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《中华肺部疾病杂志》[ISSN:1006-6977/CN:61-1281/TN]

卷:

期数:

2020年04期

页码:

461-465

栏目:

论著

出版日期:

2020-08-20

文章信息/Info

Title:

Prophylactic and therapeutic effects of small interfering RNA targeting nucleocapsid protein gene on influenza A virus

作者:

史 亮¹ 尹申慧² 任 浩² 高 荣² 马 壮¹ 孙文武¹ 曹建平¹

10016 沈阳,北部战区总医院呼吸与危重症医学科¹ 200433 上海,海军(第二)军医大学海军医学系²

Author(s):

Shi Liang¹;  Yin Shenhui²;  Ren Hao²;  Gao Rong²;  Ma Zhuang¹;  Sun Wenwu¹;  Cao Jianping¹.

¹Department of Respiratory and Critical Care Medicine, General Hospital of Northern Theater Command, Shenyang 110016, China; ²Department of Naval Medicine, Naval Medical University, Shanghai 200433, China
Corresponding author

关键词:

流感病毒;  RNA干扰;  NP;  转染;  qRT-PCR;  免疫细胞化学

Keywords:

Influenza virus;  RNA interference;  Nucleocapsid protein;  Transfection;  qRT-PCR;  Immunohistochemistry

分类号:

R563

DOI:

10.3877/cma.j.issn.1674-6902.2020.04.006

摘要:

目的 探讨靶向核壳蛋白(nucleoprotein, NP)的siRNA对甲型流感病毒(influenza virus A, IAV)的预防及治疗作用。方法 在对IAV测序的基础上设计对NP保守区特异的小干扰RNA(small interfering RNA, siRNA),并观察在犬肾细胞(Madin-Darby canine kidney, MDCK)中先转染了siRNA然后以IAV感染,或先以IAV感染,然后转染siRNA时MDCK细胞中IAV载量的变化。结果 1. 转染了siRNA的MDCK细胞再进行IAV感染,不同剂量siRNA组均较转染剂对照组IVA病毒载量显著降低(20 pmol siRNA组,P<0.05; 40 pmol siRNA组,P<0.05; 80 pmol siRNA组,P<0.01); 2. 当细胞在转染siRNA之前感染IAV,siRNA组的病毒载量显著低于对照组(P<0.01),也低于转染试剂组(P<0.05); 3. 免疫细胞化学结果显示:无论MDCK细胞预先转染siRNA还是感染了IVA后再进行siRNA转染,siRNA组的NP蛋白表达均明显低于对照组(P<0.01)。结论 靶向NP的siRNA通过干扰IVA的NP蛋白合成,从而对IVA的生长产生抑制作用; 无论MDCK细胞感染IVA前后应用靶向NP的siRNA转染均可以降低IVA的产生,说明靶向NP的siRNA对于IVA感染具有预防及治疗作用。

Abstract:

Objective To explore the prophylactic and therapeutic effects of small interfering RNA(siRNA)targeting nucleocapsid protein(NP)gene on influenza A virus(IAV). Methods On the basis of sequencing IAV, the siRNA targeting NP segment of IAV was synthesized. The changes of IAV loads in the Madin-Darby canine kidney(MDCK)were observed after siRNA transfection and IAV infection or after IAV infection and siRNA transfection. Results The virus load was lower or much lower in the siRNA-transfected cells than the transfection agent controls(P<0.05 for 20 pmol siRNA group and 40 pmol siRNA group, and P<0.01 for 80 pmol siRNA group). When the cells were infected with IAV prior to siRNA transfection, the virus load was significantly lower in the siRNA group than the controls(P<0.01)and lower in the siRNA group than the transfection agent group(P<0.05). The immunohistochemical results showed that the expression of NP protein was significantly lower in the siRNA group than the controls(P<0.01), no matter the MDCK cells were firstly transfected with siRNA or infected with IAV. Conclusion The production of IAV can be inhibited by siRNAs targeting NP in a dose-dependent manner and can also be inhibited by siRNA targeting NP after the cells were infected with IAV. The inhibition of IAV by siRNA targeting NP is resulted from the interference with NP protein synthesis.

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备注/Memo

备注/Memo:

基金项目: 中国人民解放军十二五科研基金项目(CSY14C005)
通信作者: 马 壮, Email: Ma_zhuang19641230@163.com

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更新日期/Last Update: 2020-08-20