超声靶向微泡破裂技术联合shRNA质粒沉默Toll样受体4基因

摘要/Abstract

摘要：

目的 Toll样受体（Toll-like receptor，TLR）4表达水平与脑缺血合并高血糖后的痫性发作及脑梗死预后相关，本文探讨超声靶向微泡破裂（ultrasound-targeted microbubble destruction，UTMD）联合短发夹核糖核酸（short hairpin ribonucleic acid，shRNA）干扰技术沉默TLR4的可行性和应用价值。方法将32只实验用Wistar大鼠分为4组：空白对照组（NS）、裸质粒组（P）、质粒联合超声辐照组（P+UTMD）、质粒与SonoVue联合超声辐照组（P+S+UTMD），每组各8只。裸质粒组注入质粒，质粒联合超声辐照组注入质粒行超声辐照，质粒与SonoVue联合超声辐照组侧脑室注入质粒和SonoVue微泡并行超声辐照，处理4 d后每组取5只大鼠行Western Blot检测大脑TLR4蛋白表达，取3只大鼠行免疫组织化学染色。结果质粒联合超声辐照组和质粒与S o n oV u e联合超声辐照组抑制效果明显（P+S +U TM D： 0.223±0.009，P+UTMD：0.277±0.013，t 1，3=4.900，P＜0.01；t 1，4=8.779，P＜0.01），裸质粒组与空白对照组差异无显著性（NS：0.351±0.030，P：0.339±0.034，t 1，2=0.590，P＞0.05），而质粒与SonoVue 联合超声辐照组与质粒联合超声辐照组相比抑制效果更好（P+S+UTMD：0.223±0.009，P+UTMD： 0.277±0.013，t 3，4=7.006，P＜0.05）。免疫组织化学的平均光度值分析显示，质粒联合超声辐照组和质粒与SonoVue联合超声辐照组抑制效果明显（P+S+UTMD：0.026±0.0013，P+UTMD：0.058±0.0014， t 1，3=8.334，P＜0.01；t 1，4=21.027，P＜0.01），裸质粒组与空白对照组差异无显著性（NS：0.079±0.0048， P：0.077±0.0012，t 1，2=0.797，P＞0.05），而质粒与SonoVue联合超声辐照组与质粒联合超声辐照组相比抑制效果更好（P+S+UTMD：0.026±0.0013，P+UTMD：0.058±0.0014，t 3，4=33.254，P＜0.05）。结论超声微泡造影剂联合shRNA质粒可高效稳定沉默TLR4基因。

文章导读： TLR4与脑梗死后痫性发作及预后相关，本研究应用超声靶向微泡破裂联合短发夹核糖核酸干扰技
术沉默TLR4，有望应用于脑部基因治疗，并将为以后非侵袭性的脑部基因传输奠定基础。

关键词: 超声; 微泡; Toll样受体4; 基因; 核糖核苷酸干扰

Abstract:

Objective To determine the effect of the association of ultrasound and microbubble with short hairpin RNA ( shRNA) silencing Wistar rat brain Toll-like receptor 4 (TLR4). Methods Wistar rats were divided into 4 groups:normal sodium (NS) group, naked plasmid group (P), plasmid plus ultrasound (US) irradiation group (P+ultrasound-targeted microbubble destruction [UTMD]), plasmid plus US irradiation and SonoVue group (P+S+UTMD). Lateral ventricle injection of plasmid and SonoVue microbubble into rats' brain was performed and the brains were exposed to extracranial US, the brain TLR4 gene expression efficiency was evaluated 4 days after treatment. Results Western blot result shows that there is not statistical difference between NS group and naked plasmid group (NS:0.351±0.030, P:0.339±0.034, t 1, 2=0.590; P >0.05), but plasmid plus US irradiation group (P+UTMD) and plasmid plus US irradiation and SonoVue group (P+S+UTMD) shows statistical differences between NS group and naked plasmid group (P+S+UTMD:0.223±0.009, P+UTMD:0.277±0.013, t 1, 3=4.900, P <0.01; t 1, 4=8.779, P <0.01). Immunohistochemical analysis also shows statistical differences between Ultrasound Group (P+UTMD group and P+S+UTMD group) and non-Ultrasound group (NS and P) (P+S+UTMD:0.026±0.0013, P+UTMD:0.058±0.0014, t 1, 3=8.334, P <0.01; t 1, 4=21.027, P <0.01). There is not statistical difference between NS group and naked plasmid group (NS:0.079±0.0048, P:0.077±0.0012, t 1, 2=0.797; P >0.05) and P+S+UTMD group has better inhibition effects compared with P+UTMD group (P+S+UTMD:0.026±0.0013, P+UTMD:0.058±0.0014, t 3, 4=33.254; P <0.05). Conclusion The present study shows that this method of UTMD combined mediated shRNA can be used to deliver plasmid deoxyribonucleic acid (DNA) to the brain selectively and effectively. This noninvasive technique is a promising method for cerebral therapy and could be applied in the rapidly developing gene therapy for cerebral diseases.

Key words: Ultrasound; Microbubble; Toll-like receptor 4; Gene; RNA interference

郑 1，梁燕玲1，陈佳1，陈智毅2，李斯颖1，罗建华1，方小波1. 超声靶向微泡破裂技术联合shRNA质粒沉默Toll样受体4基因[J]. 中国卒中杂志, 2015, 10(04): 284-290.

ZHENG Xuan*, LIANG Yan-Ling, CHEN Jia, CHEN Zhi-Yi, LI Si-Ying,LUO Jian-Hua, FANG Xiao-Bo.. Ultrasound and Microbubbles Mediated shRNA Transfection Silences Toll-like Receptor 4 Gene Expression[J]. Chinese Journal of Stroke, 2015, 10(04): 284-290.

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