外泌体miRNA-222预警shRNA-PCSK9诱发脑Tau蛋白过度磷酸化  

Exosomal miRNA-222 Alerts shRNA-PCSK9 Induction Brain Tau Hyperphosphorylation

作　　者：王蕾[1] 江茜 王宏[1] 袁玲[1] 吕楠[1] 郝迪[1] 崔晓雪[1] 王梓[1] WANG Lei;JIANG Qian;WANG Hong;YUAN Ling;LYU Nan;HAO Di;CUI Xiaoxue;WANG Zi(Cardiovascular and Cerebrovascular Drugs Research and Development Center,Tianjin Institute of Medical and Pharmaceutical Sciences,Tianjin 300020,China)

机构地区：[1]天津市医药科学研究所心脑血管药物研究室,天津300020

出　　处：《中国现代应用药学》2024年第5期636-643,共8页Chinese Journal of Modern Applied Pharmacy

基　　金：天津市卫生健康委员会科技项目(MS20023)。

摘　　要：目的探究血浆外泌体携带的微小RNA-222(microRNA-222,miRNA-222)能否作为shRNA-PCSK9诱发认知障碍提供早期预警的标志物。方法高脂饲料(high-fat diet,HFD)喂饲法制备高胆固醇血症小鼠模型。再将模型小鼠分为HFD-shRNA空白对照组和HFD-shRNA-PCSK9组。构建shRNA-PCSK9慢病毒,经静脉注入体内,实时荧光定量(realtime polymerase chain reaction,RT-PCR)法检测PCSK9 mRNA表达。免疫组织化学(immunohistochemistry,IHC)观察脑组织Tau蛋白和磷酸化。Western blotting检测Tau蛋白和P-Tau蛋白。ELISA法测定血清淀粉样蛋白Aβ1-42Ab水平。试剂盒分步提取血浆外泌体,负染电镜技术对外泌体形态进行鉴定,纳米颗粒跟踪分析技术测定外泌体粒径。RT-PCR技术检测血浆外泌体中携带的miRNA-222表达水平。结果HFD喂饲13周制备模型组小鼠,血清总胆固醇(total cholesterol,TC)、低密度脂蛋白(low density lipoprotein,LDL-C)含量显著升高,同时,模型组小鼠脑组织内PCSK9 mRNA表达明显升高。经shRNA-PCSK9慢病毒干扰后,PCSK9 mRNA表达抑制,同时IHC观察到shRNA-PCSK9诱发了脑组织Tau蛋白的异常表达和过度磷酸化,表明已发生神经纤维缠结的病理改变。然而,此时血清Aβ1-42Ab尚未明显升高,尚未具备诊断认知障碍的意义。将血浆外泌体中的miRNA提取,RT-PCR结果显示,HFD-shRNA-PCSK9组外泌体中携带的miRNA-222表达量与HFD-shRNA空白对照组相比显著降低。结论血浆外泌体携带的miRNA-222可以作为shRNA-PCSK9诱发认知障碍提供早期预警的标志物。OBJECTIVE To investigate whether the microRNA-222(miRNA-222)carried by plasma exosomes can serve as an early warning marker for cognitive impairment induced by shRNA-PCSK9.METHODS The high-fat diet(HFD)was used to prepare a hypercholesterolemic mouse model group.The model group mice were divided into HFD-shRNA control group and HFD-shRNA-PCSK9 group.The shRNA-PCSK9 was constructed,injected intravenously into the body,and the expression of PCSK9 mRNA was detected by real-time PCR(RT-PCR).Tau protein and phosphorylation in brain tissue were observed by immunohistochemistry(IHC).Western blotting was used to detect Tau protein and P-Tau protein.Serum amyloid Aβ1-42Ab levels were determined by ELISA.The kits extracted plasma exosomes step by step,identify the exosome morphology by negative staining electron microscopy,and determined the size of exosomes by NTA technology.RT-PCR technique was used to detect the expression level of miRNA-222 carried in plasma exosomes.RESULTS The model mouse were prepared by feeding HFD for 13 weeks,whose total cholesterol(TC)and low-density lipoprotein(LDL-C)contents in serum were significantly increased.At the same time,the expression of PCSK9 mRNA in the brain tissue of model group was significantly increased.After shRNA-PCSK9 lentivirus interference,PCSK9 mRNA expression was inhibited,and IHC observed that shRNA-PCSK9 induced abnormal expression and hyperphosphorylation of Tau protein in brain tissue,indicating that the pathological changes of neurofibrillary tangles had occurred.However,at this time,serum Aβ1-42Ab had not been significantly increased,and it had not yet been of significance for the diagnosis of cognitive impairment.The miRNA in plasma exosomes was extracted,and RT-PCR results showed that the expression of miRNA-222 carried in the exosomes of the HFD-shRNA-PCSK9 group was significantly lower than that of the HFD-shRNA control group.CONCLUSION Plasma exosomes carried miRNA-222 provides an early warning marker for shRNA-PCSK9-induced cognitive impairment.

关 键 词：高胆固醇血症 PCSK9 认知障碍 miRNA-222 

分 类 号：R965.1[医药卫生—药理学]