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目的:初步探讨金刚烷胺修饰物(NAM)抗禽流感病毒的作用机制,为抗禽流感病毒新药NAM的开发提供实验依据。方法:对狗肾细胞(MDCK)进行体外培养,分为正常对照组,病毒对照组和受试物组。①采用细胞病变法结合MTT法检测NAM对禽流感病毒(H5N1)的抑制作用,受试物组分为先加入NAM后感染病毒、先感染病毒后加入NAM和感染病毒的同时加入NAM,观察3种方式NAM对禽流感病毒的半数抑制浓度(IC50)和治疗指数(TI);②采用神经氨酸酶抑制实验检测NAM对神经氨酸酶的活性影响。结果:①3种不同的实验方式NAM剂量对数与细胞保护率均呈正相关关系(r分别为0.95、0.95和0.99,P均<0.05),且NAM与保护率存在量效依赖关系。先加入NAM后感染病毒,NAM的IC50为15.32 mg.L-1,TI为103.31;先感染病毒后加入NAM,NAM的IC50为30.78 mg.L-1,TI为28.10;感染病毒的同时加入NAM,NAM的IC50为203.92 mg.L-1,TI为4.24。②NAM具有一定的流感病毒神经氨酸酶抑制活性,其IC50为87.36 mg.L-1。结论:NAM对穿入细胞后的禽流感病毒有较好的抑制作用,同时干扰病毒吸附和侵入细胞的脱壳过程也有一定的作用,能够在一定程度上抑制病毒的神经氨酸酶活性。
OBJECTIVE: To investigate the mechanism of amantadine modification (NAM) against avian influenza virus and provide experimental evidence for the development of NAM against new bird flu virus. Methods: Dog kidney cells (MDCK) were cultured in vitro and divided into normal control group, virus control group and test group. (1) Cytotoxicity assay and MTT assay were used to detect the inhibitory effect of NAM on H5N1. The test substance was infected by adding NAM first, NAM was added after the virus was infected, and NAM was also added while the virus was infected. Observation 3 The median inhibitory concentration (IC50) and the therapeutic index (TI) of avian influenza virus by the way of NAM were measured. (2) Neuraminidase inhibition test was used to detect the effect of NAM on the activity of neuraminidase. Results: ① There was a positive correlation between dose logarithm of NAM and cell protection rate (r = 0.95, 0.95 and 0.99, respectively, P <0.05) in three different experimental ways. There was a dose-dependent relationship between NAM and protection rate. After adding NAM, the virus was infected with NAM with the IC50 of 15.32 mg.L-1 and TI of 103.31. The first infection was NAM, the IC50 of NAM was 30.78 mg.L-1, TI was 28.10. At the same time, the virus was added with NAM , ICAM of NAM was 203.92 mg.L-1, TI was 4.24. ② NAM has a certain influenza virus neuraminidase inhibitory activity, the IC50 was 87.36 mg.L-1. CONCLUSION: NAM has a good inhibitory effect on bird flu virus penetrated into cells, and at the same time, it also plays an important role in interfering with virus adsorption and inactivating cell dehulling processes and can inhibit the virus’s neuraminidase activity to a certain extent.