DOCK2在缺血性卒中大鼠小胶质细胞除极和神经发生中作用研究.pdf
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1、论著(收稿日期:2 0 2 3-0 1-10)503脑与神经疾病杂志2 0 2 3年第31卷第8 期参考文献1Graus F,Vogrig A,Muniz-Castrillo S,et al.Updated diagnosticcriteria for paraneoplastic neurologic syndromesJ.NeurolNeuroimmunol,2021,8(4):e1014.2 冉艳萍.儿童中枢神经系统副肿瘤综合征的研究进展.国际儿科学杂志,2 0 2 0,47(8):552-555.3 Grativvol RS,Cavalcante WCP,Castro LHM,et a
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9、ssociatedwith anti-N-methyl-D-aspartate(NMDA)receptor encephalitisJ.Pathol Int,2018,68(12):677-684.16Guan HZ,Ren HT,Cui LY.Autoimmune encephalitis:anexpanding frontier of neuroimmunologyJJ.Chin Med J(Engl),2016,129(9):1122-1127.DOCK2在缺血性卒中大鼠小胶质细胞除极和神经发生中作用研究张华霖费长东毛琪刘慎周大伟【摘要】目的探究靶向胞质分裂作用因子蛋白(dedicato
10、rof cytokinesis2DOCK2)蛋白在缺血性卒中(IS)大鼠脑组织中对小胶质细胞除极与神经发生的影响。方法将大鼠随机分为假手术(Sham)组,缺血性卒中(MCAO)组,IS+LC-shNC(M CA O+LV-s h NC)组,IS+LV-shDOCK2(M CA O+LV-s h D O CK 2)基金项目:辽宁省科学技术基金(2 0 19-ZD-1076)作者单位:116 0 0 0辽宁,中国人民解放军联勤保障部队第九六七医院急诊医学科(张华霖、费长东、毛琪、周大伟),神经外科(刘慎)通信作者:周大伟,Email:d r a g o n 7 6 10 2 8 12 6.c o
11、m504脑与神经疾病杂志2 0 2 3年第31卷第8 期组,每组15只。7 d后开展神经功能评分与Y型迷宫检测,取脑组织进行TTC染色、免疫荧光染色,取缺血侧海马组织开展RT-PCR检测、Westernblot检测。结果与Sham组相比,MCAO组与MCAO+LV-shNC组大鼠脑组织中DOCK2表达增加(P0.05),出现梗死区域,神经功能评分增加(P0.05)。Y型迷宫中正确反应次数减少(P0.05),正确反应时间增加(P0.05)。此外,与Sham组相比,MCAO组与MCAO+LV-shNC组大鼠海马DG区BrdU阳性细胞数、DCX表达增加(P0.05),小胶质细胞活化标记物ibal表达
12、升高(P0.05)。小胶质细胞极化标记物中,MCAO组与MCAO+LV-shNC组CD16、T NF-、IL-1、i NO SmRNA 表达明显增加(P0.05),且CD206、T CF-、A r g l、Yml mRNA 表达增加。然而,与MCAO组相比,MCAO+LV-shDOCK2组大鼠脑组织中DOCK2表达降低(P0.05),伴随着梗死面积减少(P0.05),神经功能评分减少(P0.05)。Y型迷宫中正确反应次数增加(P0.05),正确反应时间减少(P0.05)。与此同时,海马DG区BrdU阳性细胞数、DCX表达进一步增加(P0.05),但ibal表达降低(P0.05)。此外,M1型小
13、胶质细胞标记物CD16、T NF-、IL-1、i NO SmRNA 表达减少(P0.05),M2型TGF-、A r g l、Ym l m RNA 表达进一步增加(P0.05)。结论敲低DOCK2蛋白表达有利于抑制小胶质细胞活化,逆转M1型小胶质细胞除极,提高M2型小胶质细胞除极化,最终起到促进神经发生,发挥神经功能与学习、记忆能力改善作用。【关键词】DOCK2;缺血性卒中;小胶质细胞除极;神经发生;神经功能;学习和记忆能力中图分类号:R743.32文献标识码:A文章编号:10 0 6-351X(2 0 2 3)0 8-0 50 3-0 8The role of DOCK2 in microgl
14、ia polarization and neurogenesis in ischemic stroke ratsZhang Hualin,Fei Changdong,Mao Qi,Liu Shen,Zhou DaweiDepaitment of Emergency Medical,the 967 Hospital of the Joint Service Support Force of the Chinese Peoples LiberationArmy,Liaoning 116000,ChinaCorresponding author:Zhou Dawei,Email:Abstract O
15、bjective To investigate the effect of DOCK2 protein on microglial polarization andneurogenesis in rats with ischemic stroke(IS).Methods All rats were randomly divided into Group Shamoperation(Sham),Group IS(MCAO),Group ischemic stroke+LC-shNC(MCAO+LV-shNC),Group IS+LV-shDOCK2(MCAO+LV-shDOCK2),15 in
16、each group.7d later,neurological function test and Y-maze test werecarried out,brain tissue was collected for TTC staining and immunofluorescence staining,and hippocampus from ISside was collected for RT-PCR and Western blot.Results Compared with Group Sham,the expression of DOCK2in the brain of the
17、 rats in Group MCAO and Group MCAO+LV-shNC increased,the infarct area appeared,and theneurological function score increased(P0.05).In the Y-maze,the number of correct response times decreased(P0.05),and the correct response time increased(P0.05).In addition,compared with Group Sham,the number of Brd
18、U positive cells and the expression of DCX in the hippocampal DG area of the rats in Group MCAO and GroupMCAO+LV-shNC increased(P0.05),and the expression of the microglia activation marker ibal increased(P0.05).Among the microglia polarization markers,the expressions of CD16,TNF-,IL-1,and iNOS mRNA
19、in GroupMCAO and Group MCAO+LV-shNC were significantly increased(P0.05),as well as,the expressions of CD206,TCF-,Argl,and Yml mRNA increased.However,compared with Group MCAO,the expression of DOCK2 inrats of Group MCAO+LV-shDOCK2 decreased(P0.05),which accompanied with a decrease in the infarct size
20、(P0.05)and neurological score(P0.05).In addition,compared with Group MCAO,in the Y-maze,the number ofcorrect response times increased(P0.05)and the correct response time decreased(P0.05)of Group MCAO+LV-shDOCK2.At the same time,the number of BrdU-positive cells and the expression of DCX in the hippo
21、campal DGarea were further increased,but the expression of ibal decreased of Group MCAO+LV-shDOCK2.Furthermore,theexpressions of M1-type microglia markers CD16,TNF-,IL-1,and iNOS mRNA decreased(P0.05),and theexpressions of M2-type TGF-,Argl,and Yml mRNA were further increased(P0.05).Conclusion Knock
22、downof DOCK2 protein expression is beneficial to inhibit the activation of microglia,which could reverse the polarizationof M1-type microglia and increase the polarization of M2-type microglia.Finally,it promote neurogenesis,exertimprovement of neurological function and learning and memory ability.K
23、ey words1 DOCK2;Ischemic stroke;Microglia polarization;Neurogenesis;Neurological function;Learningand memory ability505脑与神经疾病杂志2 0 2 3年第31卷第8 期小胶质细胞是脑内主要免疫细胞,发挥免疫调节和免疫监视作用,维持大脑内环境稳态。传统研究认为,小胶质细胞活化造成众多细胞因子释放,极大抑制了大脑组织修复,且会进一步造成损伤。然而,缺血性卒中(ischemic stroke,IS)治疗并非简单抑制小胶质细胞活化,更需要提高神经发生,恢复缺血区微环境。最近证据表明,小
24、胶质细胞活化后M1/M2型之间除极在IS脑损伤的修复中起关键作用7 。M1型小胶质细胞与促炎性细胞因子释放相关,而M2型小胶质细胞能诱发包括神经发生、血管生成、轴突重塑及髓鞘再生等生理过程8 。阐明M1/M2型小胶质细胞除极与神经发生的影响因素有助于进一步了解IS诱发脑损伤机制。材料与方法1.实验动物60只SD大鼠,SPF级别,6 w龄雄性,由大连医科大学实验动物中心提供,生产许可证号:SCXK(辽)2 0 18-0 0 0 3。饲养于SPF级动物房中,控制饲养环境恒温2 2 2,恒湿6 0 5%,自由饮食摄水。2.实验试剂与仪器2,3,5-三苯基氯化四氮唑(TTC,北京索莱宝生物科技公司,T
25、8170);RT-PCR引物(南京金斯瑞生物科技公司);Lipofectamine3000试剂盒(美国Invitrogen公司,12 8 2 31);5-溴脱氧尿嘧啶核苷(5-Bromodeoxyuridinc,Br d U,美国Sigma公司,1352 10);Anti-mouseBrdU抗体(英国abcam公司,ab7231);A n t i-m o u s e 钙接头蛋白(ionizedcalcium binding adapter molecule 1,ibal,英国 abcam公司,ab7843);A n t i-m o u s e 又双皮质素(DCX,doublecortin,美国
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- DOCK2 缺血性 大鼠 胶质 细胞 神经 发生 作用 研究
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