽管抑制CETP可使高密度脂蛋白胆固醇升高而抑制动脉粥样硬化,但是斑马鱼cetp敲除后激活机体炎性反应而促进动脉粥样硬化,提示CETP在动脉粥样硬化发生发展的过程中具有双重调节作用。

Objective To investigate the transcriptomic changes in atherosclerosis after cetp knockout of zebrafish.Methods The gene editing technology CRISPR/Cas9 was used to knock out cetp of zebrafish to construct a homozygous mutant model.Total RNA of livers from wild type and cetp^-/- model was extracted by the Trizol method,and gene expression profiles of cetp knockout were observed by transcriptomics.KEGG enrichment analysis was performed on significantly different expressed genes to explore the signal pathways it may affect.Results Homozygous mutant zebrafish that has a frameshift mutation of adding 5 base pairs was created through CRISPR/Cas9 technology.Transcriptomics analysis showed that the gene expression profile of zebrafish liver changed significantly after cetp knockout.A total of 3808 different expressed genes were screened(P<0.05),of which 1918 genes were up-regulated and 1890 genes were down-regulated.Genes related to lipid metabolism such as abca1 and abcg2a were significantly up-regulated and lipg were significantly down-regulated.Genes related to autophagy signaling pathways such as atg13 were significantly up-regulated,and down-regulated genes were enriched in related pathways such as DNA and RNA synthesis and the tricarboxylic acid cycle.Conclusions A zebrafish model with cetp knockout is established to study the mechanism of cholesteryl ester transfer protein(CETP)in atherosclerotic diseases.Although increased high-density lipoprotein cholesterol due to the inhibition of CETP prevents atherosclero-sis,inflammation is activated after cetp knockout in zebrafish and causes atherosclerosis.It suggests that CETP has a dual regulation mechanism in the development of atherosclerosis.