【摘要】 【目的】探讨信号转导和转录激活因子1(STAT1)在狂犬病病毒（RABV）感染过程中的作用机制，为后续STAT1功能研究提供理论依据。【方法】以RABV感染小鼠小脑星形胶质细胞（C8-D1A），于接毒后12、24和48 h分别采用实时荧光定量PCR和蛋白免疫印迹（Western blotting）检测病毒基因及宿主细胞STAT1和细胞因子的表达变化；针对STAT1基因靶序列设计合成不同的siRNA序列，通过脂质体法瞬时转染C8-D1A细胞，筛选出干扰效率较高的STAT1-siRNA序列；然后以筛选出的STAT1-siRNA序列转染C8-D1A细胞，转染24h后接种0.1MOI的RABV，分别从转录水平和蛋白水平检测干扰STAT1基因表达对RABV复制及细胞因子表达的影响。【结果】RABV感染C8-D1A细胞后能引起STAT1蛋白的表达大幅度上升；无论是弱毒株rRC-HL还是标准强毒株CVS-11感染，都能引起C8-D1A细胞内IFN-α、IFN-β、TNF-α和IL-6等细胞因子基因上调表达。经STAT1-siRNA干扰效果验证，发现在最高浓度200 nmol/L下作用24 h后，STAT1-1616的干扰效率为81%、STAT1-2271的干扰效率为82%，干扰效果达极显著水平（P<0.01）；作用48h后，STAT1-1616的干扰效率为83%,STAT1-2271的干扰效率为75%，干扰效果达显著水平（P<0.05），故选择这2对STAT1-siRNA进行后续试验。干扰STAT1基因表达后，无论是接种弱毒株rRC-HL还是接种标准强毒株CVS-11，其N和P基因的表达均呈上调趋势，病毒N、P蛋白在接毒后24和36 h均呈上调表达趋势，说明干扰STAT1基因表达有利于上调病毒蛋白合成，而促进RABV复制；但干扰STAT1基因表达对RABV感染引起宿主细胞产生IFN-α、IFN-β、TNF-α和IL-6等细胞因子无显著影响（P>0.05）。【结论】合成的2对STAT1-siRNA序列（STAT1-1616和STAT1-2271）对C8-D1A细胞的STAT1基因有显著干扰效果。干扰STAT1基因表达有利于上调病毒蛋白合成，而促进RABV复制。更多还原

【Abstract】 【Objective】To explore the mechanism of signal transducer and activator of transcription 1( STAT1) in the process of rabies virus( RABV) infection, which could provide theoretical basis for subsequent research on STAT1 function.【Method】After murine-derived astrocytes( C8-D1A) infected with RABV, the expression changes of virus genes,host cell STAT1 and cytokines were detected by real-time fluorescence quantitative PCR and western blotting at 12, 24and 48 h post infection. Different siRNA sequences were designed and synthesized according to the STAT1 gene target sequence, and the liposome was instantaneously transfected into C8-D1A cells. The STAT1-siRNA sequences with the higher interference efficiency were screened for further experiments. The selected STAT1-siRNA sequences were transfected into C8-D1A cells, and then the cells were inoculated with 0.1 MOI RABV at 24 h post infection, the effects of interference with STAT1 gene expression on RABV replication and cytokine expression were detected at transcription level and protein level respectively. 【Result】RABV infection on C8-D1A cells could cause large increase in the expression of STAT1 protein. Both the attenuated strain rRC-HL and the standard virulent strain CVS-11 infection could cause upregulation of cytokines genes including IFN-α, IFN-β, TNF-α and IL-6 in C8-D1A cells. Through the verification of the interference effect of STAT1-siRNA, it was found that after 24 h of action at the highest concentration of 200 nmol/L, the interference efficiency of STAT1-1616 was 81%, and the interference efficiency of STAT1-2271 was 82%, with extremely significant level of interference effect(P<0.01). After 48 h of action, the interference efficiency of STAT1-1616 was83%, and the interference efficiency of STAT1-2271 was 75%, with significant level of interference effect(P<0.05).Therefore, these 2 pairs of STAT1-siRNA were selected for subsequent experiments. After interfering with the expression of STAT1 gene, both attenuated strain rRC-HL and the standard virulent strain CVS-11 showed an up-regulation trend in the expression of N and P genes. The virus N and P proteins showed an up-regulation trend at 24 and 36 h post infection,indicating that interfering with STAT1 gene expression was beneficial for up-regulating of virus protein synthesis and promoting RABV replication. However, interfering with STAT1 gene expression had no significant effect on the production of IFN-α, IFN-β, TNF-α and IL-6 cytokines induced by RABV infection( P>0.05).【Conclusion】The synthesized 2 pairs of STAT1-siRNA sequences( STAT1-1616 and STAT1-2271) have significant interference effects on the STAT1 gene in C8-D1A cells. Interference with STAT1 gene expression is beneficial for up-regulating virus protein synthesis and promoting RABV replication.更多还原