纳米材料Ac-αCD包装的Rab5a siRNA对肺微血管内皮细胞β-AR的调控作用  

Regulation of Nanomaterial Ac-αCD-packed Rab5a siRNA on β-AR of Pulmonary Microvascular Endothelial Cells

作　　者：孙欢[1] 朱伦刚[1] Sun Huan;Zhu Lungang(Department of Critical Care Medicine,Mianyang Central Hospital,Mianyang,Sichuan 621000,China)

机构地区：[1]绵阳市中心医院重症医学科,四川绵阳621000

出　　处：《四川医学》2020年第3期262-266,共5页Sichuan Medical Journal

基　　金：绵阳市卫计委课题(编号:201624)。

摘　　要：目的研究纳米材料缩醛化环糊精(acetalatedα-cyclodextrin,Ac-αCD)包装的Rab5a siRNA对肺微血管内皮细胞(Pulmonary Microvascular Endothelial cells,PMECs)β肾上腺素受体(β-Adrenergic Receptor,β-AR)的调控作用。方法设计合成5′端标记FITC的Rab5a siRNA序列,并由Ac-αCD组装成Ac-αCD-Rab5a siRNA的纳米颗粒处理大鼠肺微血管内皮细胞24h,同时用常规细胞转染试剂进行Rab5a siRNA转染细胞24h,激光共聚焦显微镜下比较细胞对Rab5a siRNA的摄取情况;分别利用RT-PCR、Western blotting检测Ac-αCD-Rab5a siRNA处理细胞0h、24h、48h后Rab5a的mRNA和蛋白表达;利用放射性配体结合受体试验检测Ac-αCD-NC siRNA组和Ac-αCD-Rab5a siRNA处理细胞48h后细胞表面β-AR表达情况;转染GFP标记的β2-AR质粒至内皮细胞,激光共聚焦显微镜下观察纳米系统对β2-AR细胞内定位的作用。结果纳米颗粒Ac-αCD-Rab5a siRNA可被细胞有效摄取,细胞内可见大量呈颗粒状均匀分布的绿色荧光颗粒;Ac-αCD-Rab5a siRNA可显著下调目的基因mRNA和蛋白表达水平;Ac-αCD-Rab5a siRNA可增加细胞膜表面β-AR表达;Ac-αCD-Rab5a siRNA可使β2-AR在细胞内定位发生改变。结论纳米材料Ac-αCD包装的Rab5a siRNA能被PMECs有效摄取,从而调控β-AR在细胞膜表面的表达和细胞内的定位。Objective To study the regulation of nanomaterial acetalatedα-cyclodextrin(Ac-αCD)-packed Rab5a siRNA onβ-adrenergic receptor(β-AR)of pulmonary microvascular endothelial cells(PMECs).Methods Design and synthesis of a 5′-end FITC-labeled Rab5a siRNA sequence and assembly of Ac-αCD into Ac-αCD-Rab5a siRNA nanoparticles to treat rat pulmonary microvascular endothelial cells for 24h.Transfect cells with Rab5a siRNA with conventional cell transfection reagents for 24h,simultaneously.Comparison of Rab5a siRNA uptake by cells under a laser confocal microscope.RT-PCR and western blotting were used to detect the mRNA and protein expression of Rab5a after Ac-αCD-Rab5a siRNA treated cells at 0h,24h and 48h,respectively.Radioligand binding receptor test was used to detect the expression ofβ-AR on the cell surface of Ac-αCD-NC siRNA group and Ac-αCD-Rab5a siRNA treated cells after 48h.GFP-labeledβ2-AR plasmid was transfected into endothelial cells,and the effect of the nanosystem on the localization ofβ2-AR cells was observed under a laser confocal microscope.Results Nanoparticles of Ac-αCD-Rab5a siRNA can be effectively taken up by cells,and a large number of green fluorescent particles are evenly distributed in the cells.Ac-αCD-Rab5a siRNA can significantly reduce the mRNA and protein expression levels of target genes.Ac-αCD-Rab5a siRNA can increaseβ-AR expression on cell membrane surface and changeβ2-AR localization in cells.Conclusion The nanomaterial Ac-αCD-packaged Rab5a siRNA can be effectively taken up by PMECs,thereby regulating the expression ofβ-AR on the cell membrane surface and the intracellular localization.

关 键 词：纳米颗粒 RAB5A SIRNA Β-AR 肺微血管内皮细胞 

分 类 号：R563.8[医药卫生—呼吸系统]