大鼠血管内皮细胞siRNA转染条件的优化
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摘要
目的:旨在优化siRNA转染大鼠血管内皮细胞的转染条件.方法:将0.75μL和1.5μL的脂质体LipofectamineTM3000分别与20、40、60、80nmol带荧光标记的FAMsiRNA组合,转染6、12、18、24h后,用荧光显微镜计数阳性细胞率、MTT法检测各浓度条件下内皮细胞的存活率,筛选最优转染条件.结果:(1)转染12h后,用荧光显微镜检测20、40、60、80nmol各组,均可观察到绿色荧光(2)siRNA浓度为60nmol/L,脂质体为1.5μL的组合转染效率最高,继续增加siRNA的浓度,转染效率提高不明显.(3)转染时间超过24h,各组细胞荧光减弱,细胞死亡率显著增加.结论:结果表明,以1.5μL LipofectamineTM3000与60nmol/L的siRNA浓度组成转染混合物转染12h可以实现对大鼠血管内皮细胞高效转染并保持较高的细胞活性.
Objective:This work aims to optimize the transfection parameters for transfecting siRNA(small interfering RNA)to rats vascular endothelial cells(EC).Methods:Using the 0.75μL and 1.5μL of LipofectamineTM3000 diluted with FAM-siRNA(20nmol、40nmol、60nmol、80nmol)were mixed with different concentrations of fluorescin-labeled siRNA to form a series of mixtures to transfect EC cells.At the time point of 6h,12 h,18h,24 hafter transfection,under fluorescent microscope to observe and determine the percentage of the cells transfected under each condition.The cell viability was determined by using MTT assay to achieve the optimal conditions.Results:(1)Green fluorescence was discovered under the fluorescence microscope after transfection 12 h.(2)The highest transfection efficiency can be obtained in 60nmol/L siRNA with the1.5μL of Lipofectamine transfected EC cells.No differences were found in transfection efficiency and cell viability whether using the higher concentration of siRNA.(3)Twenty-four hours later,the transfection efficiency could not be further increased by elevating the concentration of siRNA,and the cell death rate increased significantly.Conclusions:The results showed that the transfection efficiency and cell viability are dependent on the correct LipofectamineTM3000(1.5μL)-siRNA(60nmol/L)ratio after transfection 12 h.
Objective:This work aims to optimize the transfection parameters for transfecting siRNA(small interfering RNA)to rats vascular endothelial cells(EC).Methods:Using the 0.75μL and 1.5μL of LipofectamineTM3000 diluted with FAM-siRNA(20nmol、40nmol、60nmol、80nmol)were mixed with different concentrations of fluorescin-labeled siRNA to form a series of mixtures to transfect EC cells.At the time point of 6h,12 h,18h,24 hafter transfection,under fluorescent microscope to observe and determine the percentage of the cells transfected under each condition.The cell viability was determined by using MTT assay to achieve the optimal conditions.Results:(1)Green fluorescence was discovered under the fluorescence microscope after transfection 12 h.(2)The highest transfection efficiency can be obtained in 60nmol/L siRNA with the1.5μL of Lipofectamine transfected EC cells.No differences were found in transfection efficiency and cell viability whether using the higher concentration of siRNA.(3)Twenty-four hours later,the transfection efficiency could not be further increased by elevating the concentration of siRNA,and the cell death rate increased significantly.Conclusions:The results showed that the transfection efficiency and cell viability are dependent on the correct LipofectamineTM3000(1.5μL)-siRNA(60nmol/L)ratio after transfection 12 h.
引文
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[13]Gottumukkala S N,Dwarakanath C D,Sudarsan S.Ribonucleic acid interference induced gene knockdown[J].J Indian Soc Periodontol,2013,17(4):417-422.
[14]张园,李志樑,邱健,等.优化阳离子脂质体介导的组织因子小干扰RNA转染HEK-293的转染条件[J].广西医科大学学报,2009,26(2):176-179.
[15]杨萍,严金川,刘培晶.siRNA反向转染法提高原代悬浮细胞转染效率的应用[J].江苏大学学报(医学版),2010,20(3):267-269.
[16]崔烨,王璐,孙琼,等.寡肽阳离子脂质体的体外研究[J].中国药科大学学报,2013,44(4):328-333.
[17]Cseh B,Doma E,Baccarini M.The’RAF’neighborhood:Protein-protein interaction in the Raf/Mek/Erk pathway[J].FEBS Lett,2014,Aug 1;588(15):398-406.
[2]Shiha S K.RNAi-mediated gene silencing[J].Physiol Mol Biol Plants,2010,16(4):321-322.
[3]Ilarraza-Lomeli R,Cisneros-Vega B,Cervanles Gonez J,e1al.Dp71,ulrophin and beta-dyslroglycan expression and dislribulion in PC12/L6cell cocuhures[J].Neurorepor1,2007,18(16):1657-1661.
[4]Tesfaye S,Selvarajah D.Advances in the epidemiology,pathogenesis and management of diabetic peripheral neuropathy[J].Diabetes Melab Res Rev,2012,28(1):8-14.
[5]El Boghdady N A,Badr G A.Evaluation of oxidative stress markers and vascular risk factors in patients with diabetic peripheral neuropathy[J].Cell Biochem Funct,2012,30(4):328-334.
[6]杨翠燕,王金凤,张艳萍,等.大鼠胸主动脉血管内皮细胞的分离培养和纯化鉴定[J].解放军医学杂志,2010,35(2):195-197.
[7]Ehrlich S F,Quesenberry C J,Van Den Eeden S K,eI al.PaLienls diagnosed wish diabetes are all increased risk for asthma,chronic obstructive pulmonary disease,pulmonary fibrosis,and pneumonia bluntly lung cancer[J].Diabetes Care,2010,33(1):55-60.
[8]Deng S,Wang J,Hou L,et al.Annexin A1,A2,A4and A5play important roles in breast cancer,pancreatic cancer and laryngeal carcinoma,alone and synergistically[J].Oncol Lett,2013,5(1):107-112.
[9]李连喜,胡仁明.RNA干扰在疾病治疗中的研究及应用进展[J].国际内科学杂志,2007,34(7):400-403,431.
[10]Koga T,Xiang S,Park J S,et al.Differential effects of myocilin and optineurin,two glaucoma genes on neurite out-growth[J].Am J Pathol,2010,176(1):343-352.
[11]Honma K,Mishima E,Sharma.A.Role of Tannerella forsythia NanH sialidase in epithelial cell attachment[J].Infect Immun,2011,79(1):393-401.
[12]Lee J T,Lee S D,Lee J Z,et al.Expression analysis and clinical significance of CXCL16/CXCR6in patients with bladder cancer[J].Oncol Lett,2013,5(1):229-235.
[13]Gottumukkala S N,Dwarakanath C D,Sudarsan S.Ribonucleic acid interference induced gene knockdown[J].J Indian Soc Periodontol,2013,17(4):417-422.
[14]张园,李志樑,邱健,等.优化阳离子脂质体介导的组织因子小干扰RNA转染HEK-293的转染条件[J].广西医科大学学报,2009,26(2):176-179.
[15]杨萍,严金川,刘培晶.siRNA反向转染法提高原代悬浮细胞转染效率的应用[J].江苏大学学报(医学版),2010,20(3):267-269.
[16]崔烨,王璐,孙琼,等.寡肽阳离子脂质体的体外研究[J].中国药科大学学报,2013,44(4):328-333.
[17]Cseh B,Doma E,Baccarini M.The’RAF’neighborhood:Protein-protein interaction in the Raf/Mek/Erk pathway[J].FEBS Lett,2014,Aug 1;588(15):398-406.