摘要
目的探讨脂肪间充质干细胞(adipose-derived mesenchymal stem cells,ADMSCs)来源外泌体对脑缺血再灌注大鼠神经元凋亡及炎症因子影响。方法取10只健康雄性8周龄SD大鼠附睾旁脂肪组织,提取ADMSCs进行细胞培养,取第3代ADMSCs分别在常氧(21%)和低氧(1%)条件培养,24 h后取培养皿上清液,试剂盒提取法分离外泌体。60只健康雄性8周龄SD大鼠按随机数字表法等分为5组:假手术组、缺血再灌注组、PBS组、低氧外泌体组、常氧外泌体组(n=12),后4组采用Longa改良线栓法建立脑缺血再灌注模型,其中缺血再灌注组仅进行造模,不进行干预,PBS组经尾静脉注射PBS 2 mL,低氧外泌体组和常氧外泌体组分别同时给予等量低氧外泌体和常氧外泌体100μg。在大鼠脑缺血再灌注损伤后24 h,采用Longa法测定各组大鼠神经功能缺损评分,TTC染色测量脑梗死体积,TUNEL法检测神经元凋亡情况,HE染色观察组织病理学变化,免疫组化检测细胞色素C(CytC)和白细胞介素-1β(IL-1β)的表达。结果与PBS组相比,低氧外泌体组和常氧外泌体组病理学改变减轻,神经功能缺损评分、脑梗死体积百分比、细胞色素C和IL-1β的表达及神经元凋亡指数均明显降低(P<0.05),低氧外泌体组较常氧外泌体组降低更为明显(P<0.05)。结论 ADMSCs来源外泌体能减少脑缺血再灌注大鼠神经元凋亡和炎症因子IL-1β的表达,有神经保护作用,其机制可能是外泌体抑制了线粒体介导的细胞凋亡和IL-1β参与的炎症级联反应。
Objective To investigate the effect of exosomes derived from adipose-derived mesenchymal stem cells(ADMSCs) on neuron apoptosis and inflammatory cytokines in cerebral ischemia reperfusion(I/R) mice. Methods ADMSCs were extracted from adipose tissue adjacent to the epididymis in 10 8-week-old healthy male SD rats, and the cells of the third generation were cultured under normal oxygen(21%) and hypoxia(1%) conditions, respectively. After 24 h, the culture supernatant was collected and exosomes were separated by kit extraction method. Sixty 8-week-old healthy male SD rats were randomly divided into 5 groups, that is, sham-operation group, I/R group, PBS group, hypoxic exosomes group, and normoxic exosomes group(n=12). Rat model of cerebral I/R was established by insertion of a thread through the internal carotid artery in the latter 4 groups. The rats of PBS group were injected intraperitoneally with 2 mL PBS, while those of the hypoxic and normoxic exosomes groups were given 100 μg of hypoxic and normoxic exosomes, respectively. In 24 h after cerebral I/R injury, neurological function was evaluated by Longa score, infarct volumes were measured by TTC staining, neuronal apoptosis was detected by TUNEL assay, the expression of cytochrome C(CytC) and interleukin-1β(IL-1β) was detected by immunohistochemical staining. HE staining was employed to observe the morphological changes of the injury. Results Compared with the PBS group, the hypoxic and normoxic exosomes groups demonstrated significant decreases in pathological changes, neurological functional score, percentage of infarct volumes, expression levels of Cyt C and IL-1β, and neuronal apoptosis index(P<0.05). While, the decreases were more significant in the hypoxic than the normoxic exosomes group(P<0.05). Conclusion ADMSCs-derived exosomes can reduce the apoptosis of neurons and the expression of IL-1β after cerebral I/R injury and show neuroprotective effects, which may be due to exosomes’ inhibition of mitochondrial-mediated apoptosis and IL-1β-involved inflammatory cascade reactions.
作者
郝海珍
郭铁
余丹
HAO Haizhen;GUO Tie;YU Dan(Department of Neurology, the Affiliated Haikou Hospital of Xiangya Medical School, Central South University, Haikou, Hainan Province, 570208, China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2019年第17期1656-1665,共10页
Journal of Third Military Medical University
基金
海南省重点科技计划项目(ZDXM20130066)~~
