PADI4-siRNA-MSN的构建及其在RA防治中的初步应用

Preparation of PADI4-siRNA-MSN and its preliminary application in prevention and treatment of RA

目的:制备介孔二氧化硅纳米载体(MSN)并包载肽酰基精氨酸脱亚胺酶4(PADI4)小干扰RNA(PADI4-siRNA-MSN),阐明PADI4-siRNA-MSN对类风湿关节炎成纤维滑膜细胞(RA-FLS)凋亡的影响。方法:采用模板法合成MSN,包被阳离子聚合物聚乙烯亚胺(PEI)及荧光标记分子罗丹明B(Rh B),检测其表征,包括粒径、Zeta电势、荧光和形貌。设计并合成2对FAM标记的针对PADI4基因编码区的干扰核苷酸链(si-944、si-1225)及阴性对照si-NC,制备PADI4-siRNA-MSN和siNC-MSN,转染RA-FLS,流式细胞术(FCM)鉴定转染率。采用荧光定量PCR、蛋白印迹技术检测转染48 h后,干扰组和阴性对照组细胞PADI4表达量,FCM检测细胞凋亡率,激光共聚焦扫描显微镜(CLSM)观察PADI4-siRNA-MSN胞内分布。所有数据采用配对t检验进行分析。结果:MSN经表征检测显示已成功制备并包被Rh B、PEI,FCM结果显示转染率为93. 3%(si-944)和91. 9%(si-1225)。si-NC组中的PADI4 mRNA水平为1. 23±0. 27; si-944、si-1225干扰组中分别为0. 35±0. 12、0. 44±0. 12,均显著低于阴性对照组(P<0. 05)。蛋白印迹结果与PCR结果一致。与si-NC组细胞凋亡率(8. 83±0. 15)相比,si-944和si-1225组细胞凋亡率(11. 72±0. 82,13. 00±1. 42)显著增加(P<0. 05)。CLSM结果显示PADI4-siRNA-MSN分布于核周。结论:PADI4-siRNA-MSN成功制备,并转染RA-FLS,有效沉默PADI4基因,显著增加RA-FLS的凋亡率。PADI4-siRNA-MSN在抑制RA-FLS增殖、促进凋亡中具有一定的应用前景。

Objective： To elucidate the effect of PADI4-siRNA-MSN on the apoptosis of fibroblast-like synoviocytes（FLS） isolated from rheumatoid arthritis（RA） patients（RA-FLS） by preparing mesoporous silica nanoparticle（MSN） carried peptidylarginine deiminase 4（PADI4） siRNA（PADI4-siRNA-MSN）. Methods： MSN was synthesized by a template method,and then coated with cationic polymer polyethyleneimine（PEI） and fluorescent label Rhodamine B（Rh B）. Its characterizations were determined,including particle size,Zeta potential,fluorescence,and morphology. We designed and synthesized FAM-labeled si-RNA chains（si-944,si-1225） against PADI4 and negative control si-NC to prepare PADI4-siRNA-MSN and si-NC-MSN. Transfection rates were identified by flow cytometry（FCM）. The expression of PADI4 in the interference group and the negative control group was detected by fluorescence quantitative PCR and Western blot after transfection for 48 h. The apoptosis cells were detected by FCM. The intracellular distribution of PADI4-siRNA-MSN was observed by laser confocal scanning microscopy（CLSM）. All data were analyzed using the paired t-test. Results： The detection results of MSN showed that Rh B and PEI were successfully prepared and coated. The results of FCM showed that the transfection rates were 93. 3%（si-944） and 91. 9%（si-1225）. The level of PADI4 mRNA in the si-NC group was 1. 23± 0. 27,the si-944 and si-1225 groups were 0. 35±0. 12 and 0. 44±0. 12,respectively,which were significantly lower than those in the negative control group（P〈0. 05）. Western-blot results are consistent with PCR results. The apoptotic rates of si-944 and si-1225（11. 72± 0. 82,13. 00 ± 1. 42）were significantly increased compared with the si-NC group（8. 83 ± 0. 15）（P〈0. 05）. CLSM results showed that PADI4-siRNA-MSN distributed in the perinuclear. Conclusion： PADI4-siRNA-MSN has been successfully prepared and transfected into RA-FLS,and effectively silenced PADI4 gene and significantly increased the apoptosis rate of RA-FLS. PADI4-siRNA-MSN has some potential applications in inhibiting RA-FLS proliferation and promoting apoptosis.