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ID:9890934
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时间:2018-05-14
《dsrna 阻断大鼠骨髓源性神经干细胞hes5 表达的实验研究》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
1、dsRNA阻断大鼠骨髓源性神经干细胞Hes5表达的实验研究作者:王海燕1;徐如祥1;姜晓丹1;罗深秋2 (第一军医大学1珠江医院全军神经医学研究所,广东 广州510282;2细胞生物学教研室学,广东 广州 510515)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ds
2、RNA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;免疫印迹中图分类号:Q189 文献标识码:A 文章编号:1000-2588(2004)01-0
3、035-04Blockingwithdouble-strandedRNAoftheexpressionofHes5inratbonemarrow-derivedneuralstemcellsWANGHai-yan1;XURu-xiang1;JIANGXiao-dan1;LUOShen-qiu21InstituteofNeuromedicineofPLA,ZhujiangHospital,FirstMilitaryMedicalUniversity,Guangzhou510282,China;2DepartmentofCel
4、lularBiology,FirstMilitaryMedicalUniversity,Guangzhou510515,ChinaAbstract:Objective Toexaminetheefficiencyofexogenoussmalldouble-strandedRNA(dsRNA)inknockingdownthegeneexpressionatthepost-transcriptionlevel,andinvestigatethefactorsthatmayinfluencethetransfection.
5、Method ThebonemarrowstromalcellsofSDratwereseparatedandculturedinvitro,followedbyinductionofthecellstoevolveintoneuralstemcellsusingspecialculturemediumpreparedbyourlaboratory.SyntheticdsRNAwasthentransferredintothecellsatvariedconcentrations,andtheresultswereana
6、lyzedbyWesternblotting.Results Theconcentrationsrangingfrom200to300nmol/LwereoptimalforspecificallyblockingtheexpressionofHes5,whereasthesuitableconcentrationsforthecellsurvivalwerebetween50and200nmol/L.Conclusion dsRNAiscapableoftriggeringRNAinterferenceinneura
7、lstemcells,andatappropriateconcentration,itmayspecificallyandeffectivelyknockdownendogenousgeneexpressionwithoutsacrificingtheviabilityofthecells.Keywords:double-strandedRNA;hairyandenhancerofsplit;neuralstemcell,bonemarrow-derived;RNAinterference;Westernblotting收
8、稿日期:2003-07-11基金项目:国家自然科学基金(3027049);军队及广东省科技厅“十五”重大项目(01z054)ThestudyissupportedbyNationalNaturalScienceFoundationofChina(3027049),andisakeyprojectofth
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