miR-181b通过靶向PTEN调控缺血性脑卒中后血管新生的作用和机制研究.pdf

Correspouxlman生物工程359.BME&ClinMed,May2023,Vol.27,No.3生物医学工程与临床2 0 2 3年5月第2 7 卷第3期网络出版时间：2 0 2 3-0 4-2 116：13:30 D0I：10.13339/j.c n k i s g l c.2 0 2 30 42 0.0 19网络出版地址：https:/ 5只，鼠龄1012周，体质量50 2 8 0 g。随机分为4组，即假手术组、模型组、miR-NC组和miR-181b组。除假手术组外，其他3组采用大脑中动脉线栓法建模，miR-NC组和miR-181b组大鼠分别尾静脉注射miR-NC及miR-181b质粒。治疗2 周后，评价大鼠改良神经功能缺损评分（mNSS）。进行脑组织2,3,5-氯化三苯基四氮唑（TTC)染色，免疫荧光检测脑组织簇分化抗原34(CD34）、血管内皮生长因子（VEGF)表达。对人脐静脉血管内皮细胞（HUVEC)进行转染，分为Control组（正常培养）、miR-NC组（转染miR-NC空质粒）及miR-181b组（转染miR-181bmimics质粒）。CCK-8检测细胞增殖能力。划痕实验检测细胞迁移能力。成管实验检测成管能力。荧光素酶报告实验检测miR-181b与磷酸酯酶-张力蛋白同源物基因（PTEN)的靶向关系。实时荧光定量PCR(qRT-PCR)检测细胞、组织miR-181b表达水平。Westernblot检测细胞、组织中PTEN蛋白表达水平。结果与假手术组比较，模型组大鼠mNSS、脑梗死面积升高（t=17.420,34.000,P0.05）。与miR-NC组比较，miR-181b组大鼠mNSS、脑梗死面积降低（t=14.730,21.160,P0.05）。与假手术组比较，模型组大鼠脑组织中miR-181b表达（0.140.0 2），CD34阳性细胞率（45.0 3%5.2 2%），VEGF阳性细胞率（2 5.0 6%5.11%），CD34、VEG F蛋白表达水平（0.2 50.0 6、0.2 10.0 4）下降,PTEN蛋白表达水平（0.930.0 5）升高（P0.05）。与miR-NC组比较，miR-181b组大鼠脑组织中miR-181b表达（3.0 2 0.45），CD34阳性细胞率（6 8.7 3%7.0 4%），VECF阳性细胞率（0.2 3%0.0 6%），CD34、V ECF蛋白表达水平（0.8 6 0.12、0.7 2 0.0 9）升高PTEN蛋白表达水平（0.37 0.02）下降（P0.05）。m iR-18 1b 组细胞miR-181b表达水平，细胞2 4h、48 h 及7 2 hOD值、迁移率，节段长度、分支长度、连接数和网孔数高于miR-NC组（P0.05）。m i R-18 1b 可负性调控PTEN表达。结论过表达miR-181b可靶向PTEN改善脑缺血再灌注损伤大鼠的神经功能，促进血管新生。关键词：miR-181b；人张力蛋白同源物基因（PTEN)；缺血性脑卒中；血管新生中图分类号：R743.3文献标识码：A文章编号：10 0 9-7 0 9 0(2 0 2 3)0 3-0 359-0 8Effect and mechanism of miR-181b in regulating angiogenesis after ischemic stroke via targeting PTENZHANGYe,ZHUANG Xue-ming,WANG Jing,XU Hai-ting,WANG Zhong-xiang,WANG Shi-bo,ZHANG Li,TANG Guang-(Department of Emergency,the 904th Hospital of Joint Support Force of the Chinese Peoples Liberation Army,W214000,Jiangsu,China)nding author:TANG Guang-man.E-mail:.Abstract:Objective To investigate the effect and mechanism of miR-181b on angiogenesis after ischemic stroke.MethodsA total of 65 SPF grade male SD rats were sacrificed,with age of 10-12 weeks and body mass of 50-280 g.All of the ratswere randomly divided into 4 groups:sham group,model group,miR-NC group and miR-181b group.Except control group,other three groups were modeled by middle cerebral artery occlusion method,the rats in miR-NC group and miR-181b groupwere injected with miR-NC and miR-181b plasmid through tail vein,respectively.After treatment for 2 weeks,the modifiedneurological severity score(mNSS)was evaluated.The expression of cluster differentiation antigen 34(CD34)and vascular en-dothelial growth factor(VEGF)in brain tissue was detected by immunofluorescence.The human umbilical vein endothelial cell(HUVEC)were transfected and divided into control group(normal culture),miR-NC group(transfected miR-NC empty plasmid)and miR-181b group(transfected miR-181b mimics plasmid).The cell proliferation ability was detected by cell counting kit-8(CCK-8)assay;cell migration ability was detected by scratch assay;tube formation assay was used to detect tube formationaility;the targeting relationship between miR-181b and human tensin homologue gene(PTEN)was detected by luciferase re-porter assay;quantitative real-time fluorescence polymerase chain reaction(qRT-PCR)was used to detect expression level ofmiR-181b in cells and tissues;Western blot was used to detect expression levels of PTEN,CD34 and VEGF protein in cells and作者单位：中国人民解放军联勤保障部队第九四医院急诊科，江苏无锡2 140 0 0作者简介：张烨（198 4一），男，江苏徐州市人，本科，主治医师，主要从事急诊脑卒中、胸痛中心和外周血管手术治疗工作。电话：0 510-8 5142 16 0。E-mail:。基金项目：无锡市卫生健康委员会科研项目（Z201909）通信作者：唐广满（198 5），男，山东泰安市人，研究生，主治医师，主要从事脊柱侧弯治疗工作。电话：158 52 50 6 96 3。E-mail：40 9536 37 0 q q.c o m。版权保护，不得翻录。360-BME&Clin Med,May 2023,Vol.27,No.3生物医学工程与临床2 0 2 3年5月第2 7 卷第3期tissues.Resultss Compared with sham group,mNSS and cerebral infarction area of model group were increased(t=17.420,34.000,P 0.05).Compared with miR-NC group,mNSS and cerebral infarction area in miR-181b group were decreased(t=14.730,21.160,P 0.05).Compared with sham group,the expression of miR-181b(0.14 0.02),CD34 positive rate(45.03%5.22%),VEGF positive rate(25.06%5.11%),CD34 and VEGF protein expression levels(0.25 0.06,0.21 0.04)in braintissue of model group were decreased,and PTEN protein expression level(0.93 0.05)increased(P 0.05).Compared with miR-NC group,miR-181b expression(3.02 0.45),CD34 positive rate(68.73%7.04%),VEGF positive rate(0.23%0.06%),CD34 and VECF protein expression levels(0.86 0.12,0.72 0.09)in brain tissue of miR-181b group were increased,whilePTEN protein expression level(0.37 0.02)decreased(P 0.05).MiR-181b negatively regulated PTEN expression.The expres-sion level of miR-181b,optical density(OD)value,migration rate,segment length,branch length,connection number and meshesof cells at 24-hour,48-hour and 72-hour in miR-181b group were higher than those in miR-NC group(P 0.05).The miR-181b negatively regulated PTEN expression.Conclusion It is demonstrated that overexpression of miR-181b could target PTENto improve neural function and promote vascularization in rats with cerebral ischemia-reperfusion injury.Key words:miR-181b;human tensin homologue gene;ischemic stroke;vascular neuro