| Objectives To investigate the effect of Micro RNA-150 on bacterial infection in mice through neutrophils.Methods The following experiment was conducted in two groups of mice,one group of mi R-150 gene deletion(mi R-150ko)and the other group of normal wild-type WTC57BL/6J mice(WT):1.The tail genomes of mi R-150 ko mice and wild-type WT mice were extracted and the genotypes were detected by PCR amplification.2.Real-time PCR was used to identify the knockout efficiency of mi R-150 gene in mice.3 mi R-150 knockout mice and C57 mice were determined by blood routine and flow cytometry.4.Preparation of bacterial infection model and selection of matched male mi R-150 ko and WT mice(6-10 weeks): 150 KO control group,150 KO experimental group,C57 control group and C57 experimental group,respectively.The experimental group was given 100 ul normal saline solution of escherichia coli by tail vein,and the control group was given100 ul sterile normal saline by tail vein.The survival rate of mi R-150 knockout mice was compared with that of C57 mice after infection.5.Bone marrow and peripheral blood PMN flow cytometry analysis of mi R-150 gene knockout mice and wild-type WT mice after infection;6.Blood culture counts of mi R-150 gene knockout mice and wild-type WT mice after infection;7.After infection,mi R-150 gene knockout mice and wild-type WT mice were stained with PMN in peripheral blood.8.In vitro phagocytic assay of peripheral blood PMN of mi R-150 knockout mice and wild-type WT mice;9.Lung tissue sections and HE staining of mi R-150 knockout mice and wild-type WT mice after infection.Results 1.The tail genomic DNA of mi R-150 knockout mice and wild-type WT mice were amplified by PCR,and 262 bp and 866 bp length fragments were produced,respectively,to confirm the correct genotype.2.real-time PCR was performed on thymus and spleen cells of mi R-150 knockout mice and C57 mice.The expression of mi R-150 gene in knockout mice was significantly lower than that in wild-type WT mice.3.Blood cell count and flow cytometry results showed that the ratios of leukocytes,neutrophils and granulocytes in peripheral blood of mi R-150 knockout mice were significantly higher than those in C57 mice,and bone marrow flow cytometry showed no significant difference in neutrophils between the two groups.4.Infection survival test results showed that all the mi R-150 knockout mice and C57 mice in the control group survived,and the survival rate was 100%.The survival rate of mi R-150 knockout mice in the experimental group was 73.3%±3.3%,and that of C57 mice in the experimental group was 46.7%±2.5%,with the former significantly higher than the latter.5.Results of flow cytometry analysis in peripheral blood and bone marrow after infection,peripheral blood PMN of mi R-150 knockout mice in the experimental group was significantly higher than that of C57 mice,while bone marrow neutrophils were significantly lower than that of C57 mice.6.There was no colony formation in the blood of the two types of mice in the control group.The peripheral bacterial blood of the mice in the experimental group was cultured for 12 h to produce obvious white moist,neat edges,and round,with special odor colonies.The number of colonies formed in the bacterial blood culture of C57 mice was 4.20×103±0.18×103/20 ul in peripheral blood.The count colony of mi R-150 knockout mice was0.48 × 103± 0.01 × 103/20 ul in peripheral blood.The amount of viable bacteria in peripheral blood per unit volume of mi R-150 knockout mice was significantly lower than that of C57 mice.7.Reigh’s staining results showed that the peripheral blood of mi R-150 knockout mice in the control group had more PMN than that in C57 mice,with no significant difference in morphology,and most of them were 3-5 lobulated nuclear neutrophils.The number of PMN in peripheral blood of mi R-150 knockout mice in the experimental group was higher than that of C57 mice.The former were mostly lobulated neutrophils,while the latter were rod-shaped nuclei in PMN and PMN granules.8.the results of in vitro phagocytosis experiments showed that the phagocytosis percentage of PMN in mi R-150 ko mice was 95.0±5.8 %,and that of PMN in C57 mice was 87.0±3.6 %.The phagocytosis capacity of neutrophils in mi R-150 ko mice was higher than that in C57 mice.9.HE staining lung tissue sections of the control group showed no edema and no bleeding in the lung,complete alveolar wall structure,normal morphology,clear alveolar cavity,no obvious exudate,and no inflammatory cell infiltration in the alveoli and stroma.In the experimental group,the alveolar volume of mi R-150 ko mice increased,the alveolar septa widened,and inflammatory cells appeared in the alveolar cavity,but the integrity was not damaged.The alveolar cavities of WT-C57BL/6J mice in the experimental group were significantly different in size,with wider alveolar septa,thinner alveolar wall,and damaged integrity.More inflammatory cells were infiltrated around the alveoli.Conclusions 1.Loss of mi R-150 significantly increased the number of neutrophils in mice.2.The deletion of mi R-150 can reduce the number of live bacteria in the blood after bacterial infection and improve the survival rate of mice.3.Loss of mi R-150 can reduce lung damage caused by bacterial infection.4.The absence of mi R-150 increased the resistance of mice to the onset and death of bacterial infection.Figure13;Table7;Reference 142... |
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