维生素K代谢相关基因CYP4F2参与凝血因子FⅨ活化的研究

Vitamin K metabolize associated gene CYP4F2 is involved in activation of coagulation factor Ⅸ

摘要:

目的 探索维生素K代谢相关基因CYP4F2对维生素K依赖性凝血因子活化(凝血因子FⅨ为例)的影响。方法 构建同时含有CYP4F2-cDNA片段与FⅨ-cDNA片段的pIRES质粒,通过酶切验证。使用Lipofectamine 2000 转染试剂,将构建成功的pIRES-FⅨ-CYP4F2质粒以及pIRES-FⅨ质粒、pIRES空白对照质粒瞬时转染LO2细胞。在不同质粒转染的细胞中,通过Western blotting验证CYP4F2和FⅨ蛋白的表达,应用FⅨa Assay检测试剂盒检测 FⅨa活性。结果 经酶切证实,pIRES-FⅨ-CYP4F2质粒构建成功。Western blotting证实,pIRES-FⅨ及pIRES-FⅨ-CYP4F2质粒成功转染LO2细胞。FⅨa Assay检测发现,在过表达FⅨ的LO2细胞培养上清和细胞裂解液中,FⅨa活性较对照组显著升高；当同时过表达CYP4F2和FⅨ后,LO2细胞培养上清和细胞裂解液中的FⅨa活性较单独过表达FⅨ组明显降低。结论 维生素K代谢相关基因CYP4F2的过表达对维生素K依赖性FⅨ的活化具有抑制作用。

Abstract:

Objective To determine the effect of vitamin K metabolize associated gene CYP4F2 on the activation of vitamin K dependent coagulation factor Ⅸ (FⅨ). Methods The pIRES plasmid containing CYP4F2-cDNA fragment and FⅨ-cDNA fragment was constructed, and enzyme digestion was used to verify the products. The plasmids pIRES-FⅨ-CYP4F2, pIRES-FⅨ and pIRES(control plasmid) were transfected into LO2 cells respectively with Lipofectamine 2000 transfection reagent. Western blotting was used to detect the expression of CYP4F2 and FⅨ.The activity of FⅨa was detected by FⅨa activity assay kit. Results The results of enzyme digestion indicated that the pIRES-FⅨ-CYP4F2 plasmid was successfully constructed. The plasmids pIRES-FⅨ and pIRES-FⅨ-CYP4F2 were successfully transfected into LO2 cells. FⅨa activity was significantly higher in the supernatants and cell lysates from the FⅨ-overexpressed LO2 cells than the control cells, but the activity was significantly reduced in the LO2 cells with the over-expression of FⅨ and CYP4F2 than the cells with the over-expression of FⅨ. Conclusion The over-expression of CYP4F2 exerts inhibitory effect on the activation of vitamin K dependent FⅨ.