| Objective To establish a mouse model of acute interstitial pneumonitis with murine cytomegalovirus (MCMV) after allogeneic skin transplantation.Methods (1) Established a mouse model of allogeneic skin transplantation:21 of C57BL/6 donor and 84 of BALB/c recipient, female mice, were underwent back skin graft. CyclosporineA (CsA,15mg/kg) was subsequently given for 14 days. During this period, transplanted mice were observed daily living conditions and survival status of the skin.After 14 days, to detect the levels of IFN-γin plasma of recipients by ELISA. (2) Established a recipient mouse model of interstitial pneumonitis by MCMV infection:○1 All recipient animals were randomly divided into five groups (n=16), four experimental groups were infected with MCMV by intranasally with 1×102 PFU/ml(group A),1×103 PFU/ml(group B),1×104 PFU/ml (group C)and 1×105 PFU/ml(group D), and the control group(group E) was inoculated with 50μl of MEF cell suspension.○2 Detection methods: On post infection days 5,9,14 and 21, mice were Anesthesia sacrificed and lung samples were collected for HE staining, viral isolation, PCR,RT-PCR, Real time PCR, transmission electron microscope(TEM),In situ hybridization, and Immunohistochemistry detection. Results (1) After transplantation 14d, some recipient mice activity slowed, weight were reduced, and the skin allografts in some mice have hair growth, the most of transplanted mice of subcutaneous vascular formation was noted, all skin allografts with a mean survival time(MST)of 13.5±0.3days. Detected the IFN-γlevels in mouse plasma can be seen IFN-γwere significantly lower in the transplanted mice than in normal(p<0.05).So recipient mice were immunesuppressed at postgrafting 14 days, the allogenic skin transplantation were successed.(2)○1 After infection,to observe the survival status of mice, group D compared with others were significantly lethargy, anorexia, weight loss; using statistical software to analyze body weight of all mice ,displayed group D compared with others were significantly differences (P <0.01).○2 Observed pathological changes in lung tissue of all groups by HE staining ,and found Focal pathological abnormality in the lung in the mice with high dose of virus ( group C and D ), after infection14,21d, lung tissue damage in group D were the most serious(alveolar hemorrhage , pulmonary interstitial widening, formation of bullae rupture alveoli, enlargement of pulmonary interstitial cells, pulmonary interstitial inflammatory cell infiltration, alveolar cavity smaller, and the edge of the lung generated compensatory emphysema);○3 Forviral isolation: after MCMV infection 5,9,14 and 21d, the lung homogenates were inoculated in MEF cells,after blind 3 passages, group C and D at 5,9 and 14d were visibled cytopathic effect (CPE), positive rates were 37.5% and 62.5%,others had not CPE ;○4 After infection 5,9,14,and 21 d, to detect MCMV IE and M55 DNA and RNA transcripts in infection groups. The results displayed MCMV IE and M55 DNA was positive by PCR in infected groups, MCMV IE mRNA of each infected groups were positive for transcripts, after infection 9,14,and 21 d, the positive rate of M55 mRNA transcripts were 43.8%,62.5%,75%, showed the virus is active infection state;○5 Detected the MCMV IE DNA copies in the lung tissue of infected mice by Real timePCR, found in the group D and C with infected high dose of virus, the number of copies in the 21d were significant changes,it more high in 5,9d than in14, 21d, the peak at 9d, at this point, the numberof copies in group C and Dwere (1.35±0.10×104copies/μg and 5.57±0.15×104copies/μg), and the D group of the highest copy number of virus ,after infection 14d, the number of virus copies in group C and D were(1.81±0.24×102copies/μg and 2.05±0.05×103copies/μg), the viral copies were decreased, relative to the MCMV IE DNA copies in low dose groups (groupA and B) did not change significantly;○6 Herpes virus particles were found by TEM in the epithelial of the lung tissue in group C and D;○7 The virus nucleic acid was localized in lung epithelial cells by in situ hybridization. MCMV protein was revealed in the interstitial lung epithelial cells in the group of high dose by immunohistochemistry,the control were negative.Conclusions (1) From the skin graft survival time and the immunosuppression status of transplant recipients can be sure the model of allogeneic skin transplantation was established;(2) At different times of MCMV infection, used HE staining, transmission electron microscopy, in situ hybridization and immunohistochemistry, to detecte change of lung tissue of recipients in each group, showed that the presence of virus within the epithelial cells, the high dose group produced a typical lung interstitial pneumonia-like lesions, and found that 14 and 21d after infection is more severe injury in lung. By virus isolation, PCR, RT-PCR and Real timePCR to detecte MCMV DNA and mRNA, IE and M55 genes were Exist, namely, the virus were active infection status,found after infection 9d,the virus copies were highest, relative in14, 21d the number of viral copies were reducted, so we think that the virus may become latent state after 21d..Above methods to sum up evidence, the study demonstrated that a mouse model of acute interstitial pneumonitis was successfully established in the mouse after allogeneic skin transplantation in natural MCMV infection. |
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