DMRIE-C脂质体介导CLEC2B转染Jurkat细胞及其转染率的影响因素

DMRIE-C liposome mediated CLEC2B transfects into Jurkat cells and the influence factors of transfection rate

目的探讨由DMRIE-C脂质体介导将CLEC2B siRNA转染入Jrukat细胞的过程中,对Jurkat细胞的影响,并分析其转染率的影响因素。方法通过DMRIE-C转染CLEC2B siRNA入Jrukat细胞,台盼蓝染色法观察细胞活性,流式细胞仪检测siRNA转染率,RT-PCR检测CLEC2B基因沉默效率。结果 DMRIE-C脂质体对细胞的损伤程度与两者比例相关,对转染体系600μL(其中200μL密度为2×106/mL淋巴细胞)进行转染时,脂质体达3μL时可损伤细胞,不利于后续试验进行;siRNA∶DMRIE-C比例为3μL∶2μL,siRNA终浓度约80 nmol/L时,转染率最高,可达30.7%,转染后CLEC2B基因抑制率可达(35±1.9)%(t=15.9,P<0.05)。结论 DMRIE-C脂质体可实现目的 siRNA对Jurkat细胞有效转染,同时转染中脂质体与细胞比例、血清、操作方式等因素均可对转染效率产生一定的影响。

Objective To observe the process of transfecting CLEC2B siRNA mediated by DMRIE-C liposome into the Jurkat cells and to analyze the factors of influencing transfection rate.Methods We transfected CLEC2B siRNA into the Jurkat cells by DMRIE-C.Trypan blue staining was used to observe the cell activity.siRNA transfection efficiency was detected by flow cytometry,and the CLEC2B gene silencing efficiency was detected by RT-PCR.Results The cell damage degree in the transfection process was influenced by the cell members /transfection system lipo concentration ratio,3 μl lipo in the transfection system（ 600 μl total liquid,200 μl 2 × 106/ml lymphocytes） would damage cells,and go against the following experiment.The transfection efficiency could increase to 30.70% when the siRNA： DMRIE-C ratio was adjusted to 3 μl∶ 2 μl,and then,the CLEC2B gene expression inhibition ratio was about 35 ± 1.9%（ t = 15.9,P〈 0.05）.Conclusion DMRIE-C lipo cloud successfully transfer aim siRNA to Jurkat cells,and meanwhile,lipo /lymphocyte ratio,serum interference and operation skills may influence the transfection efficiency.