发动蛋白(dynamin)抑制剂Dyngo-4a在大鼠骨髓间充质干细胞内吞葡聚糖过程中存在脱靶效应  

The off-target effects of dynamin inhibitor Dyngo-4a during the internalization of dextran by bone marrow mesenchymal stem cells in rat

作　　者：张颖 朱静[1,2] 许皓[2,3] 易勤[1,2] 颜亮 叶亮[1,2] 张心愿[1,2] 谭彬 ZHANG Ying;ZHU Jing;XU Hao;YI Qin;YAN Liang;YE Liang;ZHANG Xinyuan;TAN Bin(Department of Pediatric Research Institute,Children’s Hospital of Chongqing Medical University;National Clinical Research Center for Child Health and Disorders,Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of Pediatrics;Department of Clinical Laboratory,Children’s Hospital of Chongqing Medical University,Chongqing 400014,China)

机构地区：[1]重庆医科大学附属儿童医院儿科研究所 [2]国家儿童健康与疾病临床医学研究中心,儿童发育疾病研究教育部重点实验室,儿科学重庆市重点实验室 [3]重庆医科大学附属儿童医院检验科,重庆400014

出　　处：《细胞与分子免疫学杂志》2023年第1期57-62,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：重庆市科技局面上项目(cstc2020jcyj-msxmX0363);重庆市科技局社会事业与民生保障科技创新专项(一般)(cstc2017shmsA130091)

摘　　要：目的研究发动蛋白(DNM)抑制剂Dyngo-4a在依赖DNM的内吞途径中可能存在的脱靶作用。方法分离培养SD大鼠骨髓间充质干细胞(BMSC)并进行流式细胞术鉴定,将细胞分为抑制剂对照组、Dyngo-4a处理组、转染阴性对照(si-NC)组、DNM2小干扰RNA(si-DNM2)转染组、转染和Dyngo-4a共处理组。反转录PCR和Western blot法验证DNM2基因沉默效率,CCK-8法检测Dyngo-4a处理后细胞活性变化,共聚焦显微镜检测内吞的Dylight649标记的转铁蛋白(transferrin-Dylight649)和四甲基罗丹明标记的葡聚糖(dextran-TMR)阳性的囊泡数量以及平均荧光强度。结果使用小干扰RNA能显著下调BMSC的DNM2的mRNA和蛋白表达,转染组内吞的转铁蛋白囊泡数量与转染阴性对照组比较明显降低,si-DNM2转染组内吞的葡聚糖囊泡数量和平均荧光强度与si-NC组比较变化不明显,转染si-DNM2和Dyngo-4a共处理组与单独si-DNM2转染组比较,Dyngo-4a处理组与抑制剂对照组比较,葡聚糖囊泡数量和平均荧光强度均明显降低。结论Dyngo-4a作为靶向DNM的抑制剂在葡聚糖内吞进BMSC过程中存在脱靶效应。Objective To investigate the possible off-target effects of dynamin(DNM)inhibitor Dyngo-4a in dynamin-dependent endocytic pathways.Methods Bone marrow mesenchymal stem cells(BMSCs)obtained from SD rats were isolated and cultured,and identified by flow cytometry.The cells were divided into inhibitor control group,Dyngo-4a-treated group,negative control siRNA(si-NC)transfection group,DNM2 siRNA transfection(si-DNM2)group,si-DNM2 and Dyngo-4a co-treated group.Real time quantitative PCR and Western blot analysis were used to verify the silencing efficiencies of DNM2 gene and CCK-8 assay were used to detect the cell viability after Dyngo-4a treatment.Confocal microscopy was used to detect the number and mean fluorescence intensity(MFI)of transferrin-Dylight649-positive and dextran-TMR-positive vesicles.Results The mRNA and protein expression levels of DNM2 were down-regulated using small interfering RNA.The number of transferrin-Dylight649-positive vesicles significantly decreased in si-DNM2 group compared with si-NC group.For the number and MFI of dextran-TMR-positive vesicles,no significant change was observed between the si-DNM2 group and the si-NCgroup,but there was a significant reduction in the si-DNM2 and Dyngo-4a co-treated group compared with the si-DNM2 group.A significant decrease was also found in the Dyngo-4a-treated group compared with the inhibitor control group.Conclusion The off-target effects of dynamin inhibitor Dyngo-4a presents in the internalization of dextran by BMSCs.

关 键 词：脱靶效应 发动蛋白 Dyngo-4a 内吞作用 葡聚糖 骨髓间充质干细胞(BMSC) 

分 类 号：R965[医药卫生—药理学] R392-33[医药卫生—药学] Q25[生物学—细胞生物学]