摘要
目的:探究周围神经损伤(PNI)后施万细胞的死亡机制,使用纳米凝胶搭载的siRNA靶向抑制施万细胞死亡.方法:下载GEO数据库中PNI的转录组数据,依次进行数据处理、差异分析、GO功能富集分析、铁死亡通路鉴定以及靶基因的筛选.通过蛋白印记和qPCR实验验证靶基因的差异性.自组装单宁酸(TA)-siRNA纳米凝胶,通过丁达尔效应实验、粒径和电位测量、细胞吞噬实验、细胞骨架染色和CCK-8细胞活力实验鉴定纳米凝胶的物理学和生物学特性.通过划痕实验、免疫荧光染色实验、蛋白印记和qPCR实验验证TA-siRNA纳米凝胶抑制施万细胞铁死亡的有效性.结果:PNI后施万细胞出现明显的铁死亡现象,Bex1是调控施万细胞铁死亡的关键基因.TA-siRNA纳米凝胶具有优良的物理学和生物学特性,能够将siRNA成功地携带到损伤的施万细胞中并沉默靶基因,从而有效地抑制施万细胞损伤后的铁死亡.结论:纳米凝胶搭载的siRNA可以靶向抑制施万细胞铁死亡,为临床治疗PNI提供新的方向.
Abstract
Objective To investigate the mechanism of Schwann cell death after peripheral nerve injury(PNI),and to inhibit Schwann cell death by nanogel-delivered siRNA.Methods The transcriptome data of PNI in GEO database were downloaded for data processing,followed by difference analysis,GO functional enrichment analysis,ferroptosis pathway identification,and target gene screening.The differences of target genes were verified through protein imprinting and qPCR experiment.The physical and biological properties of self-assembled tannic acid(TA)-siRNA nanogels were identified by Tyndall effect,particle size and potential measurements,phagocytosis assay,cytoskeleton staining,and CCK-8 cell viability assay.The effectiveness of TA-siRNA nanogel in inhibiting ferroptosis in Schwann cells was verified by scratch experiment,immunofluorescence staining,protein imprinting,and qPCR experiment.Results After PNI,obvious ferroptosis was observed in Schwann cells,and Bex1 was identified as the key gene regulating ferroptosis in Schwann cells.With excellent physical and biological properties,TA-siRNA nanogel could successfully carry siRNA into damaged Schwann cells,and silence their ferroptosis genes,thus effectively inhibiting ferroptosis after Schwann cell damage.Conclusion Nanogel-delivered siRNA can inhibit ferroptosis in Schwann cells,providing a new direction for PNI treatment.
基金项目
军队医学科技青年培育计划(19QNP005)
南方医科大学南方医院临床研究专项(2022CR006)
南方医科大学南方医院院长基金(2020B028)
NETL
NSTL
维普
万方数据