摘要
目的探讨血液miRNA-548ah在慢性乙型肝炎病毒(HBV)感染不同时期的表达,并分析其临床价值。方法选取2015年1月至2019年1月恩施土家族苗族自治州中心医院收治的108例慢性HBV感染者为研究对象(HBV感染组),根据分期不同将108例患者分为免疫耐受期组(32例)、免疫清除期组(28例)、低复制期组(25例)和再活动期组(23例);另外选取同期100例健康体检者为对照组。采用采用Exo Quick试剂盒提取血液外泌体,并采用Western blot蛋白质印记法验证外泌体的真实性;采用实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测血液外泌体中miRNA-548ah水平,并收集其血清HBV DNA和丙氨酸氨基转移酶(ALT)水平。比较HBV感染组患者和对照组血液外泌体miRNA-548ah差异,并绘制ROC曲线探讨血液外泌体miRNA-548ah在慢性HBV感染不同时期中的应用价值,采用Pearson相关法分析血液外泌体miRNA-548ah与HBV DNA和ALT的关系。结果蛋白质免疫印迹法检测外泌体表面特异性标志物CD63和Tsg101蛋白阳性,提示外泌体提取成功。HBV感染组和对照组血液外泌体miRNA-548ah水平分别为(3.46±0.83)和(0.72±0.15),差异有统计学意义(t=32.518、P <0.001);ROC曲线分析显示,外泌体miRNA-548ah在慢性HBV感染诊断中具有一定价值(AUC=0.785、95%CI:0.723~0.877、截断值:1.04、敏感性为84.5%、特异度为82.6%)。免疫耐受期组、免疫清除期组、低复制期组和再活动期组血液外泌体miRNA-548ah分别为(4.18±1.34)、(1.41±0.45)、(1.32±0.31)和(1.48±0.42),差异有统计学意义(F=27.435、P <0.001);其中免疫耐受期组和免疫清除期组患者差异有统计学意义(t=10.427、P <0.001),免疫清除期组和低复制期组患者差异无统计学意义(t=0.838、P=0.406);低复制期组和再活动期组差异无统计学意义(t=1.510、P=0.138)。Pearson相关分析表明,HBV感染免疫耐受期miRNA-548ah与HBV DNA、ALT均呈显著正相关(r=0.857、P <0.001,r=0.763、P <0.001);HBV感染再活动期患者miRNA-548ah与HBV DNA、ALT均呈显著正相关(r=0.776、P=0.001,r=0.711、P=0.001);HBV感染免疫清除期mi RNA-548ah与HBV DNA、ALT呈正相关,但相关性不显著(r=0.572、P=0.035,r=0.531、P=0.042);HBV感染低复制期miRNA-548ah与HBV DNA、ALT呈正相关,但相关性不显著(r=0.541、P=0.039,r=0.502、P=0.048)。结论血液外泌体mi RNA-548ah在慢性HBV感染者高表达,有望成为诊断慢性HBV感染的新型标志物,且其在HBV感染自然史不同阶段的表达存在差异,与血清HBV DNA和ALT亦相关,可用于指导临床用药并进行及时调整。
Objective To investigate the expression of microRNA-548 ah in blood at different stages of chronic hepatitis B virus(HBV) infection and to analyze its clinical value. Methods Total of 108 patients with chronic HBV infection admitted to the Central Hospital of Enshi Tujia and Miao Autonomous Prefecture from January 2015 to January 2019 were selected as HBV infection group. According to their different stages, 108 patients were divided into immune tolerance period group(32 cases), immune clearance period group(28 cases), low replication period group(25 cases) and reactivation period(23 cases). While 100 healthy people taking physical examination were selected as healthy control group. Exo Quick kit was used to extract blood exosomes, and protein imprinting was used to verify the authenticity of exosomes. The serum levels of HBV DNA and alanine aminotransferase(ALT) were measured by fluorescence quantitative RT-PCR. The difference of mi RNA-548 ah in blood exosomes between HBV infection group and healthy control group were compared, respectively, and the value of serum exosomes mi RNA-548 ah for chronic HBV infection and staging were analyzed by receiver operating curve(ROC). The relationship between serum exosome mi RNA-548 ah and HBV DNA and ALT were analyzed by Pearson correlation method. Results The positive expression of CD63 and Tsg101 protein were detected by Western blot, which indicated that exosome was successfully extracted. The levels of micro RNA-548 ah in blood exosomes of HBV infection group and control group were(3.46 ± 0.83) and(0.72 ± 0.15), respectively, with significant difference(t = 32.518, P < 0.001). ROC analysis showed that exosome micro RNA-548 ah had a certain value in the diagnosis of chronic HBV infection(AUC: 0.785, 95%CI: 0.723-0.877, truncation value: 1.04, sensitivity: 84.5%, specificity: 82.6%). The levels of micro RNA-548 ah in blood exosomes of the immune tolerance period group, the immune clearance period group, the low replication period group and the reactivation period group were(4.18 ± 1.34),(1.41 ± 0.45),(1.32 ± 0.31) and(1.48 ± 0.42), respectively, with significant difference(F = 27.435, P < 0.001);there was significant difference between immune tolerance period group and immune clearance period group(t = 10.427, P < 0.001), but without significant difference between immune clearance group and low replication period group(t = 0.838, P = 0.406), neither between low replication group and reactivation group(t = 1.510, P = 0.138). Pearson correlation analysis showed that micro RNA-548 ah was positively correlated with HBV DNA(r = 0.857, P < 0.001) and ALT(r = 0.763, P < 0.001). There were significant positive correlations between micro RNA-548 ah and HBV DNA(r = 0.776, P = 0.001) and ALT(r = 0.711, P = 0.001);and micro RNA-548 ah in the immune clearance stage of HBV infection was positively correlated with HBV DNA(r = 0.572, P = 0.035) and ALT(r = 0.531, P = 0.042), but the correlation was not significant;micro RNA-548 ah in the low replication stage of HBV infection was positively correlated with HBV DNA(r = 0.541, P = 0.039) and ALT(r = 0.502, P = 0.048), but the correlation was not significant. Conclusions High expression of micro RNA-548 ah in blood exosome of patients was expected to become a new marker for the diagnosis of chronic HBV infection. There were significant differences in the expression of micro RNA-548 ah in different stages of natural history of HBV infection. It was also related to serum HBV DNA and ALT levels, which could guide the clinical medication and timely adjustment.
作者
刘蜜
向田
李叶静
朱焦
Liu Mi;Xiang Tian;Li Yejing;Zhu Jiao(Department of Laboratory Medicine,The Central Hospital of Enshi Tujia and Miao Autonomous Prefecture,Enshi Tujia and Miao Autonomous Prefecture 445000,China;Department of Pathology,The Central Hospital of Enshi Tujia and Miao Autonomous Prefecture,Enshi Tujia and Miao Autonomous Prefecture 445000,China)
出处
《中华实验和临床感染病杂志（电子版）》
CAS
2021年第5期337-343,共7页
Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)
作者简介
通信作者:朱焦,Email:pumelo2025@163.com。
