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Clinical Analysis of HCMV Infection in Colitis Patients and a Murine Model with Murine Cytomegalovirus Infection Resulting in Acute Colitis after Allogenetic Skin Transplantation

Author: ZhangZuo
Tutor: WangMingLi
School: Anhui Medical University,
Course: Microbiology
Keywords: human cytomegalovirus murine model murine cytomegalovirus acute colitis
CLC: R574.62
Type: Master's thesis
Year: 2011
Downloads: 2
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Abstract

Objective: To provide a reliable animal model for study of human CMV acute colitis, we tried to infect with murine cytomegalovirus (MCMV) in mice that were received allogenetic skin transplantation under immunosuppression. So the model become a reliable animal model for study pathopoiesis and diagnosis/therapy of human CMV acute colitis.Method: Part1: Analysis of clinical samples: We take 36 colon samples of acute colitis patients from center of endoscopy of anhui traditional chinese medical college to test between January and April in 2011. There are 23 male patients and 13 female patients and the age range from 45 years old to 82 years old. The sample is in marginal part of pars affecta. (1) HCMV isolation is performed by laboratory diagnosis of HCMV infection according to infection design. After trituration and centrifugation, supernatant of samples was inoculated in HELF, and cytopathic effect(CPE) of HCMV was observed every day ; meanwhile indirect immunofluorescence is performed for pp65 antigen. (2) HCMV IE gene was chosed to detection in samples by polymerase chain reaction(PCR). These can determine infection rate of HCMV and infection state in acute colitis patients by record and analysis.Part 2: Construction of a murine model①Skin transplantation was performed between 18 donor C57BL/6 mice and 72 recipient BALB/c mice, and the size of skin graft is about (1.0cm×1.0cm).②All recipient mice were then given Cyclosporine at 15mg/kg daily for 2 weeks by intraperitoneal injection. Mice were randomly divided into 3 groups. Two infection groups were received MCMV-infected mouse embryonic fibroblasts (MEF) at 104PFU and 105PFU respectively, and the control group received MEF only. We observed any possibly pathophysiological behavior changes and recorded the changes in body weight. The mice were sacrificed at 5d, 9d, 14d, 21d post infection and colon tissue was collected for analysis by virus isolation, HE stain, transmission electron microscope, in situ hybridization, MCMV M55 gene RT-PCR and immunohistochemistry. The purpose is to observe the correlation between MCMV and pathological change in colon of mice.Results: Part 1: We find that HCMV infects intestinal tissues in all patients by PCR for IE gene and virus isolation, and the positive rate is up to 91.67%. CPE was observed after blind passage, and samples is present positive by PCR for IE gene. The positive rate is 36.1% by virus isolation. There is positive signal of apple green in positive samples of virus isolation by indirect immunofluorescence.Part 2: Mice infected with MCMV at 105PFU group showed anorexia, lethargy and degression in locomotor activity(p<0.01). This group of mice showed significant decrease in body weight than that of other groups. Colon tissues were collected 14 days after infection. Histological examination revealed that the mucous layer became thinner in the proximal colon and increased number of lymphoid follicles in distal colon in infected animals.The structure of mucosa layer was chaotic and there was a large area of infiltration of inflammatory cells in the group 105PFU MCMV. Electron microscopy showed that there were herpes virus particles in the colon tissue. Viral DNA was present in the colon by in situ hybridization for IE1 gene, and viral M55 gene transcript was positive by RT-PCR. One of the viral major proteins, pp65 antigen, was widely distributed in the colon by immunohistochemistry. These data demonstrated that MCMV established infection in colon of the mice after allogenetic skin transplantation. All index above are negative.Conclusion: (1) Intestinal tissues infected with HCMV is common, and the positive rate is up to 91.67%. The proliferatation rate is about 36.1% in patients from center of endoscopy of anhui traditional chinese medical college. These evidences show that HCMV infection is correlation with acute colitis closely and provide new diagnostic strategy for of HCMV acute colitis. Anti-viral treatment is executed in acute colitis patients, especially steroid-refractory colitis patients.(2) Infection with MCMV in mouse after allogenetic skin transplantation by nasal cavity inoculation resulted in the pathological changes in colon tissue similar to that of inflammation in human colon. We successfully construction a murine model with murine cytomegalovirus infection resulting in acute colitis after allogenetic skin transplantation. The model provides a important platform for pathogenesis of HCMV acute colitis and medicine intervention furtherly, meanwhile it adds datas for treatment of virus-induced inflammation clinically. So the model become a reliable animal model for study of human CMV acute colitis.

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