甲型H1N1流感病毒感染小鼠肺共信号分子变化研究

黄锐; 黄霞; 车霄; 王乐霄; 李佳颖; 王志杰; 高蓉; 王福生; 焦艳梅; 白冰珂; 徐哲

摘要:

背景甲型流感病毒传染性强，重症感染死亡率高。共信号分子是参与免疫应答的重要分子。探究甲型流感病毒感染后肺病毒载量与共信号分子的变化特点及相关性可为病毒感染后的治疗提供参考依据。

目的探究甲型流感病毒A/Puerto Rico/8/34(H1N1)(PR8株)感染小鼠肺病毒载量和共信号分子的变化特点及相关性。

方法 72只7周龄BALB/c小鼠随机分为高剂量病毒组(10⁸TCID₅₀/mL，50 uL/只，32只)、低剂量病毒组(10⁴TCID₅₀/mL，50 uL/只，32只)和对照组(PBS，50 uL/只，8只)，三组分别于感染后24 h、48 h、72 h和96 h每个时间点各处死8、8、2只小鼠；解剖取左肺行HE染色和实时定量逆转录PCR(qRT-PCR)检测肺病毒载量、共信号分子CD28、CD226、ICOS、CTLA-4、TIGIT、PD-1 mRNA相对表达量。将高、低剂量病毒组所有病毒感染小鼠(n=64)肺病毒载量与共信号分子mRNA相对表达量行相关性分析。

结果感染后96 h内，高剂量病毒组相比低剂量病毒组小鼠同一时间肺损伤较重，且共刺激分子CD28、CD226、ICOS mRNA相对表达量均减少(P均＜0.05)。高剂量病毒组小鼠感染后24 h PD-1 mRNA相对表达量增加(P＜0.01)，感染后48 h CTLA-4 mRNA相对表达量增加(P＜0.01)且病毒复制达到峰值。低剂量病毒组小鼠感染后72 h PD-1 mRNA相对表达量增加(P＜0.05)且病毒复制达到峰值，感染后96h CTLA-4 mRNA相对表达量增加(P＜0.01)。病毒感染小鼠(n=64)肺病毒载量与PD-1 mRNA相对表达量存在正相关关系(r=0.540，P＜0.001)，与CD226 mRNA相对表达量存在负相关关系(r=-0.340，P=0.006)。

结论甲型流感病毒感染小鼠肺组织中CTLA-4、PD-1 mRNA相对表达量增加等现象的早期出现可能提示小鼠感染的重症化。肺组织中的PD-1和CD226可能是与肺病毒载量相关的共信号分子靶点。

Abstract:

Background Influenza A virus is highly infectious with a high mortality rate in severe infection cases. Co-signaling molecules are important molecules involved in immune response. Exploring the change characteristics and correlation between pulmonary viral load and co-signaling molecules after influenza infection can provide reference for the treatment of influenza A virus infection.

Objective To explore the change characteristics of pulmonary viral load and its association with co-signaling molecules mRNA expression in mice infected with influenza A virus A/Puerto Rico / 8/34 (H1N1) (PR8 strain).

Methods Totally 72 BALB/c mice aged 7 weeks were randomly divided into high dose virus group (10⁸TCID₅₀/mL, 50uL/ mouse, 32 mice), low dose virus group (10⁴TCID₅₀/mL, 50uL/ mouse, 32 mice) and control group (PBS, 50uL/ mouse, 8 mice), 8, 8 and 2 mice were sacrificed in the three groups at each time point of 24h, 48h, 72h and 96h after infection, respectively. All mice were dissected for the left lung which used for HE staining, qRT-PCR was applied to detect the pulmonary virus load and the relative mRNA expression levels of co-signaling molecules CD28, CD226, ICOS, CTLA-4, TIGIT and PD-1. The correlation between pulmonary viral load and mRNA expression of co-signaling molecules were analyzed in all mice infected with high and low dose viruses (n=64).

Results Within 96h after infection, the lung injury of high dose virus group was more severe than that of low dose virus group, and the mRNA expression of co-stimulatory molecules CD28, CD226 and ICOS was decreased (all P＜0.05). In the high dose virus group, the relative expression of PD-1 mRNA increased at 24h after infection (P＜0.01), the relative expression of CTLA-4 mRNA increased (P＜0.01) and the viral replication reached to the peak at 48h after infection. In the low dose virus group, the relative expression of PD-1 mRNA increased (P＜0.05) and the viral replication reached to the peak at 72h after infection, the relative expression of CTLA-4 mRNA increased (P＜0.01) at 96 hours after infection. The pulmonary viral load of infected mice (n=64) was positively correlated with the relative expression level of PD-1 mRNA (r= 0.54, P＜0.001), and negatively correlated with the relative expression level of CD226 mRNA (r=-0.34, P=0.006).

Conclusion After infected with influenza A virus, the early appearance of the increased mRNA expression of CTLA-4 and PD-1 may indicate the severe infection of mice. PD-1 and CD226 in mice lung tissue may be the co-signaling molecular targets correlated with pulmonary viral load.

甲型H1N1流感病毒感染小鼠肺共信号分子变化研究

Changes of pulmonary co-signaling molecules in influenza A (H1N1) virus infected mice