miR-34a调控Notch-1／NF-κB信号通路对脓毒症小鼠脑功能的保护作用  

Protective effects of miR-34a on brain function of mice with sepsis by regulating Notch-1/NF-κB signaling pathway

作　　者：葛赟[1] 黄曼[1] 颜成敏 陈芬[1] 马岳峰[1] 

机构地区：[1]浙江大学医学院附属第二医院综合ICU,杭州310052 [2]杭州市西溪医院ICU,杭州310023

出　　处：《中华急诊医学杂志》2017年第1期76-81,共6页Chinese Journal of Emergency Medicine

基　　金：浙江省自然科学基金（LY14H150003）

摘　　要：目的观察脓毒症小鼠脑组织TNF-α、IL-1β、IL-6、IL-10及其Notch-1、NF-κB mRNA及其蛋白表达的动态变化，通过构建miR-34a过表达慢病毒载体及包装，并进行鞘内注射，研究其调控Notch-1／NF-κB信号通路对脓毒症小鼠脑组织各指标的影响。方法将清洁级小鼠54只随机（随机数字法）分为假手术组（sham组，n=9，仅行腹腔开关术），模型组（CLP组，n=15，采用盲肠结扎穿孔模型），阴性对照组[NC组，n=15，鞘内注射阴性对照慢病毒（滴度为5×10^8TU／mL）5μL／d，共3d，第7天行CLP术]，干预组[n=15，鞘内注射miR-34a慢病毒（滴度为5×10^8TU／mL）5μL共3d，第7天行CLP术]，各组小鼠均于术后24h，麻醉后处死，留取脑组织标本。观察小鼠行为学变化，术后24h进行神经功能评分；用ELISA法分别测定脑组织TNF-α、IL-1β、IL-6、IL-10的水平；用实时荧光定量-PCR（real-time fluorogenic quantitative-PCR）法测定脑组织Notch-1、NF-κB mRNA表达；用Western Blot法测定脑组织Notch-1、NF-κB蛋白表达；并观察脑组织病理学改变。结果与sham组比较，CLP组在术后24h点神经功能评分、TNF-α、IL-6水平、NF-κB mRNA及其蛋白表达（P〈0．01）、IL-1β显著升高（P〈0．05），IL-10水平、Notch-1 mRNA及其蛋白表达显著下降（P〈0．01），NC组在术后24h点神经功能评分、TNF-α、IL-1β水平、NF-κB蛋白表达（P〈0．01）、IL-6水平显著升高（P〈0．05），IL-10水平、Notch-1 mRNA及其蛋白表达显著下降（P〈0．01），NF-κB mRNA表达差异无统计学意义（P〉0．05）；与CLP组比较，干预组在术后24h点TNF-α水平（P〈0．01）、NF-κB mRNA（P〈0．05）及其蛋白表达（P〈0．01）显著下降，神经功能评分、IL-1β、IL-6水平差异无统计学意义（P〉0．05），IL-10、Notch-1 mRNA（P〈0．05）及其蛋白表达（P〈0．01）显著升高，NC组各指标差异均无统计学意义（P〉0．05Objective To observe the dynamic changes of TNF-α, IL-1β, IL-6, IL-10, and expressions of Notch-1, NF-κB mRNA and their protein levels in the brain lissue of septic mice and intervention effects of intrathecal injection of lentiviral vector of miR-34a gene for regulating Notch-1/NF-κB signaling pathway. Methods A total of 54 mice of clean grade were divided randomly （random number） into four groups, namely sham group （ n = 9）, in which sham-operated laparotomy was performed; CLP group （n=lS）, in which the cecum ligation operation （CLP） was performed; NC group （n = 15）, in which intrathecal injection of lentivirus 5μL （ concentration 5×10^8 TU/mL）, one time per day, for 3 days, then CLP was performed on the seventh day; intervention group （ n = 15 ）, in wihch intrathecal injection of miR-34a lentivirus 5μL （ concentration 5×10^8 TU/mL）, one time per day, for 3 days, then CLP was performed on the seventh day. The mice of four groups were sacrificed 24 h after modeling or operation. The changes of behavior of mice was observed and the neurological scores were assessed 24 h after CLP. The levels of TNF-α, IL-1β, IL-6 and IL-10 in the brain were measured by ELISA method. The mRNA expression and protein levels of Notch-1 and NF-κB in the brain tissue were measured by real-time PCR and Western blot method, respectively. Pathological changes of brain tissue were observed under light microscope. Results The neurological scores, the cerebral TNF-α, IL-6 levels, the mRNA expression and protein level of NF-κB （P 〈0. 01 ）, and IL-113 levels （P 〈 0. 05 ） in CLP group 24 h after modeling were higher than those in sham group. The cerebral IL-10 level and the mRNA expression and protein level of Notch-1 （ P 〈 0. 01 ） in CLP group 24 h after modeling were lower than those in sham group. The neurological scores, the cerebral TNF-α and IL- 113 levels, and the protein level of NF-κB （P 〈0.01 ） , and IL-6 （ P 〈 0.05 ） in NC group 24 h after modeling were high

关 键 词：MIR-34A NOTCH-1 NF-ΚB 慢病毒载体 小鼠 

分 类 号：R459.7[医药卫生—急诊医学]