The Role Of Cytomegalovirus In The Pathogenisis Of Intestinal Dysganglionsis

Part Ⅰ. The Correlation research between human intestinal neuronal dysplasia and human cytomegalovirusObjective:To investigate the correlation between human intestinal neuronal dysplasia and human cytomegalovirus.Methods:From September2012to September2014,30children with intestinal neuronal dysplasia(Hirschsprung’disease and allied disease) underwent hirschsprung’ disease radical operation were considered as studied group, at the same time,10babies with with intussusception received colectomy and anastomosis were considered as control group. Blood samples and colon tissues from the two groups were analyzed by different methods for detecting HCMV, including ELISA for CMV-IgG and IgM in blood samples, immunohistochemical staining for CMV late protein and in situ hybridization for CMV specific DNA sequences in colon tissues.Results:The rate of the cases with positive CMV-IgG in the studied group was higher than that in the control group (26.7%vs0%, P<0.05), but there is no difference about CMV-IgM between the two groups. There were a total of60colonic samples including proximal normal section and distal abnormal section of the colon in studied group, and the control group contained10colonic samples. The positive rate of CMV late protein in studied group is higher than that in the control group (21.7%vs0%, P<0.05). The result of CMV-DNA in colonic samples was same to that of CMV late protein in the studied group, but the locations of between late protein and CMV-DNA in the colonic tissues were different. CMV late protein primarily was focused in mucosa, submucosa and capillary endothelial cells in muscle layer of distal abnormal colonic tissues, and not in the proximal normal sections. CMV-DNA expressed in both normal and abnormal colon sections, especially strongly in intestinal ganglionic cells.Conclusions:HCMV may be one of the causes for intestinal neuronal dysplasia, but the pathogenesis is still not clear. Part Ⅱ. A mouse model of intestinal neuronal dysplasia induced by congenital MCMV infectionObjective:To establish of a murine model of intestinal neuronal dysplasia by congenital MCMV infection.Methods:Balb/c mice at age of6weeks without CMV infection were caged, and different concentrations of MCMV were injected into the intraperitoneum of pregnant mice on11th day post mating plug. Each5mice were inoculated with the same concentration of MCMV, and the effects of different concentrations of virus on pregnant and fetal mice were collected. Then the concentration of MCMV was determined for the murine model of intestinal neuronal dysplasia.10pregnant balb/c mice inoculated with adequate concentration of MCMV on11th day post mating plug were considered as experimental model group, and the5pregnant mice received without MCMV were considered as control group. The survival rates of murine embryo and the weight of newborn murine in the two groups were collected. Immunohistochemical staining, in situ hybridization and PCR were used to detect MCMV in the two groups. The number of intestinal ganglion cells in the distal colon was used to evaluate the murine model.Results:The pregnant mice were inoculated with1×102PFU/ul MCMV, and fetal and newborn mice were not affected. The pregnant mice inoculated with1×10PFU/ul MCMV all died during36h after inoculation. Then the concentration of1×103PFU/ul was determined for the model of intestinal neuronal dysplasia. The pregnant mice inoculated with the concentration of1×103PFU/ul were considered as experimental group, and they showed decreased appetite and decreased activity after inoculation. The mean weight of newborn murine in the experimental group was significantly lower than that in the control group (P<0.05), at the same time, CMV was only detected in fetal and newborn mice in experimental group. Comparing with control group, the number of the intestinal ganglion cell in distal colon of the murine model was less (P<0.05). The rate of murine model of intestinal neuronal dysplasia accounted for14%of the all live newborn murine in the experiment group.Conclusions:The murine model of intestinal neuronal dysplasia is successfully established, which is good for the further study on the correlation between CMV infection and intestinal neuronal dysplasia. Part Ⅲ. Preliminary study on the role of cytomegalovirus in the pathogenisis of intestinal dysganglionsisObjective:To study on the role of cytomegalovirus infection in the pathogenisis of intestinal dysganglionsis.Methods:The culture of embryonic gut in vitro was executed in between murine model group and control group, furthermore, the peristaltic frequency and survival time of the gut were collected respectively. The numbers of CD8+CTL and effective DC were noted by flow cytometry in the two groups, at the same time, the amounts of between perform and particle enzyme were evaluated by western blot (WB). After coculture gut neural crest stem cells (GNCSCs) and MCMV. the mounts of MHC-I.Fas and TNFR1in GNCSCs were analysed respectively.Results:Compared with control group, the peristaltic frequency and survival time of the gut decreased with significant difference (P<0.05). In model group, the proportions of CD8+CTL and effective DC in peripheral blood of newborn mice increased significantly (P<0.05). The expression of perforation and granular enzyme in the colon of model mice increased significantly, higher than that in the control group. After coculture GNCSCs and MCMV, the expressions of the above three molecules in GNCSCs increased with significant difference (P<0.05).Conclusion:After congenital CMV infection, cellular immunity was activated. The numbers of effective DC and CD8+CTL increased, while the expressions of MHC-I, Fas and TNFR1from GNCSCs with MCMV infection also increased. GNCSCs were specially recognized by CD8+CTL by the above three molecules and damaged by perforation and granular enzyme, leading to intestinal dysganglionsis.