摘要
目的探讨瞬时转染miR-155mimics和miR-155 inihibitor化学合成物在足细胞中表达变化及其稳定情况。方法体外培养小鼠肾小球足细胞,用脂质体法将miR-155mimics/NC、miR-155 inihibitor/NC、Cy3 miR-155 mimics/inhibitor NC转染足细胞,构建足细胞内miR-155高表达模型、低表达模型、荧光基团。然后用免疫荧光显微镜观察不同剂量LipofectamineTM 2000转染不同浓度Cy3 miR-155 NC后的荧光转染效果。用实时荧光定量PCR技术检测足细胞中miR-155mimics浓度梯度(50、80、100 nmol/mL)、miR-155 inihibitor浓度梯度(80、100、150 nmol/mL)的miRNA-155表达。结果达到一定剂量的LipofectamineTM 2000足以饱和不同浓度的miRNA,并将不同浓度Cy3 miR-155 NC成功转入足细胞内,且转染效率达80%以上。qRT-PCR结果显示转染miR-155mimics、miR-155 inihibitor 24 h后,miR-155在足细胞中的表达明显改变,与阴性对照组(NC)比较差异有统计学意义,表明转染成功。转染80 nmol/mL浓度的miR-155mimics,miR-155在足细胞中的表达明显上调(P<0.001),与其他浓度对比表达量升高,差异有统计学意义。转染150 nmol/mL浓度的miR-155 inihibitor,miR-155在足细胞中的表达明显下调(P<0.001),与其他浓度对比表达量降低,差异有统计学意义。结论 LipofectamineTM 2000达到适当剂量时将各工作浓度的miRNA-155转入足细胞内的转染效果高。80 nmol/mL的miR-155mimics和150 nmol/mL的miR-155 inhibitor可能较适合用于实验研究。
Objective To investigate the expression and stability of transiently transfected miR-155 mimics and miR-155 inhibitor chemical compounds into podocytes.Methods Mouse glomerular podocytes were cultured in vitro,and miR-155 mimics/NC,miR-155 inhibitor/NC,Cy3 miR-155 mimic/inhibitor NC were transfected into podocytes by liposome method to construct miR-155 high expression model or low expression model and fluorophore in podocytes.The transfection effect of different doses of LipofectamineTM 2000 transfecting different concentrations of Cy3 miR-155 NC was observed by immunofluorescence microscopy.miRNA-155 expression transfected with miR-155 mimics at concentration gradient(50,80,100 nmol/mL) or miR-155 inhibitor at concentration gradient(80,100,150 nmol/mL) in podocytes was detected by qRT-PCR.Results A fixed dose of LipofectamineTM 2000 was sufficient to saturate different concentrations of miRNA,and different concentrations of Cy3 miR-155 NC were successfully transferred into podocytes,and the transfection efficiency was over 80%.qRT-PCR results showed that the expression of miR-155 in podocytes was significantly changed after transfection of miR-155 mimics or miR-155 inhibitor for 24 h,and the difference was statistically significant compared with the negative control group(NC),indicating a successful transfection.When transfected with miR-155 mimics at a concentration of 80 nmol/mL,the expression of miR-155 in podocytes was significantly up-regulated compared with other concentrations(P<0.001).And when transfected with miR-155 inhibitor at 150 nmol/mL,miR-155 expression in podocytes was significantly down-regulated compared with other concentrations(P<0.001).Conclusion With appropriate dose of LipofectamineTM 2000,the transfection efficiency of working concentration of miRNA-155 into podocytes is high.Concentrations of 80 nmol/mL miR-155 mimics and 150 nmol/mL miR-155 inhibitor may be suitable for experimental studies.
作者
凌霄雁
林栩
郑心彤
古贤君
梁钊
覃卿
杜秀日
LING Xiaoyan;LIN Xu;ZHENG Xintong;GU Xianjun;LIANG Zhao;QIN Qing;DU Xiuri(Nephrology Department of Affiliated Hospital,Youjiang Medical University for Nationalities,Baise 533000,China;Graduate School,Youjiang Medical University for Nationalities,Baise 533000,China)
出处
《右江医学》
2019年第12期886-891,共6页
Chinese Youjiang Medical Journal
基金
国家自然科学基金(81860131)
广西自然科学基金(2017 GXNSFAA198288)
百色市科学研究与技术开发计划项目(百科20160608)
