Establisment Of The Models Of BALB/c Mice Infected With Helicobacter Hepaticus And The Preliminary Effects On Immune Responses

Helicobacter hepaticus is a gram-negative, microaerophilic and spiral-shaped bacterium, which was isolated for the first time by FOX in1994. It is described as one of the pathogenic factors that can cause chronic active hepatitis, cirrhosis, hepatocellular neoplasms and typhlocolitis among some strains of mice. In adult immunocompetent mice, H.hepaticus infection is usually asymptomatic and it was found that the rate of H.hepaticus infection was high among experimental animals. The aim of this research is to establish the H. Hepaticus infection model by using BALB/c mice, investigating the pathological feature, determining whether functional characteristics of dendritic cells (DC) are altered in hepaticus-infected mice and whether H.hepaticus infection can influence the experimental results or not.First, we recovered H.hepaticus standard strain ATCC51450purchased from American Type Culture Collection. After amplification, suspected bacterial colony was identified by Gram’s staining, biochemical characterization and gene sequencing by16S rRNA gene amplification.200specific-pathogen-free (SPF) male mice without H.hepaticus infection were used in this experiment. These mice were randomly divided into two groups on average with one as the experimental group and another one as the control group. Mice of the experimental group were inoculated with0.2mL H.hepaticus bacterial suspension for3times with48hours intervals, and the control group was fed with PBS. Bateria DNA from feces of all the mice were extracted at2,3,4and5month post last inoculation with H.hepaticus and submitted to16S rRNA gene amplification of H.hepaticus specific. The results indicated that the infection rates increased over time. At the second month since the last H.hepaticus inoculation the infection rate of mice of experimental group increased to100%nearly. And15mice were executed at2,3,4and5month since the last time of H.hepaticus inoculation. Mice liver, stomach, cecum and colon were taken for H.hepaticus specific16S rRNA gene amplification and histopathology examination. The result showed that hepaticus could colonized in liver, stomach, cecum and colon and the colonization rates of H.hepaticus increased over time. In liver tissues, liver cells steatosis around the central vein, lobular focal necrosis, lobular focal necrosis and inflammatory infiltration with different degrees around focal necrosis was observed. With the extension of time the severity of the lesion strengthened. The stomach and caecum lesions were not significant. A small amount of inflammatory infiltration in gastric submucosa, gastric mucosa epithelial necrosis areas, and different degrees of inflammatory infiltration cecumshowed were observd.8mice which infected with H.hepaticus for5months and8noninfected mice were used. Murine bone marrow-derived dendritic cells (DC) were co-cultured which were stimulated by GM-CSF and IL-4. Then DCs were subjected to FACS analysis for surface markers (CDllc, CD40, CD80and MHCⅡ) detection. The results showed that the expression rates of CD40and MHCⅡ on DCs of mice in exprimental group were higher than that in control group. On this basis, we choosed15H.hepaticus-infected mice and non-infected mice respectively which were inoculated0.2mL virus suspension of Newcastle disease virus (NDV) ZJ1strian. Serum was collected by orbital venous blood sampling for the detection of Newcastle disease antibody to explore the difference of antibody titer between the two groups. The results showed that in different periods, H.hepaticus infection could promote NDV antibody levels, which may be associated with the results above that H.hepaticus infection could stimulate the expression rates of CD40and MHCⅡ on DCs.In conclusion, we have established stable models of BALB/c mice infected with H.hepaticus, investigated the colonization rates in various organs of mice and the pathogenic characteristics of H.hepaticus and explored whether H.hepaticus infection could effect the surface expression of the maturation-associated markers and immunology experiments. These studies have some significance for the further study of H.hepaticus.