miRNA对Fontan术后血小板活化程度及血栓形成影响的精确预测价值

VALUE OF microRNA IN PREDICTING PLATELET ACTIVATION AFTER FONTAN PROCEDURE AND ITS EFFECT ON THROMBOSIS

目的探讨微小核糖核酸(miRNA)对Fontan术后血小板活化程度及血栓形成影响的精确预测价值。方法选取单心室行Fontan术后病儿6例作为实验组(TG组),体检正常儿童6例作为对照组(CG组),分别提取两组血清、血小板和白细胞内miRNA;选取和心血管病相关的miRNA(miR-126、miR-150、miR-197、miR-223及miR-21),通过qRT-PCR检测其表达情况;通过流式细胞仪检测血小板活化程度;通过Western Blot方法检测经慢病毒转染抑制miR-223表达后对细胞核因子κB(NF-κB)信号通路蛋白表达的影响。结果 CG组和TG组血清中miR-126、miR-223表达水平比较,差异有显著性(t=3.22、3.45,P<0.05)。两组外周血血小板和白细胞中miR-223表达水平比较,差异有显著性(t=5.62、4.32,P<0.05)。流式细胞仪检测结果显示,TG组血小板中CD62p、CD41a的表达率较CG组显著升高(t=17.57、87.78,P<0.01);Western Blot检测结果显示,TG+病毒无转录抑制因子组(TC组)白细胞Iκb蛋白表达下调,p-p65蛋白表达上调;CG+病毒含转录抑制因子组(CI组)白细胞中Iκb蛋白表达上调,p-p65蛋白表达下调;与CI组组比较,TG+病毒含转录抑制因子组(TI组)白细胞中Iκb蛋白表达下调,p-p65蛋白表达上调;Ikkb和NF-κB蛋白表达在不同处理组间无明显变化。结论 Fontan术后病儿血清中miR-223表达水平升高,血小板活化率增加;miR-223表达升高后可能通过NF-κB信号路径来促进血小板活化和血管内皮损伤,从而影响血栓形成。

Objective To investigate the value of microRNA(miRNA)in predicting platelet activation after Fontan procedure and its effect on thrombosis.Methods Six children who underwent Fontan procedure for a single ventricle were enrolled as trial group(TG group),and six normal children who underwent physical examination were enrolled as control group(CG group).Serum,platelet,and leukocyte miRNAs were extracted.To Identify the miRNAs(miR-126,miR-150,miR-197,miR-223,and miR-21)associated with cardiovascular diseases,and qRT-PCR was used to measure their expression.Flow cytometry was used to evaluate the degree of platelet activation,and Western Blot was used to investigate the effect of miR-223 inhibition by lentiviral transfection on the nuclear factor-κB(NF-κB)signaling pathway.Results There were significant differences in the expression of miR-126 and miR-223 in serum between the two groups(t=3.22,3.45,P<0.05).There were also significant diffe-rences in the expression of miR-223 in platelets and leukocytes between the two groups(t=5.62,4.32,P<0.05).According to the Results of flow cytometry,the TG group had significantly higher expression of CD62p and CD41a in platelets than the CG group(t=17.57,87.78,P<0.01).According to the results of Western Blot,the TG+no transcription inhibitor group had downregulated expression of Iκb protein and upregulated expression of p-p65 protein in leukocytes;the CG+transcription inhibitor group had upregulated expression of Iκb protein and downregulated expression of p-p65 protein in leukocytes;compared with the CG+transcription inhibitor group,the TG+transcription inhibitor group had downregulated expression of Iκb protein and upregulated expression of p-p65 protein in leukocytes;there were no significant differences in the expression of Ikkb and NF-κB proteins between treatment groups.Conclusion There are increases in the expression of miR-223 in serum and platelet activation rate after Fontan procedure.Increased expression of miR-223 may promote platelet activation and vascular endothelial injury via the NF-κB signaling pathway and thus affects thrombosis.