【摘要】 目的 探讨小鼠短散布核元件B1反义RNA(B1as RNA)抗小鼠衰老的分子机制及是否通过激活老年小鼠转录因子(TF)EB进而提高小鼠的自噬水平。方法 将老年BALB/c小鼠(≥14月龄)分成3组：B1as RNA治疗(治疗组),LacZ3F3R RNA无关RNA对照(无关对照)组和生理盐水对照(空白对照)组；每周注射1次，注射4 w,然后二氧化碳安乐处死小鼠。6～8周龄、同品系年轻正常小鼠不用任何药物处理作为实验对照(年轻对照)组。透射电镜观察小鼠肝脏细胞中自噬溶酶体。Western印迹检测小鼠肝脏和脾脏细胞中微管相关蛋白轻链(LC)3-Ⅰ和LC3-Ⅱ蛋白表达；Western印迹检测小鼠肝脏和脾脏细胞质与细胞核中TFEB蛋白表达。实时定量聚合酶链反应(RT-qPCR)方法检测小鼠肝脏和脾脏细胞中TFEB基因及自噬相关基因Beclin-1,Sqstm1/p62,Map1lc3b, Atg10,Atg12和Ppargc1A mRNA水平。结果 透射电镜检测到治疗组及年轻对照组肝脏细胞自噬溶酶体的形成。治疗组肝脏及脾脏LC3-Ⅱ/LC3-Ⅰ比值、细胞核TFEB蛋白表达明显高于空白对照组(P<0.05)。治疗组TFEB mRNA与对照组无统计学差异(P>0.05),而治疗组肝脏和脾脏细胞中Beclin-1、Sqstm1/p62、Map1lc3b、Atg10、Atg12和Ppargc1A mRNA表达水平明显高于空白对照组及无关对照组(P<0.05),接近年轻对照组水平。结论 促进老年小鼠细胞TFEB的核转位进而提高TFEB依赖的自噬水平是B1as RNA抗小鼠衰老的一种分子机制。更多还原

【Abstract】 Objective To investigate the molecular mechanism of short interspersed nuclear element B1 antisense RNA(B1as RNA) against mouse aging and whether it could increase the autophagy level of mice by activating transcription factor(TF)EB.Methods The aging BALB/c mice(≥14 months) were divided into 3 groups: B1as RNA treatment(treatment) group, LacZ3F3R RNA unrelated RNA control(unrelated control) group and normal saline control(blank control) group. The mice were injected once a week and euthanized with carbon dioxide after 4 w of injection. 6～8 week-old young normal mice of the same strain were treated without any drug as the experimental control(young control) group.The autolysosomes in mouse liver cells were observed by transmission electron microscope. The expressions of microtubule-associated protein light chain(LC)3-Ⅰand LC3-Ⅱprotein in mouse liver and spleen cells were detected by Western blot. Western blot was used to detect TFEB protein expression in cytoplasm and nucleus of liver and spleen cells of mice. Reverse transcription-quantitative polymerase chain reaction(RT-qPCR) method was used to detect the mRNA levels of TFEB gene and autophagy related genes Beclin-1, Sqstm1/p62, Map1lc3b, Atg10, Atg12, Ppargc1A in mouse liver and spleen cells.Results Transmission electron microscopy showed that autolysosomes formation in liver cells in the treatment group and young control group. The LC3-Ⅱ/LC3-Ⅰratio and necleu TFEB protein expression of liver and spleen in the treatment group were significantly higher than those in the black control group(P<0.05).There was no significant difference in TFEB mRNA between the treatment group and the control groups(P>0.05). The mRNA expression levels of Beclin-1, Sqstm1/p62, Map1lc3b, Atg10, Atg12 and Ppargc1A in the liver and spleen cells of treatment group were significantly higher than those in the blank control group and the unrelated control group(P<0.05), and close to the levels of the young control group.Conclusions One of the molecular mechanisms of anti-aging of B1as RNA is that it promotes nuclear translocation of TFEB in aged mouse cells and increases TFEB-dependent autophagy level.更多还原