微小RNA-216a在急性胰腺炎并发肺损伤患者中的表达水平及其对内皮细胞通透性的影响

MiR-216a expression in acute pancreatitis patients with lung injury and its effect on the permeability of endothelial cell

目的:探讨微小RNA-216a(miR-216a)在AP并发肺损伤患者中的表达水平及其对内皮细胞通透性的影响。方法:收集2015年12月至2016年3月间海军军医大学第一附属医院消化内科收治的40例AP患者,根据患者是否并发急性肺损伤(ALI),分为AP伴ALI组(AP-ALI组,13例)和AP不伴ALI组(AP组,27例)。以8名健康志愿者为对照组。抽血分离血浆并抽提RNA,采用RT-PCR法检测3组血浆miR-216a水平。使用外泌体抽提试剂盒提取血浆中的外泌体,并通过电镜观察进行外泌体鉴定。抽提外泌体RNA,检测外泌体中miR-216a的水平。将AP-ALI组患者血浆外泌体分别与人脐静脉内皮细胞(HUVEC,AP-ALI-HUVEC组)、转染anti-miR-216a的HUVEC(AP-ALI-anti-miR-216a HUVEC组)共培养24 h,以未做任何处理的HUVEC作为对照组,用Millicell ERS-2上皮伏特欧姆计测量3组跨内皮细胞电阻,以表示其通透性。结果:AP-ALI组血浆miR-216a水平为14.45±1.64,显著高于AP组的11.08±1.60和对照组的5.37±1.54,差异有统计学意义(P值均<0.01)。电镜下可见血浆外泌体呈杯状膜泡样,大小50~90 nm。AP-ALI组血浆外泌体miR-216a水平为14.03±1.58,显著高于AP组的10.86±1.31和对照组的5.01±0.79,差异有统计学意义(P值均<0.01)。以对照组HUVEC细胞的电阻值为1,AP-ALI-HUVEC组跨内皮细胞的电阻比值显著低于AP-ALI-anti-miR-216a HUVEC组(0.74±0.04比1.02±0.08),差异有统计学意义(P<0.05)。结论:miR-216a水平在AP并发ALI患者血浆外泌体中显著增加。miR-216a可增加HUVEC细胞的通透性,可能与AP时肺损伤的发生相关。

Objective To explore the expression of miR-216a in patients with acute pancreatitis(AP)associated with acute lung injury(ALI)and its influence on endothelial cells permeability.Methods 40 AP patients admitted in Department of Gastroenterology of the First Affiliated Hospital of Navy Medical University from December 2015 to March 2016 were collected and were classified into AP with ALI(AP-ALI group,n=13)and AP without ALI(AP group,n=27)according to the presence or absence of ALI.8 normal volunteers were enrolled in the control group.Blood samples were collected and the plasma samples were separated.Plasma RNA was extracted.miR-216a level in plasma was detected by RT-PCR.Plasma exosomes were extracted by exosome extraction kit and identified by the electron microscopy.Exosome RNA was extracted.miR-216a level in exosome was detected by RT-PCR.Plasma exosomes of AP-ALI patients were co-cultured with human umbilical vein endothelial cells(HUVEC,AP-ALI-HUVEC group)and anti-miR-216a transfected HUVECs(AP-ALI-anti-miR-216a HUVEC group)for 24 hours,respectively,and untreated HUVECs served as control group.Trans-endothelium electrical resistance(TEER)was measured by Millicell Ers-2 epithelial volt-ohmmeter to evaluate the cell permeability.Results RT-PCR results showed that the expression level of plasma miR-216a in AP-ALI group(14.45±1.64)was significantly higher than that in AP group(11.08±1.6)and the control group(5.37±1.54)(P<0.01).Under electron microscope,plasma exosomes were goblet like vacuoles,with the size of about 50-90 nm.The plasma exosomal miR-216a level in the AP-ALI group(14.03±1.58)was significantly higher than that in the AP group(10.86±1.31)and the control group(5.01±0.79),and the difference was statistically significant(P<0.01).The resistance value of HUVEC in the control group was referred as 1,and the resistance ratio of HUVEC in AP-ALI-HUVEC group was 0.74±0.04,which was significantly lower than that of HUVEC in AP-ALI-anti-miR-216a HUVEC group(1.02±0.08),the difference was statistically significant(P<0.01).Conclusions miR-216a was highly expressed in plasma exosomes of AP patients with ALI.miR-216a can increase endothelial cell permeability,which may be associated with ALI during AP.