摘要
目的探究外泌体(Exo)转运miR-223对创伤性脑损伤(TBI)大鼠脑组织损伤与小胶质细胞活化的影响及机制。方法通过脂质体法分别将miR-NC质粒、miR-223 mimic质粒转染至HEK293细胞,实时荧光定量PCR测定细胞中miR-223表达水平;提取转染后HEK293细胞Exo,通过透射电子显微镜、纳米颗粒跟踪分析及Western blot对Exo进行鉴定,并采用实时荧光定量PCR测定Exo中miR-223表达水平;将40只SD大鼠随机分为假手术组、模型组、NC-Exo组、miR-223-Exo组,每组10只。除假手术组外的3组大鼠均通过改良Feeney自由落体法制备TBI模型,NC-Exo组与miR-223-Exo组大鼠分别经尾静脉注射转染miR-NC质粒细胞来源的Exo、转染miR-223 mimic质粒细胞来源的Exo;2周后,苏木精-伊红(HE)染色观察各组大鼠脑组织病理学变化,尼氏(Nissl)染色检测各组大鼠尼氏体改变与分布情况,酶联免疫吸附实验(ELISA)测定各组大鼠血清肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)水平,免疫荧光双染法观察各组大鼠脑组织内Nod样受体蛋白3(NLRP3)与离子钙接头蛋白(Iba-1)表达,Western blot测定各组大鼠脑组织内NLRP3、凋亡相关斑点样蛋白(ASC)、半胱氨酸蛋白酶-1(Caspase-1)的蛋白表达水平。结果细胞转染后,与对照组和miR-NC组比较,miR-223组细胞中miR-223相对表达量显著增加(P<0.05);分离的颗粒物具有典型的Exo形态,粒径峰值大约处于120 nm,Exo标志蛋白CD9、CD63及CD81均明显高表达,且miR-223相对表达量显著增加(P<0.05)。与模型组比较,miR-223-Exo组大鼠脑组织损伤现象得到改善,尼氏体形态恢复且数目增加,血清TNF-α、IL-1β、IL-6水平均降低(P<0.05),脑组织内NLRP3与Iba-1荧光染色强度减少(P<0.05),同时,脑组织内NLRP3、ASC、Caspase-1蛋白相对表达量均下调(P<0.05)。结论Exo转运miR-223能够明显改善TBI大鼠脑组织损伤,抑制小胶质细胞激活,该作用可能与抑制NLRP3炎性小�
Objective To investigate the effect and mechanism of exosome(Exo)transported miR-223 on brain tissue injury and microglial activation in rats with traumatic brain injury(TBI).Methods The miR-NC plasmid and miR-223 mimic plasmid were transfected into HEK293 cells by liposome method,and the expression level of miR-223 in the cells was determined by quantitative real-time PCR.Exo was extracted from transfected HEK293 cells and identified by transmission electron microscopy,nanoparticle tracking analysis and Western blot,the expression level of miR-223 in Exo was determined by quantitative real-time PCR.Forty SD rats were randomly divided into sham group,model group,NC-Exo group and miR-223-Exo group,with 10 rats in each group,TBI model was prepared by modified Feeney free fall method in all groups except sham group,rats in NC-Exo group and miR-223-Exo group were injected with cell-derived Exo transfected with miR-NC plasmid and cell-derived Exo transfected with miR-223 mimic plasmid via tail vein,respectively.Two weeks later,hematoxylin-eosin(HE)staining was used to observe the pathological changes of brain tissue in each group,Nissl staining was used to detect the changes and distribution of Nissl bodies in each group,enzyme-linked immunosorbent assay(ELISA)was used to measure the serum levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6),immunofluorescence double staining was used to observe the expression of nod-like receptor family pyrin domain containing 3(NLRP3)and ionized calcium binding adaptor molecule 1(Iba-1),Western blot was used to detect the protein expression of NLRP3,apoptosis-associated speck-like protein containing(ASC)and Caspase-1.Results After transfection,compared with control group and miR-NC group,the relative expression of miR-223 in miR-223 group significantly increased(P<0.05).The isolated particles had typical Exo morphology,the peak particle size was about 120 nm,the Exo marker proteins CD9,CD63 and CD81 were significantly overexpressed,and the relati
作者
孙衍昶
徐鹏翔
何青龙
欧阳一彬
莫业和
Sun Yanchang;Xu Pengxiang;He Qinglong;Ouyang Yibin;Mo Yehe(Dept of Neurosurgery,The Second Affliated Hospital of Hainan Medical College,Haikou 570311)
出处
《安徽医科大学学报》
CAS
北大核心
2023年第7期1111-1118,共8页
Acta Universitatis Medicinalis Anhui
基金
海南省卫生健康行业科研项目(编号:21A200314)。
