Optimization of transfect conditions for cathepsin B-RNAi-lentivirus to the brain micro vascular endothelial cells
10.3760/cma.j.issn.1008-6315.2012.11.003
- VernacularTitle:慢病毒介导的小RNA干扰组织蛋白酶B转染小鼠脑微血管内皮细胞的条件
- Author:
Guohong ZHOU
- Publication Type:Journal Article
- Keywords:
RNAi;
Lentivirus;
Cathepsin B;
Mice brain micvascular endothelial cells;
Transfection rate
- From:
Clinical Medicine of China
2012;28(11):1128-1131
- CountryChina
- Language:Chinese
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Abstract:
ObjectiveTo Research on the best condition of transfecting RNAi plasmid into the mice brain micro vascular endothelial cells to interfere the tissue enzymes and protease through the cathepsin B-RNAi-lentivirus.MethodsThe brain micro vascular endothelial cells ( bEND.3 ) were divided into three groups at random:culture medium completely group (NP group),culture medium with 5 mg/L polybrene group ( P1 group) and culture medium with 8 mg/L polybrene group ( P2 group),each group has five different dimensional MOI (1,5,10,15,20),after transfected,48 h,72 h,96 h,the fluorescence rate of GFP protein were counted by a fluorescence microscope to obtain the transfected rate,the MOI of the best optimization condition were observed;After one week,the effect of the most appropriate of MOI to cytoactive and survival rate calculated through MTT.ResultsA small amount of green fluorescent protein(GFP) in each group were observed after transfected for 48 h,the transfection rate of each group were 1％ -9％ ;In the same MOI groups,there was no significant difference between the 72 h and 96 h ( P ＞ 0.01 ) ; In groups of MOI ( 1,5,15 ),the prevalence transfection rate of each group were lower than 20％ and in MOI(10,20) group,the transfection rate were between 71％ -97％ and 60％ - 84％,and the transfection rate in group 10 was significantly higher than group 20 ( P ＜ 0.01 ) ; P1 group showed significantly higher survival rate than that in P2 group by MTT assay [ ( 81.38 ± 6.69 ) ％ vs (62.03 ± 5.13)％,t =15.795,P ＜ 0.001 ].ConclusionThe most optimal conditions of CathepsinB-RNAi-Lentivirus infection bEND.3 depend on MOI(10),polybrene(5 mg/L) and the transfection time of 72 h.
