dsRNA阻断大鼠骨髓源性神经干细胞Hes5表达的实验研究.doc

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dsRNA阻断大鼠骨髓源性神经干细胞Hes5表达的实验研究

摘要：目的??观察外源性短双链RNA(dsRNA)在转录后水平即mRNA水平降低基因表达的效率，并对其影响因素进行初步探讨。方法??将体外分离培养的大鼠骨髓基质细胞，通过由本实验室配制的特殊培养基诱导成神经干细胞，应用人工合成的dsRNA于不同浓度转染神经干细胞，Westernbloting检测dsRNA是否能阻断转录因子Hes5的表达，0.5%锥虫蓝法测定各浓度组中培养细胞活力。结果??200~300nmol/L浓度的dsRNA能特异有效地阻断Hes5的表达，而50~200nmol/L浓度的dsRNA有利于干细胞存活。结论??dsRNA能在神经干细胞内启动RNA干扰作用，适宜浓度的dsRNA能在特异有效地阻断内源性基因的表达的同时，最大限度地保持细胞活力。

关键词：双链RNA；Hes5；神经干细胞，骨髓源性；RNA干涉；免疫印迹

Blockingwithdouble-strandedRNAoftheexpressionofHes5inratbonemarrow-derivedneuralstemcells

WANGHai-yan1；XURu-xiang1；JIANGXiao-dan1；LUOShen-qiu2

1InstituteofNeuromedicineofPLA,ZhujiangHospital,FirstMilitaryMedicalUniversity,Guangzhou510282,China;2DepartmentofCellularBiology,FirstMilitaryMedicalUniversity,Guangzhou510515,China

Abstract:Objective??Toexaminetheefficiencyofexogenoussmalldouble-strandedRNA(dsRNA)inknockingdownthegeneexpressionatthepost-transcriptionlevel,andinvestigatethefactorsthatmayinfluencethetransfection.Method??ThebonemarrowstromalcellsofSDratwereseparatedandculturedinvitro,followedbyinductionofthecellstoevolveintoneuralstemcellsusingspecialculturemediumpreparedbyourlaboratory.SyntheticdsRNAwasthentransferredintothecellsatvariedconcentrations,andtheresultswereanalyzedbyWesternblotting.Results??Theconcentrationsrangingfrom200to300nmol/LwereoptimalforspecificallyblockingtheexpressionofHes5,whereasthesuitableconcentrationsforthecellsurvivalwerebetween50and200nmol/L.Conclusion??dsRNAiscapableoftriggeringRNAinterferenceinneuralstemcells,andatappropriateconcentration,itmayspecificallyandeffectivelyknockdownendogenousgeneexpressionwithoutsacrificingtheviabilityofthecells.

Keywords:double-strandedRNA;hairyandenhancerofsplit;neuralstemcell,bonemarrow-derived;RNAinterference;Wes