【摘要】 目的 分析小鼠短散布核元件B1反义RNA（B1 antisense RNA,B1as RNA）静脉注射正常小鼠后，在小鼠血液及不同组织中的生物分布；以及转染培养的正常小鼠胚胎细胞后，细胞摄取B1as RNA的效率。方法 取6只8～12周龄BALB/c小鼠，雌雄各3只，经尾静脉注射20μg B1as RNA，分别于注射后不同时间经眼眶静脉丛采血；取54只8～12周龄BALB/c小鼠，经尾静脉注射20μg B1as RNA，注射后不同时间分别安乐处死6只小鼠，雌雄各3只，解剖后取心脏、肝脏、脾脏、肺、肾脏和胸腺；将B1as RNA转染入培养的小鼠胚胎细胞，转染后不同时间取一定量的细胞，采用RT-q PCR法检测B1as RNA在小鼠血液及不同组织的生物分布以及胚胎培养细胞中B1as RNA的存留水平。将30只自然衰老的BALB/c小鼠（≥14月龄）分成3组：治疗组（经尾静脉注射20μg B1as RNA，每周1次）、无关RNA对照组（经尾静脉注射20μg Lac Z3F3R RNA，每周1次）和盐水对照组（注射相同体积的盐水），每组10只，另设年轻对照组（正常年轻的8～12周龄BALB/c小鼠，雌雄各5只），给药4周后，安乐处死各组小鼠，取肝脏组织，RT-q PCR法检测衰老相关基因(Sirt1、p21、p16^INK4a、p15^INK4b、p19^Arf)的表达水平。结果 尾静脉注射后，B1as RNA在小鼠血液中可存留约30 min，在大多数检测到的组织中可存留2～4 h，在肺中可存留约48 h;B1as RNA转染小鼠胚胎培养细胞后45 min，细胞摄取B1as RNA的效率约为每个细胞摄取400个B1as RNA分子。与盐水对照组比较，B1as RNA处理可显著下调p21、p16^Ink4a、p15^Ink4b、p19^Arf基因m RNA的表达（t分别为10.01、4.461、4.420和5.309,P均<0.05），显著上调Sirt1基因mRNA的表达（t=4.579,P<0.05）。结论 B1as RNA转染细胞可被细胞有效摄取。静脉注射小鼠后，B1as RNA可在血液和组织中停留一定时间，并调节衰老相关基因的表达，使其接近年轻正常小鼠的表达水平。更多还原

【Abstract】 Objective To investigate the biological distribution of B1 antisense RNA（Blas RNA） of mouse short interspersed nuclear element in blood and tissues of normal mice after vein injection and detect the cell uptake efficiency of B1 as RNA using cultured normal mouse embryo cells after transfection.Methods Six 8～12-week-old BALB/c mice,three males and three females,were injected with 20 μg Blas RNA via tail vein,and blood samples were collected at different times after injection.54 BALB/c mice of 8～12-weeks were injected with 20 μg Blas RNA via tail vein,of which six mice,three males and three females,were euthanized at different times after injection,and various tissues,including the heart,liver,spleen,lung,kidney and thymus were harvested.Blas RNA was transfected into cultured mouse embryonic cells,and a certain amount of cells were taken at different time after transfection.The biological distribution of B1as RNA in mouse blood and different tissues and the persistence of Blas RNA in cultured embryo cells were detected by RT-qPCR.30naturally senescent BALB/c mice（≥ 14 months old） were divided into three groups:treatment group（20 μg Blas RNA injected via tail vein,once a week）,irrelevant RNA control group（20 μg LacZ3F3R RNA injected via tail vein,once a week） and saline control group（injected with the same volume of saline）,with 10 mice in each group,and a young control group（normal young 8～12-week-old BALB/c mice,five males and five females） was set.Four weeks after administration,mice in each group were euthanized,the liver tissues were taken,and the expression levels of aging-related genes(Sirtl,p21,p16^Ink4a,p15^Ink4b,p19^Arf) were detected by RT-qPCR.Results After tail vein injection,Blas RNA was available in the blood of mice for approximately 30 min,persisted for approximately 2～4 h in most detected tissues and persisted for approximately 48 h in lungs.The efficiency of cellular uptake of Blas RNA was approximately 400 molecules per mouse embryo culture cell 45 min after transfection with B1as RNA.Compared with the saline control group,Blas RNA treatment significantly down-regulated the mRN A expression of p21,p16^In4a,p15^In4b and p19^Arf genes（t=10.01,4.461,4.420 and 5.309 respectively,each P <0.05）,and significantly up-regulated the mRNA expression of Sirt1 gene（t=4.579,P <0.05）.Conclusion Blas RNA was efficiently taken up by cells after transfection.After intravenous injection,Blas RNA stayed in the blood and tissues for a certain period of time and regulated the expression of aging-related genes in aged mice so as to make them approach to the expression level of young normal mice.更多还原