靶向核壳蛋白基因的siRNA对流感病毒的预防和治疗作用

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。

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

ObjectiveTo explore the prophylactic and therapeutic effects of small interfering RNA (siRNA) targeting nucleocapsid protein (NP) gene on influenza A virus (IAV). MethodsOn 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. ResultsThe 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. ConclusionThe 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.