摘要： 目的·提取和鉴定腹膜透析滤出液（peritoneal dialysis effluent，PDE）外泌体，比较不同腹膜转运特性患者PDE外泌体miR-200a的表达水平，预测其潜在靶基因及参与的生物学过程。方法·将20例腹膜透析（peritoneal dialysis，PD）患者按照腹膜平衡实验（peritoneal equilibrium test，PET）值分为H组（高转运/高平均转运）和L组（低转运/低平均转运）。每位患者收集500 mL留腹过夜PDE，浓缩后超速离心，透射电子显微镜（transmission electron microscope，TEM）、纳米粒子跟踪分析（nanoparticle tracking analysis，NTA）和Western blotting鉴定外泌体。提取外泌体miRNA，实时荧光定量PCR检测2组患者PDE外泌体miR-200a的表达水平，分析其与PET值和超滤量的相关性。利用Targetscan、miRmap和miRWalk数据库预测miR-200a的靶基因，用DAVID数据库进行基因功能（gene ontology，GO）富集分析。结果·浓缩的PDE超速离心后提取到的外泌体，TEM下呈双层膜包裹的圆形囊泡，NTA显示其直径大多在50~150 nm，表面具有外泌体标志蛋白CD9和CD63。L组中PDE外泌体miR-200a的相对表达量显著高于H组，并且与PET值呈负相关（
r=-0.871，
P=0.000），与4 h超滤量呈正相关（
r=0.448，
P=0.048），但与24 h超滤量无相关性（
r=0.355，
P=0.125）。生物信息学分析发现共有679种基因可由3个数据库共同预测到；GO分析显示这些靶基因不仅参与正向调控间充质细胞增殖，也在金属离子结合方面有一定富集。结论·PDE中存在外泌体，腹膜转运特性较低的患者PDE外泌体miR-200a相对表达量较高，其与PET值呈负相关，与4 h超滤量呈正相关。miR-200a的潜在靶基因众多，可能通过不同生物学过程影响腹膜转运特性。
关键词: 腹膜透析滤出液, 外泌体, miR-200a, 腹膜转运特性, 生物信息学 Abstract: Objective·To extract and identify the exosomes in peritoneal dialysis effluent (PDE), compare the different expression levels of PDE exosomal miR-200a in patients with different peritoneal transport characteristics, predict the potential target genes of miR-200a and analyze the related biological processes.
Methods·Twenty stable peritoneal dialysis (PD) patients were divided into two groups, i.e., H group (high/high average) and L group (low/low average) according to the values of peritoneal equilibration test (PET). Overnight PDE 500 mL from each patient was collected, concentrated, and ultracentrifuged to obtain exosomes. Transmission electron microscope (TEM), nanoparticle tracking analysis (NTA) and Western blotting were used to identify and characterize the exosomes. Exosomal miRNAs were extracted and the expression levels of PDE exosomal miR-200a in the two groups were determined by real-time quantitative PCR. The relations between exosomal miR-200a levels and PET values or ultrafiltration volume (UF) were analyzed. The target genes of miR-200a were predicted by Targetscan, miRmap and miRWalk, and DAVID was used to perform gene ontology (GO) enrichment analysis.
Results·The exosomes extracted from concentrated PDE by ultracentrifugation exhibited a round and bilayer membrane-enveloped vesicle structure under TEM with the diameters ranging from 50-150 nm. The exosomes also expressed the particular molecular marker CD9 and CD63. The relative expression level of PDE exosomal miR-200a in L group was higher than that of H group. The expression levels were negatively correlated with PET values (r=-0.871, P=0.000) and positively correlated with 4 h UF (r=0.448, P=0.048). But there was no relationship between miR-200a and 24 h UF (r=0.355, P=0.125). Bioinformatic results showed that there were 679 target genes of miR-200a that could be predicted by all the three databases. GO analysis suggested that these genes not only participated in positive regulation of mesenchymal cells proliferation, but also focused on ion binding, especially metal cation binding.
Conclusion·Exosomes indeed exist in PDE. The relative expression level of exosomal miR-200a is higher in the PDE from the patients with low peritoneal dialysis transport characteristics. The relative quantity of miR-200a is negatively correlated with the values of PET and positively correlated with 4 h UF. There are abundant potential target genes of miR-200a, indicating that it may affect the peritoneal transport characteristics by different biological processes.
Key words: peritoneal dialysis effluent, exosome, miR-200a, peritoneal dialysis characteristics, bioinformatics PDF全文下载地址:
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