疾病动物模型,H7N9禽流感病毒 z-bio 2021-03-08 391

　　method

　　1 Animal grouping

　　The mice were randomly divided into normal saline control group, high, medium, and low infection dose groups according to their body weight. Each group had 10 mice, and were inoculated with 1×108, 1×107 or 1×106 TCID50 virus to observe clinical symptoms and mortality. . Another 30 mice were inoculated with 1 × 106 TCID50 virus, and 6 mice were randomly selected for euthanasia on 1, 2, 3, 5, and 7 days after challenge for viral load detection and histopathological observation.

　　2 Attack poison

　　mice were anesthetized by intramuscular injection of ketamine hydrochloride injection at a concentration of 20 mg/mL, and the anesthesia dose was 0.1 mL per mouse. 50μL of 1 × 108, 1 × 107 or 1 × 106 TCID50 H7N9 avian influenza virus stock solution was instilled into the nasal cavity of mice with a pipette; mice in the control group were instilled in 50 μL of normal saline. After inoculation, the mice are bred normally and fed

　　The amount of feed is the standard ration, and the drinking water is supplied with autoclaved water in sufficient quantity.

　　3 Observation index

　　The time before the start of the challenge is recorded as 0 days, and observations will be made every 24 hours within 14 days after the challenge, including the mouse's activity, reactivity, diet, respiration, and the presence or absence of arched back, erect hair, curling up, trembling, and death Wait. Use an electronic scale to measure your weight every day.

　　2.4 Pathological examination

　　Three mice were randomly selected for euthanasia 1, 2, 3, 5, and 7 days after the challenge. The heart, liver, spleen, lung, kidney, brain, and intestine were taken for pathological specimen preparation. The pathological specimen preparation method was fixed with formaldehyde and paraffin wax. Embedding, sectioning, H. E and immunohistochemical staining. Immunohistochemical staining adopts SP method: primary antibody dilution 1:200, overnight at 4℃, wash with PBS; add secondary antibody, incubate at room temperature for 10 min, wash with PBS; DAB color development, after staining with hematoxylin, dehydrated, transparent and neutral gum Cover film.

　　5 Viral load and antibody determination

　　Three mice were randomly selected for euthanasia 1, 2, 3, 5, and 7 days after the challenge. The lung tissues were lavaged with normal saline, and bronchoalveolar lavage fluid (BALF) and lung, heart, liver, Spleen, kidney, brain, intestine and other tissues were inoculated into MDCK cells, and the cytopathic changes were observed. On the 14th day after the challenge, the eyeballs were taken to collect blood, centrifuged to take the supernatant, and the antibody titer was tested by hemagglutination inhibition test (HI) [3].

　　6 White blood cell count

　　1, 2, 3, 5, and 7 days after infection, the mouse eyeballs were taken to take anticoagulant blood for the total number of white blood cells and the classification count.

　　2.7 Statistical methods

　　The data is expressed as (珋x ± s), and the data shall be processed statistically using the SPSS 13. 0 software package. The comparison between groups uses the pairwise q test of analysis of variance; the comparison of rates uses the chi-square test. P ＜0.05 and P ＜0.01 indicate statistical significance.