Establishment And Indentification Of A Mice Model Of Chronic Graft-verse-hoet Disease With Oral Lesions

Objective:(1)To establish murine chronic graft-versus-host disease models via transplanting different doses of human peripheral blood mononuclear cells(hPBMCs)into the severely immunodeficient NCG mice(NOD-Prkdcem26IL2rgem26/Nju,NCG)intravenously.To analyze the pathological features of oral tissues of mice by observing the pathological changes of oral tissues in salivary gland and tongue mucosa etc.,and detecting CD45+,CD8+,CD4+,IL17+ and FoxP3+T cells in oral tissues of mice in models.Materials and Methods:1.Forty-eight NCG mice were divided into six groups:A,B,C,D,E,and F,eight in each group.The body weight was recorded before treatment.Peripheral venous blood of healthy volunteers was collected,and hPMBCs was separated by gradient density centrifugation.Then 300 ?1 of RPMII Medium 1640 containing 2.5×106,5.0×106,7.5×106,10.0×106 hPBMCs and with no cells were injected into Mice in group A,B,C,D,and E were injected with 300 ?l of 2.5×106,5.0×106,7.5×106,10.0×106 hPBMCs and RPMI1640 medium without cells,and the F group did not do any treatment.and Group F was not treated.Body weight and clinical signs were recorded once every three days.Then all mice were sacrificed on the 75th after transplantation,and the tissues of parotid gland,submandibular gland,tongue,buccal,labium,palate,liver,lung,kidney and so on were stained with HE to observe the histopathological changes.Whether or not oral cGVHD occurs in mice is defined based on the oral histopathological score of mice.At the same time,immunofluorescence was used to detect the expression of human CD45+,CD8+,CD4+,IL17+and FoxP3+cells in oral tissues of mice with oral cGVHD.2.Another twelve 8-10 weeks old female NCG mice were injected with 5.0×106hPBMCs via the tail vein,and 3 mice were randomly sacrificed on the 15th,30th,45th and 75th day after transplantation,and the parotid gland,submandibular gland,tongue,buccal mucosa,labium mucosa,and palate mucosa were collected and stained with HE.The submandibular glands were stained with Masson.The histopathological changes were observed and scored.Results1.After transplantation of hPBMCs,hair and weight loss,arch back,eyelid closure,swelling of the ankle joint,and mild congestion of the tongue mucosa were observed in some mice in 2.5×106hPBMCs,5.0×106hPBMCs,7.5×106hPBMCs and 10.0×106hPBMCs dose modeling groups.The mean survival time of mice in 2.5 ×106 and 5.0 ×106 hPBMCs dose modeling group was longer than that in 7.5 ×106 and 10.0 ×106 hPBMCs dose modeling group,and the discrete trend of 5.0×106 hPBMCs dose modeling was smaller than that in 7.5 ×106 hPBMCs dose modeling group.The mean clinical scores ± standard error(x±SEM)of 2.5×106,5.0×106,7.5×106,10.0×106h PBMCs dose modeling group and control group were 2.23 7±0.627,3.275±0.792,3.7000±0.661,4.925±0.862 and 0 respectively.The clinical scores of the mice in each modeling group were significantly different from those in the RPMI1640 medium control group(p<0.05).2.Lymphocytes infiltrated in the parotid gland,submandibular gland,tongue,buccal,labium,palate,liver,lung,kidney and gastrointestinal tissues of transplanted mice.And no significant changes were observed in clinical signs and histopathology of mice in control group.In the transplanted mice,acinar cells atrophy,destruction,ductal dilation,collagen deposition,interstitial and acinar infiltration of plasma cells in the salivary glands were observed,and salivary glands fibrosis causes structural damage.And stratum granulosum,stratum spinosum,stratum basale,submucosal lymphocyte infiltration and epithelial cells vacuolar degeneration,basal cells liquefaction denaturation and unclear basement membrane were observed in tongue mucosa.Similar changes can be seen in the buccal,labium,and palate mucosa of mice.3.The x± SEM of oral pathology scores of mice in each dose modeling group and RPMI1640 control group were 5.125±3.351,10.500±4.070,12.833±3.790,18±5.066 and 0,respectively,and the oral pathological scores of the mice in the 5.0×106,7.5 ×106 and 10.0×106hPBMCs dose modeling groups were statistically significant compared with the RPMI1640 medium control group(p<0.05)respectively.The incidence of oral tissue lesions in mice was 37.50%,50.00%,62.50%,75.00%,and 0,respectively,which was positively correlated with the number of infused hPBMCs(R=1.000,p=0.000).4.The human CD45+,CD8+FoxP3+,CD4+FoxP3+,IL17+ T lymphocytes infiltrated in the parotid gland,submandibular gland,tongue mucosa,buccal mucosa,lip mucosa and palate mucosa of mice with oral cGVHD.5.Lymphocytes infiltration and fibrosis began to occur in the salivary glands of mice in 5.0×106hPBMCs dose modeling group on 30th day after transplantation,followed by gradual increase of lymphocyte infiltration and gradual transformation to collagen deposition and plasma cell infiltration.A small amount of lymphocyte infiltration began to occur in the stratum basale of tongue mucosa on 45th day after transplantation,followed by increased lymphocyte infiltration and upward cortical erosion,and vaculation liquefaction of basal cells emerged.6.The mean values ± standard error of mean of oral pathology scores of mice in 5.0×106hPBMCs dose modeling group at 15th,30th and 45th day after transplantation were 0.33±0.58,4.33±1.15 and 15.00± 1.73 respectively.The oral pathological scores of the mice in 5.0×106hPBMCs dose modeling group increased at 15th,30th and 45thday after transplantation.The difference of the scores between on the 45th day and on the 15th day after transplantation was statistically significant(p<0.05).Oral pathological changes mainly occurred 30th day after transplantation.Conclusion:1.The mice cGVHD model involves multiple organs and tissues including oral tissues.The histopathological changes of oral tissues in mice are similar to those of clinical oral cGVHD.Oral tissue lesions in mice were concentrated after 30th day of after transplantation of hPBMCs and continued until the end of the experimental observation.A variety of T lymphocyte subpopulations were implanted in the oral tissues of model mice.2.This method can be used to establish a mouse model for oral cGVHD related research,especially the study of T cell related mechanisms.3.The larger number of transplanted hPBMCs,the higher the moldability of the mice oral cGVHD model.From the survival time of mice after transplantation,the incidence of cGVHD lesions in mice and the severity of lesions,5.0×106 hPBMCs or more can be used to establish a mice model of cGVHD with oral lesions.The dose of 5.0×106 hPBMCs can be used to establish a mouse model of cGVHD with longer survival time.The dose of 7.5×106hPBMCs can be used to establish a mice model of cGVHD with heavier oral lesions.