波形蛋白在B族链球菌引发新生儿脑膜炎中的作用及其机制  

Role and mechanism of vimentin in neonatal meningitis induced by group B streptococcus

作　　者：张沐捷 梁嘉欣 肖承东 吴红[2] 刘晨飞 张艳玲[1,3] 彭亮 ZHANG Mujie;LIANG Jiaxin;XIAO Chengdong;WU Hong;LIU Chenfei;ZHANG Yanling;PENG Liang(The Fifth Affiliated Hospital of Guangzhou Medical University,Department of Clinical Laboratory,Key Laboratory of Biological Targeting Diagnosis,Therapy and Rehabilitation of Guangdong Higher Education Institutes Guangzhou 510700,Guangdong Province,China;不详)

机构地区：[1]广州医科大学附属第五医院广东高校生物靶向诊治与康复重点实验室,广东广州510700 [2]广州医科大学附属第二医院检验科,广东广州510260 [3]广州医科大学金域检验学院,广东广州510180

出　　处：《中国生物制品学杂志》2024年第7期831-836,842,共7页Chinese Journal of Biologicals

基　　金：广东省自然科学基金面上项目(2021A1515011081);广东省普通高校特色创新类项目(2022KTSCX099);广州市卫生健康科技项目(20221A010071)。

摘　　要：目的 评价波形蛋白(vimentin,Vim)在B族链球菌(group B streptococcus,GBS)引发新生儿脑膜炎中的作用,并探讨其作用机制,以期为新生儿GBS脑膜炎预防和治疗提供实验依据。方法 以人脑微血管内皮细胞(human brain microvascular endothelial cell,HBMEC)为细胞模型,通过转染Vim小干扰RNA(small interfering RNA,siRNA)序列,构建HBMEC的Vim抑制株,同时设转染NC siRNA组(转染阴性对照序列),Western blot及免疫荧光法检测Vim抑制效果。转染48 h后,按MOI=100感染GBS,分别于感染2和3 h进行黏附及侵袭试验;感染3 h后,采用Western blot及免疫荧光法检测抑制Vim对HBMEC中NF-κB p65核转位的影响;Western blot法检测抑制Vim对HBMEC内ERK通路激活相关蛋白及炎性因子表达水平的影响。结果 与转染NC siRNA组比较,转染Vim siRNA1组HBMEC中Vim蛋白表达水平及荧光强度均显著降低(t分别为3.242和71.51,P分别<0.05和<0.001)。与NC siRNA+GBS组比较,Vim siRNA+GBS组黏附率及侵袭率均显著降低(t分别为9.949和30.050,P均<0.001);磷酸化p65水平显著下降(t=2.824,P <0.05),p65蛋白的核转位现象受到抑制,核内绿色荧光强度大幅下降;TNF-α、IL-6、磷酸化细胞外信号调节激酶1/2(extracellular signal-regulated kinase 1/2,ERK1/2)蛋白水平受到显著抑制(t分别4.000、6.367、3.872,P均<0.05)。结论 抑制Vim可减少GBS对HBMEC的黏附及侵袭,抑制NF-κB及ERK信号通路的激活及下游炎性因子的产生,从而减少GBS对HBMEC的损伤。Objective To evaluate the role of vimentin(Vim)in neonatal meningitis induced by group B streptococcus(GBS)and explore its molecular mechanism,so as to provide experimental basis for the prevention and treatment of GBS neonatal meningitis.Methods Human brain microvascular endothelial cells(HBMECs)were used as the cell model,the Vim knockdown strain was constructed by transfection of Vim small interfering RNA(siRNA)sequence into HBMECs,and NC siRNA group transfected with negative control sequence was set at the same time.The inhibitory effect of Vim was detected by Western blot and immunofluorescence assay.After 48 h of transfection,the cells were infected with GBS at a MOI of 100,and adhesion and invasion tests were performed 2 h and 3 h after infection,respectively.After 3 h of infection,the effect of Vim inhibition on NF-κB p65 nuclear translocation in HBMECs was measured by Western blot and immunofluorescence assay,and the effect of Vim inhibition on ERK pathway activation related proteins and inflammatory factors in HBMECs was detected by Western blot.Results Compared with the control group,the Vim protein level and fluorescence intensity in HBMECs of Vim siRNA group significantly decreased(t = 3.242 and 71.51,P < 0.05 and < 0.001,respectively).Compared with NC siRNA + GBS group,the adhesion rate and invasion rate of Vim siRNA + GBS group decreased significantly(t = 9.949 and 30.050,respectively,each P < 0.001);the phosphorylated p65 level decreased significantly(t = 2.824,P < 0.05),the nuclear translocation of p65 protein was inhibited,and the intensity of green fluorescence in nucleus decreased significantly;the levels of TNF-α,IL-6 and phosphorylated ERK1/2 proteins were significantly inhibited(t = 4.000,6.367 and 3.872,respectively,each P < 0.05).Conclusion Inhibition of Vim can reduce the adhesion and invasion of GBS to HBMECs,inhibit the activation of NF-κB and ERK signaling pathway and the production of downstream inflammatory factors,thus reducing the damage of GBS to HBMECs.

关 键 词：波形蛋白 B族链球菌 脑膜炎 NF-ΚB信号通路 

分 类 号：R378[医药卫生—病原生物学]