脑卒中血清培养骨髓间质干细胞移植对大鼠脑缺血再灌注的影响  

The effects of transplantation of mesenchymal stem cells preconditioned with stroke serum on cerebral ischemia-reperfusion in rats

作　　者：贾韩静 童一心 黄哲华 Jia Hanjing

机构地区：[1]浙江中医药大学附属第一医院,310006

出　　处：《浙江临床医学》2023年第5期652-655,659,共5页Zhejiang Clinical Medical Journal

摘　　要：目的观察脑卒中血清预处理的骨髓间质干细胞(mesenchymal stem cell,MSCs)移植对大鼠脑缺血再灌注的影响。方法分离培养骨髓MSCs并观察细胞形态,流式细胞术检测CD44、CD90、CD34、CD45抗原表达以鉴定细胞。分离缺血性脑卒中和正常大鼠血清,与MSCs共培养,制备SS-MSCs(脑卒中血清),NS-MSCs(正常脑组织血清)及FBS-MSCs(胎中血清)。Zea Longa线栓法制作MCAO大鼠模型,MSCs经尾静脉注射移植,分为MCAO组(等体积PBS)、MCAO+FBS-MSCs组(2×10^(6) cells FBS-MSCs)、MCAO+NS-MSCs组(2×10^(6) cells NS-MSCs)、MCAO+SS-MSCs组(2×10^(6) cells SS-MSCs),并设sham组(等体积PBS)。监测大鼠MSCs移植后第1、7、14天行为变化。TTC、HE、Tunel染色观察脑组织梗死情况、组织状态及细胞凋亡情况。ELISA检测脑组织中脑源性神经营养因子(BDNF)、神经生长因子(NGF)、肝细胞生长因子(HGF)、血管内皮细胞生长因子(VEGF)表达水平。同时行免疫组织化学染色观察BrdU、BDNF、NGF表达。结果成功分离高纯度MSCs,分组预处理移植后,与MCAO组比较,各干预组第7、14天双侧贴纸去除时间、mNSS评分均降低,MCAO+SS-MSCs组行为改善最显著(P<0.01)。TTC、HE、Tunel染色结果显示与MCAO组比较,MSCs移植干预均可降低大鼠脑梗死面积,改善脑组织损伤,抑制细胞凋亡,且MCAO+SS-MSCs组改善程度最大(P<0.05)。此外,MCAO+SS-MSC组BDNF、NGF、HGF、VEGF水平较MCAO组升高(P<0.01),且高于其他干预组(P<0.05)。免疫组化结果显示MCAO+SS-MSC组Brdu、BDNF、NGF表达水平较MCAO组升高(P<0.05),且高于其他干预组(P<0.05)。结论脑卒中血清预处理骨髓MSCs移植可有效改善MCAO大鼠脑组织损伤,可通过抑制脑组织细胞凋亡,上调BDNF、NGF、HGF、VEGF水平,发挥保护作用。Objective To observe the effects of transplantation of mesenchymal stem cells(MSCs)preconditioned with stroke serum on cerebral ischemia-reperfusion in rats.Methods MSCs were isolated and cultured to observe cell morphology and to identify CD44,CD90,CD34,and CD45 expression by flow cytometry.Serum from ischemic stroke and normal rats was isolated,and MSCs were cultured and divided into SS-MSCs(stroke serum),NS-MSCs(normal brain tissue serum),and FBS-MSCs(fetal bovine serun).Zea longa wire thrombosis method was used to create MCAO rat model which were treated with preconditioning MSCs tail vein injection transplantation intervention and were divided into the MCAO group(equal volume of PBS),the MCAO+FBS MSCs group(2×10^(6) cells FBS MSCs),the MCAO+ns MSCs group(2×10^(6) cells ns MSCs),the MCAO+SS MSCs group(2×10^(6) cells SS MSCs),and the sham group(equal volume of PBS).The behavioral changes of rats at 1,7,and 14 d after MSCs transplantation were monitored.Cerebral infarction,tissue status and apoptosis were assessed by TTC,HE and Tunel staining.The expression levels of BDNF,NGF,HGF and VEGF in brain tissues were detected by ELISA.Meanwhile,immunohistochemical staining was performed to observe BrdU,BDNF and NGF expression.Results The high purity MSCs were successfully isolated and pretreated in groups for transplantation,compared with the MCAO group,the bilateral patch removal time and mNSS score on days 7 and 14 in the intervention groups were all significantly reduced,with the most significant behavioral improvement in the MCAO+SS-MSCs group(P<0.01).TTC,HE.The Tunel staining results showed that MSCs transplantation intervention could reduce the cerebral infarction area,improve brain tissue damage and inhibit apoptosis compared with the MCAO group,and the MCAO+SS-MSCs group had the greatest improvement(P<0.05).In addition,the levels of BDNF,NGF,HGF and VEGF in the MCAO+SS-MSC group were significantly higher than those in MCAO group(P<0.01),and higher than those in other intervention groups.The immunohistochemi

关 键 词：脑缺血再灌注 骨髓间质干细胞 移植 脑卒中血清预处理 

分 类 号：R743[医药卫生—神经病学与精神病学]