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目的确定5种亚型溶血磷脂酸受体(LPAR)基因在大鼠心肌细胞中的表达,针对主要分布亚型LPAR3基因构建重组质粒。方法利用实时荧光定量PCR和Western Blot检测LPA受体亚型在原代培养乳鼠心肌细胞的表达量;克隆LPAR3基因的编码区全序列,构建重组质粒。结果在5种LPAR亚型中,LPAR3在心肌细胞中的表达最高,其次为LPAR1,LPAR4和LPAR5在心肌细胞中的表达较低,而LPAR2则未检测到;Western Blot检测显示LPAR3的表达明显高于LPAR1的表达。对LPAR3基因进行序列分析,结果显示GenBank中序列号为NM_023969.1的序列与其它物种的该序列比较,缺失了一段保守的碱基片段;根据另一条序列(AB051164.1)设计引物,成功扩增LPAR3全基因片段,构建重组质粒。结论 LPAR3是大鼠心肌细胞中主要的溶血磷脂受体亚型;GenBank中序列号为NM_023969.1的LPAR3基因序列存在碱基缺失,大鼠中编码LPAR3的正确序列为AB051164.1。
Objective To determine the expression of LPAR gene in rat cardiomyocytes and to construct a recombinant plasmid targeting LPAR3 gene. Methods Real-time quantitative PCR and Western Blot were used to detect the expression level of LPA receptor subtype in primary cultured neonatal rat cardiomyocytes. The complete coding region of LPAR3 gene was cloned and the recombinant plasmid was constructed. Results Among the five LPAR subtypes, LPAR3 had the highest expression in cardiomyocytes, followed by LPAR1, LPAR4 and LPAR5 in cardiomyocytes but not LPAR2. Western Blot showed that LPAR3 expression was significantly higher Expression of LPAR1. Sequence analysis of LPAR3 gene showed that the sequence of GenBank with sequence number NM_023969.1 was deleted from the conserved sequence of other species, and the other sequence (AB051164.1) LPAR3 whole gene fragment, construct recombinant plasmid. Conclusion LPAR3 is a major lysosomal phospholipid receptor subtype in rat cardiomyocytes. The base sequence of LPAR3 gene in GenBank is NM_023969.1, and the correct sequence of LPAR3 in rat is AB051164.1.