低氧诱导的神经干细胞外泌体对缺氧缺血性脑损伤大鼠干细胞增殖分化及神经行为影响  

Exosomes derived from hypoxia-preconditioned neural stem cells effect cell proliferation,differentiation and neural behavior in rat models of hypoxic-ischemic brain damage

作　　者：潘依依 张晓英 李青华 宋靖荣[1] 胡津津 Pan Yiyi;Zhang Xiaoying;Li Qinghua;Song Jingrong;Hu Jinjin(Department of Pediatics,Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China)

机构地区：[1]上海交通大学医学院附属第九人民医院儿科,上海200011

出　　处：《中华生物医学工程杂志》2024年第4期248-257,共10页Chinese Journal of Biomedical Engineering

摘　　要：目的探讨低氧诱导的神经干细胞(NSCs)外泌体对缺氧缺血性脑损伤(HIBD)大鼠NSCs增殖分化的影响及对HIBD大鼠神经行为功能恢复的作用。方法超速离心并鉴定低氧诱导的NSCs外泌体(H-Exo);将传至2~3代的NSCs与HIBD新生大鼠脑组织匀浆共培养以模拟HIBD脑内微环境,并分为3组:PBS组(NSCs+PBS共培养)、常氧NSCs外泌体(N-Exo)组(NSCs+N-Exo共培养)及H-Exo组(NSCs+H-Exo共培养),应用免疫荧光方法检测NSCs的增殖和分化情况;制作新生大鼠HIBD模型,并分为4组:Sham组(假手术组)、PBS组(尾静脉注射PBS)、N-Exo组(尾静脉注射N-Exo)及H-Exo组(尾静脉注射H-Exo),14 d后制作损伤大脑区域切片,利用免疫荧光检测该区域NSCs的增殖和分化情况;采用mNSS评分对PBS组、N-Exo组及H-Exo组HIBD新生大鼠第0、2、7、14天评估神经行为功能恢复情况。采用单因素方差分析各组之间的统计学差异,使用LSD检验进行后置分析。结果离体细胞水平:与PBS组和N-Exo组的新生大鼠NSCs相比,H-Exo组NSCs的增殖均显著增加(25.40±2.28比16.84±1.79、16.44±2.08,均P<0.01);与PBS组相比,H-Exo组分化为神经元和少突胶质细胞的比例增加(18.94±2.41比13.16±0.72、76.29±1.47比55.57±10.93,均P<0.05),与PBS组相比,N-Exo组分化为神经元和少突胶质细胞的比例增加,但差异均无统计学意义(均P>0.05)。动物实验:与N-Exo组相比,H-Exo组内源性NSCs的增殖显著增加(11.24±1.17比7.36±1.01,P<0.01),其向少突胶质细胞的分化显著增加(4.66±0.57比2.54±0.80,P<0.01);与PBS组相比,H-Exo组和N-Exo组分化为神经元和星形胶质细胞的数量都显著增加(15.11±1.74、13.61±1.54比6.71±1.59;16.95±2.83、17.32±1.70比11.78±0.92,均P<0.01),但与N-Exo组之间相比,H-Exo组分化为神经元和星形胶质细胞的数量差异均没有统计学意义(均P>0.05)。神经行为功能:PBS组、N-Exo组和H-Exo组第0天的mNSS评分差异均无统计学意义(P>0.05),与PBS组相比,N-Exo组和Objective The objective of this study is to investigate the effect of hypoxia-preconditioned exosomes derived from neural stem cells(NSCs)on the proliferation and differentiation of NSCs in rat models of hypoxic-ischemic brain injury(HIBD)and its effect on the recovery of neurobehavioral function in rats with HIBD.Methods Hypoxia-preconditioned NSCs exosomes(H-Exo)were identified by ultracentrifugation,and the NSCs transmitted to the 2nd to 3rd generations were co-cultured with HIBD neonatal rat brain tissue homogenate to simulate the brain microenvironment of HIBD,and were divided into three groups:PBS group(NSCs+PBS co-culture),N-Exo group(NSCs+normoxic NSCs exosomes N-Exo co-culture)and H-Exo group(NSCs+H-Exo co-culture),immunofluorescence was used to detect the proliferation and differentiation of NSCs,and the neonatal rat HIBD model was made and divided into four groups:Sham group(sham operation group),PBS group(tail vein injection of PBS),N-Exo group(tail vein injection of N-Exo)and H-Exo group(tail vein injection of H-Exo).After 14 days,sections of the damaged brain region were made.The proliferation and differentiation of NSCs in this region were detected by immunofluorescence,and the recovery of neurobehavioral function was evaluated on the 0th,2nd,7th,and 14th days of the newborn HIBD rats in the PBS group,N-Exo group and H-Exo group by using mNSS score.ANOVA was used to analyze the statistical differences between groups,and post hoc analysis was performed using the LSD test.Results The results showed that compared with the neonatal rat NSCs in the the PBS group and the N-Exo group,the proliferation of NSCs in the H-Exo group was significantly increased(25.40±2.28 vs 16.84±1.79,16.44±2.08,all P<0.01);compared with the PBS group,the proportion of differentiation into neurons and oligodendrocytes in the H-Exo group increased(18.94±2.41 vs 13.16±0.72,76.29±1.47 vs 55.57±10.93,all P<0.05).However,compared with the PBS group,the proportion of neurons and oligodendrocytes in the N-Exo group increased,b

关 键 词：神经干细胞 外泌体 低氧诱导 缺氧缺血性脑损伤 增殖分化 

分 类 号：R722.1[医药卫生—儿科]