| Proteus mirabilis is a kind of zoonotic pathogenic bacteria,which is widely distributed in nature.It can cause the alimentary tract,the respiratory tract,the urogenital tract infection,and brings a great threat to people’s health and animal husbandry industry.Outer membrane vesicles are globular vesicles with phospholipid bilayer structure secreted by Gram-negative bacteria in the process of growth.They carry outer membrane proteins,lipopolysaccharides,nucleic acids,virulence factors and other substances,which are important tools for bacteria to communicate with the external environment.Studies have shown that the OMV play an important role in the process of bacteria infection.They improve the pathogenicity and invasiveness of pathogenic microorganisms by mediating the transport process of virulence factors,thus affecting the physiological process of host cells.In this study,The OMV were isolated and extracted from P.mirabilis,on this basis,through the in vitro challenge experiment,preliminarily explores P.mirabilis OMV effect on intestinal porcine epithelial cell,for the further research of P.mirabilis pathogenic mechanism and the prevention and control of P.mirabilis indisease provides new train of thought.The main contents of the research are as follows:1.Isolation and identification of P.mirabilisIn this experiment,122 stool samples of diarrhea piglets were collected from pig farm,Chong Qing Municipality Rong Chang area,and bacteria were isolated and cultured from them.After culture characteristics of bacteria,gram staining microscopy,amplification and sequencing of 16S r RNA,22 strains of P.mirabilis were successfully Isolated and identified.Five strain were randomly selected to prepare bacterialsuspension and intraperitoneally injected into mice for challenge test,after 24 hours the intestinal mucosa of the mice had severe bleeding,and the intestinal tract was full of a large number of yellow and transparent contents,which indicated that P.mirabilis had a strong invasion and pathogenicity on the intestinal tissues.At the same time,the growth curve of P.mirabilis was determined,and the growth stability period was reached after culture at 37℃and 180rpm to 16h.During this period,the number of bacteria was the largest,and the secretion of OMV was the largest,which was the best time point for extraction of OMV.2.Isolation and extraction of OMV of P.mirabilisAfter 16 hours of bacteria culture,OMV were isolated and extracted by ultrafiltration concentration and density gradient centrifugation.The appearance and morphology were observed by transmission electron microscopy,the density and diameter were determined by Nano Sight,the protein concentration was determined by BCA Protein Assay Kit,and the protein components were analyzed by SDS-PAGE.The experimental results showed that the OMV of P.mirabilis were colorless and transparent spherical vesicles of different sizes.The diameter ranged from 80 nm to 220nm,with the maximum of 139nm.The density is 3.85×10~9/ml.The protein concentration was about 158.62ug/ml.The content of protein was distributed in 85-10 KDa,mainly concentrated around 85,50 and 10 KDa.3.The effect on the Activity of OMV on intestinal porcine epithelial cellAccording to the protein concentration measured by BCA method,the OMV was diluted to a series of concentration gradient(79.31,39.66,19.83,9.91,4.96ug/ml)and added to the intestinal porcine epithelial cells for the infection test.The cell survival rate was determined by MTT assay,The results showed that compared with the control group,the cell survival rate of the cells with the addition of OMV was significantly reduced(P<0.05),and the cell survival rate decreased with the increase of the addition concentration.The results showed OMV had inhibiting effect on intestinal porcine epithelial cell.In order to further study the effect on the activity of the influence of OMV on intestinal porcine epithelial cell.After the intestinal porcine epithelial cells were challenged by OMV,the contents of IL-8 in cell supernatant were determined,the level of ROS was determined by DCFH-DA,and the DNA damage of cell was detected by SCGE.The experimental results showed that:Compared with the control group,the content of IL-8 in the superscript increased significantly after the addition of OMV(P<0.05),and the secretion of IL-8 reached a peak when the concentration of OMV reached 39.66ug/ml at challenge 4d.Meanwhile,the level of ros increased significantly(P<0.05).According to the measured Tail DNA%,Tail DNA%of the cells increased significantly after the addition of the OMV(P<0.05).In the lowest concentration group,4.96ug/ml could cause cell DNA damage after the 2d challenge,and OMV of other concentrations could cause significant cell DNA damage during all the challenge time.These results indicated that OMV of P.mirabilis could stimulate the secretion of IL-8 by intestinal porcine epithelial cell and induce the inflammatory response of the cells.Stimulate cells to produce more reactive oxygen species,increase the oxidative pressure of cells,thus causing oxidative damage to cells;OMV indues DNA double-strand break damage in intestinal porcine epithelial cell,and shows a certain degree of concentration time dependence.In conclusion,OMV of P.mirabilis have inhibiting effect on intestinal porcine epithelial cell,resulting in decreased cell survival rate,stimulating cell inflammatory response,causing cell oxidative damage,and inducing cell DNA double-strand break damage. |
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