颈动脉超声联合血清miR-34a检测对动脉粥样硬化脑梗死发生及梗死体积的关系分析.pdf

LIU Lin-lin.WANG Xiao.WANGXin-Vi临床研究447BME&Clin Med,July 2023,Vol.27,No.4生物医学工程与临床2 0 2 3年7 月第2 7 卷第4期网络出版时间：2 0 2 3-0 6-2 8814:49:15D0I:10.13339/ki.sglc.20230626.017网络出版地址：https:/ 0 2 0 年10 月邢台市第一医院治疗的152 例脑梗死患者（脑梗死组），其中男性8 5例，女性6 7 例；年龄497 1岁，平均年龄6 0.2 3岁；病程412 h，平均病程8.0 6 h。选择同期在医院行健康体检的6 0 例（对照组）,其中男性39例，女性2 1例；年龄49 7 1岁，平均年龄6 0.0 9岁。行颈动脉超声检查，采用实时荧光定量聚合酶链式反应检测血清miR-34a水平。比较两组颈动脉超声和miR-34a水平,Pearman法分析颈动脉超声和miR-34a水平与相关性，单因素分析临床资料,Logistic回归分析确定动脉粥样硬化脑梗死发生的独立影响因素，通过受试者工作特性(ROC)曲线分析各指标及回归分析诊断效能。结果脑梗死组患者颈动脉内膜中膜厚度(IMT)和miR-34a明显高于对照组(1.57 0.18 us0.670.11,2.340.58vs1.050.30。P=0.0 0 0 0.0 0 0）。脑梗死体积与IMT和miR-34a均呈正相关（r=0.888、0.7 8 8,P=0.000)。Lo g i s t i c 回归分析表明,IMT比值比（OR)=2.726,95%可信区间（CI)1.5874.683和血清miR-34a（O R=2.199,95%CI1.2753.792)等指标是脑梗死发生的独立影响因素(P0.05）。根据R0C曲线可得,IMT诊断的临界值为1.2 3mm，其灵敏度为6 1.8 4%，特异度为7 6.6 7%,R0C曲线下面积（AUC）为0.8 0 1(95%CI0.7500.852)；血清miR-34a诊断的临界值为1.55,其对应的灵敏度为6 3.16%，特异度为7 6.6 7%,AUC为0.7 6 0(95%CI0.7070.812)。将回归预测方程作为新变量P,在最佳临界切点时，回归分析灵敏度度为8 9.47%，特异度为8 0.0 0%,AUC为0.8 6 9(95%CI0.8310.900),回归分析灵敏度和AUC显著高于各指标单独检测(P0.05。结论梗死体积与颈动脉超声和血清miR-34a成正相关，颈动脉超声和血清miR-34a可应用于单独预测动脉粥样硬化脑梗死的发生，且联合诊断预测脑梗死诊断效能更高，值得临床进一步研究和推广。关键词：颈动脉超声；微小RNA-34a；动脉粥样硬化；脑梗死；梗死体积；诊断效能中图分类号：R743.3文献标识码：A文章编号：10 0 9-7 0 9 0(2 0 2 3)0 4-0 447-0 5Correlation analysis of carotid ultrasound combined with serum miR-34a on occurrence and volume of atheroscle-roticcerebralinfarction(Department of Ultrasound,The First AfiliatedHospital of Xingtai Medical College,Xingtai054000,Hebei,China)Abstract:Objective To explore the correlation between carotid ultrasound combined with serum miR-34a and occurrenceas well as volume of atherosclerotic cerebral infarction.Methods From November 2018 to October 2020,a total of 152 pa-tients with cerebral infarction were enrolled,which included 85 males and 67 females,aged 49-71 years old with mean ageof 60.23 years old;course of disease was 4-12 days with mean course of 8.06-hour.Sixty healthy were set as control group,which included 39 males and 21 females,aged 49-71 years old with mean age of 60.09 years old.The carotid ultrasonogra-phy was performed,and serum miR-34a level was detected by real-time fluorescence quantitative polymerase chain reaction.The carotid ultrasound and miR-34a level between 2 groups was compared,and Pearman method was used to analyze correla-tion between carotid ultrasound and serum level of miR-34a.The clinical data were analyzed by univariate analysis.Logisticregression analysis was used to determine the independent influencing factors of atherosclerotic cerebral infarction.The receiv-er operating characteristic(ROC)curve was used to analyze the diagnostic efficacy of each index and regression analysis.Re-sults The carotid intima-media thickness(IMT)and miR-34a in cerebral infarction group were significantly higher than those incontrol group(1.57 0.18 vs 0.67 0.11,2.34 0.58 vs 1.05 0.30.P=0.000,0.000).The volume of cerebral infarction waspositively correlated with IMT and miR-34a(r=0.888,0.788,P=0.000).Logistic regression analysis showed that IMTodds ratio(OR)=2.726,95%confidence interval(CI)1.587-4.683)and serum miR-34a(OR=2.199,95%CI 1.275-3.792)were inde-pendent influencing factors of cerebral infarction(P 0.05).According to ROC curve,the critical value of IMT diagnosis was 1.23 mm,the sensitivity was 61.84%,the specificity was 76.67%,and area under R0C curve(AUC)was 0.801(95%CI 0.750-0.852).The critical value of serum miR-34a diagnosis was 1.55,with corresponding sensitivity of 63.16%,specificity of 76.67%,and作者单位：邢台医学高等专科学校第一附属医院（邢台市第一医院）超声医学科，河北邢台0 540 0 1作者简介：刘琳琳（198 8），女，河北邢台市人，本科，主治医师，主要从事超声波医学研究。电话：1510 32 990 55。E-mail:。基金项目：邢台市重点研发计划项目（2 0 2 0 ZC196）版权保护，不得翻录。448-BME&Clin Med,July 2023,Vol.27,No.4生物医学工程与临床2 0 2 3年7 月第2 7 卷第4期AUC of 0.760(95%CI 0.707-0.812).The regression prediction equation was used as novel variable P,the sensitivity ofregression analysis was 89.47%,specificity was 80.00%,and AUC was 0.869(95%CI 0.831-0.900)at optimal criticalpoint.The sensitivity and AUC of regression analysis were significantly higher than those of each index(P 0.05).ConclusionIt is demonstrated that infarction volume is positively correlated with carotid ultrasound and serum miR-34a,carotid ultrasoundand serum miR-34a could be used to predict occurrence of atherosclerotic cerebral infarction alone,combined diagnosis showedhigher diagnostic efficiency in predicting cerebral infarction,which is worthy of further clinical research and promotion.Key words:atherosclerosis;microRNA-34a;atherosclerosis;cerebral infarction;infarct volume;diagnostic performance脑梗死系脑部血液循环功能性障碍而诱发局部脑组织软化或坏死的心血管疾病，意识障碍、昏迷等是其常见临床表征，具有发病率高、疾病进展快及预后差等特征,威胁患者生命安全1.2 。目前临床治疗脑梗死及早恢复脑组织血液灌注为主要目标，治疗方式为脑保护、降低颅内压、溶栓及手术介人治疗等3。研究发现4,脑梗死的及早发现并对症治疗,可有效挽救因缺氧、缺血而受损的脑组织，增加神经元的合成和分泌，恢复患者神经功能，优化预后，但目前临床对疾病的预测尚无有效指标。颈动脉超声是常见筛查脑部血管的影像学,其中以颈动脉内膜中膜厚度(intima-mediathickness,IMT)最为常见，可表征血管斑块稳定性，但其具有延滞性,无法预测疾病的发生，存在局限性5,。血清学指标因其取样简便、检测成本低廉及时效性强，已成为临床常见的筛选疾病首选标志物。miRNA是常见的非编码RNA，广泛存在机体中,其可与mRNA特异性结合,发挥生理功能,在肿瘤、免疫系统、造血功能障碍和心血管疾病中具有诊断效能和疾病预测价值，其中miR-34a与机体内皮细胞损伤和功能紊乱存在相关性,而脑梗死的初始病变亦为血管内皮细胞的坏死或凋亡,可能存在关系7.8 。因而笔者分析颈动脉超声联合血清miR-34a检测对动脉粥样硬化脑梗死发生及梗死体积的关系，旨在为预测脑梗死发生和改善预后等临床提供依据。1资料与方法1.1临床资料选择2 0 18 年11月至2 0 2 0 年10 月邢台市第一医院治疗的152 例脑梗死患者,其中男性8 5例,女性67例；年龄49 7 1岁，平均年龄6 0.2 3岁（标准差4.03岁）；病程412 h，平均病程8.0 6 h（标准差2.05h）。选择同期在医院行健康体检的6 0 例（对照组）,其中男性39例,女性2 1例；年龄49 7 1岁，平均年龄6 0.0 9岁（标准差4.18 岁）。此次研究试验方案经笔者所在医院伦理委员审核并批准。选择标准：均符合中国急性缺血性脑卒中诊治指南中脑梗死诊断标准9;年龄18 7 5岁；首次发作，发病至人院时间 7 2 h，并在7 2 h内完成脑部MRI扫描和颈动脉超声等影像学检测，存在动脉粥样硬化斑块；临床病例资料齐全、完整；患者或其家属知情同意此次研究。排除标准：既往合并心肌梗死或冠心病等心血管疾病者；合并免疫系统或脑部器质性病变者；合并脑、肺、大肠等部位恶性肿瘤者；合并心脏、肾脏及肺部功能等器官衰竭者；不配合基线资料采集者。两组年龄和性别比较，差异无统计学意义(t/x=0.225、1.46 0,P=0.8 2 2、0.2 2 7)1.2方法1.2.1颈动脉超声采用德国SiemensS200彩色多普勒超声诊断仪，探头为9L4线阵，频率为4MHz。造影剂为Bracco公司的SonoVue造影剂，应用0.9%氯化钠溶液（生理盐水)稀释摇匀后使用。检测时,患者行平卧体位,并在颈部准备垫枕，头部位置偏向对侧，检测左侧从颈动脉开始，右侧从头臂动脉分叉处开始，依次、逐一检测颈动脉，正常的颈动脉血管呈现线状,IMT1.0mm;存在早期动脉粥样硬化斑块,1.0 mmIMT 10 cm,中小体积10 cm3O1.2.3血清miR-34a检测分别采集患者入院次日或对照组当日清晨空腹肘静脉血约2 mL,应用德国SIGMA台式高速冷冻离心机3-18 KS离心机分离血清，参数：2 50 0 r/min，时间15min，半径8 cm，温度4，于-7 0 保存，统一检测。实时荧光定量聚合酶链式反应（polymerasechainreaction,PCR)法检测血清miR-34a。总RNA的提出使用Trizol试剂盒盒（上海梵态生物科技有限公449BME&Clin Med,July 2023,Vol.27,No.4生物医学工程与临床2 0 2 3年7 月第2 7 卷第4期司，中国），反应条件为：95,5min;95,30 s;6 0，30s，共计循环40 次。计算miR-34a的相对表达量。miR-34a引物：上游5-TCTGTCTCTCTTGGCAGTGTCTT-3，下游 5-AATGGTTGTTCTCCACTCTCTCTC-3;U6引物：上游5-CTCGCTTCGGCAGCACA-3,下游:5-AACGCTTCACGAATTTGCGT-3。1.2.4单独及联合检测诊断效能以影像学为金标准，绘制颈动脉超声和miR-34a相对表达检测的单独和回归受试者工作特性(receiv-eroperatingcharacteristic,ROC）曲线图，血清指标诊断标准根据ROC曲线获得最佳诊断临界值，miR-34a数值高于临界值为阳性，并建立Logistic模型,评价二者联合诊断效能1.3统计学方法采用SPSS25.0统计软件。计数资料以n(%)表示，行?检验；计量资料以均数标准差表示，行t检验;Pearman相关分析梗死体积与颈动脉超声IMT值和血清miR-34a的关系,Logistic回归分析确定动脉粥样硬化脑梗死发生的独立影响因素；绘制IMT和miR-34a单独和联合检测ROC曲线，采用Han-ley-McNeil方法比较ROC曲线下面积（areaundercurve,AUC)。P 0.0 5为差异有统计学意义。2结果2.1两组患者颈动脉超声和miR-34a比较脑梗死组患者IMT和miR-34a均明显高于对照组(1.57 mm 0.18 mm vs 0.67 mm 0.11 mm;2.34 0.58vs1.050.30），差异有统计学意义（t=44.189、21.170,P=0.000,0.000)2.2脑梗死体积与颈动脉超声和miR-34a相关性分析脑梗死患者梗死体积为（9.341.0 9）cm,脑梗死体积与IMT和miR-34a均呈正相关（r=0.888、0.788,P=0.000,0.000)。见图1。2.52.03wu/LWI1.521.00.5r=0.888,P=0.000r=0.788,P=0.000006810121468101214脑梗死体积/cm3脑梗死体积/cm3图1脑梗死体积与IMT、m i R-34a 相关性Fig.1Correlation between cerebral infarction volume and IMT,miR-34a2.3Logistic 回归分析以单因素中有统计意义的两项指标为自变量，以是否发生动脉粥样硬化脑梗死为因变量，行非条件Logistic回归分析。结果表明,IMT比值比（oddsratio，OR）=2.7 2 6,95%可信区间（confidence interval,CI)1.5874.683,P=0.000）和血清miR-34a（0 R=2.199,95%CI1.2753.792,P=0.005）是脑梗死发生的独立影响因素(P0.05)。见表1。表1Logistic回归分析结果Tab.1Results of Logistic regression analysis自变量回归系数标准误WaldPOR95%CIIMT1.0030.27613.2060.0002.7261.587 4.683血清miR-34a0.7880.2788.0350.0052.1991.275 3.792常数项-0.6650.18912.3800.000一2.4颈动脉超声和miR-34a单独诊断及回归分析价值比较以IMT和miR-34a作为检验变量，有无脑梗死为状态标量，以灵敏度为Y轴，以“1特异性”为X轴绘制ROC曲线。根据ROC曲线可得（图2）,IMT临界值1.2 3mm，灵敏度为6 1.8 4%，特异度为76.67%,AUC 为 0.8 0 1(95%CI 0.750 0.852);血清miR-34a诊断的临界值为1.55，其对应的灵敏度为450-BME&Clin Med,July 2023,Vol.27,No.4生物医学工程与临床2 0 2 3年7 月第2 7 卷第4期63.16%，特异度7 6.6 7%,AUC为0.7 6 0（9 5%CI0.7070.812)。将回归预测方程作为新变量P,在最佳临界切点时，回归分析灵敏度8 9.47%，特异度80.00%,AUC为0.8 6 9(95%CI 0.8310.900),回归分析灵敏度和AUC显著高于各指标单独检测（P0.05)。见表2、3。3讨论动脉粥样硬化是脑梗死常见的病理基础,若不稳定性斑块出现破裂、脱落等情况，则可造成脑动脉堵塞、狭窄，进而诱发脑梗死；同时研究表明，颈动脉狭窄程度超7 0%的患者中脑梗死患病率高达13%12,13。临床实践显示14,脑梗死不可逆,预后和恢复情况与神经功能损伤程度相关，在溶栓、改善脑循环、抗脑水肿及降颅压等措施后,极易复发，且预后极差，造成失语、瘫痪等后遗症,对家庭和社会带来沉重负担，因而及早预测脑梗死发生,并评估危险因素,对脑梗死的治疗方案调整和改善患者生存质量具有重要价值。颈动脉超声具有经济、无创伤及便捷等优势，且IMT可定量评估斑块稳定程度，但其受限于图像分辨率和主1.0r0.80.6曲线来源0.4miR-34a-IMT一联合诊断0.2参考线000.20.40.60.81.01-特异度图2IMT和miR-34a单项检测和回归分析的ROC曲线图Fig.2ROC curves of IMT and miR-34a single detection andregression analysis表2 单项检测和回归分析ROC曲线下面积比较Tab.2 Comparison of area under ROC curve between single testand regression analysis指标AUC标准误95%CIPIMT0.8010.0360.7500.8520.000miR-34a0.7600.0330.707 0.8120.000回归分析0.8690.0290.831 0.900表3IMT和miR-34a单项检测及回归分析诊断脑梗死比较例(%)Tab.3ROC curves of IMT and miR-34a single detection and regression analysiscases(%)诊断指标灵敏度特异度阳性预测值阴性预测值IMT94/152(61.84)46/60(76.67)94/108(87.04)46/104(44.23)miR-34a96/152(63.16)46/60(76.67)94/110(85.45)46/102(45.10)回归分析136/152(89.47)48/60(80.00)136/148(91.89)48/64(75.00)36.2390.2572.03418.019P0.0000.8790.3620.000观因素,存在局限性，而在超声的基础上联合血清指标可有效提高脑梗死阳性检出率15。笔者试验显示血清miR-34a在脑梗死患者中异常表达，但具体应用于预测脑梗死研究较少，因而探讨颈动脉超声联合血清miR-34a检测对动脉粥样硬化脑梗死发生，为临床诊断提供参考价值。脑梗死的发生、发展与颈动脉粥样硬化程度、斑块稳定性及狭窄程度紧密相联，而颈动脉超声在探查颈动脉斑块体积、位置、数量、IMT等方面具有独特优势，可为临床预测、治疗脑梗死提供丰富、直观的解剖学信息。笔者研究显示，脑梗死组患者IMT明显高于对照组，且Logistic回归分析显示IMT是脑梗死发生的影响因素(P0.05),证实IMT与脑梗死的发展有关。进一步比较诊断效能,IMT临界值1.2 3mm,灵敏度为6 1.8 4%,特异度7 6.6 7%,AUC0.801(95%CI0.7500.852），具有一定的预测价值，诊断效能较低。梁蓉17 证实,颈动脉超声预测脑梗死发生的灵敏度为80.36%、特异度为7 2.0 0%，笔者研究灵敏度结果低于文献报道，可能与评判标准有关。笔者研究仅依靠定量IMT预测疾病,而文献以IMT、斑块纤维帽完整性综合判断，但单独颈动脉超声诊断效能均较低。miRNA是非编码蛋白的核糖核苷酸，在多种疾病中均具有重要作用,已成为心脑血管疾病预测和预后评估的潜在、重要血清标志物,可通过与mRNA结合调控机体细胞分裂、调亡等生物学反应，miR-34a表达于心脏、脑组织中，可调控脑卒中等疾病的进程。血管内皮细胞的功能失调与动脉粥样硬化有关，而miR-34a可介导血管内皮功能，间接参与机体斑块的形成18.19。笔者研究结果表明，脑梗死组患者血清miR-34a水平明显高于对照组，且ROC检测表明血清miR-34a诊断的临界值为1.55，其对应的灵敏度为6 3.16%、特异度7 6.6 7%,AUC为0.7 6 0(9 5%CI451.生物医学工程与临床2 0 2 3年7 月第2 7 卷第4期BME&Clin Med,July 2023,Vol.27,No.40.7070.812),单独诊断效能较低。笔者研究将IMT和miR-34a进行联合诊断并绘制ROC曲线，二者联合诊断的灵敏度显著高于单独的诊断效能，且保持较高的特异度和准确率，即颈动脉超声联合miR-34a可有效提高脑梗死检出率。此外，梗死体积的大小决定脑梗死患者的预后，笔者研究脑梗死体积与IMT和miR-34a均呈正相关（r=0.888、0.7 8 8，P=0.0 0 0），进一步论证二者应用于脑梗死发生预测的临床价值。综上所述，动脉粥样硬化脑梗死体积与颈动脉超声和血清miR-34a成正相关，颈动脉超声和血清miR-34a可应用于单独预测动脉粥样硬化脑梗死的发生，且联合诊断预测动脉粥样硬化脑梗死诊断效能更高，值得临床进一步研究和推广。参考文献：1 Jiang Q,Xiao S,Shu L,et al.Pituitary apoplexy leading tocerebral infarction:A systematic reviewJ.Eur Neurol,2020,83(2):121130.2 J Fujino Y,Kawasaki T,Kawamata H,et al.Cerebral infarctionwith pulmonary thromboembolism due to 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