Establishment Of An Animal Model Of Sheep Pulmonary Adenomatosis In Sheep

Objective: Sheep pulmonary adenomatosis(SPA),also known as jaagsiekte,is a chronic,progressive,fatal alveolar carcinoma of the fine bronchi.It has devastated the farming industry in many countries and regions,causing serious economic losses to farmers.SPA shares common features with human bronchoalveolar carcinoma(BAC),including a similar histological appearance and common activation of cellular signalling pathways.The size and organisation of the human lung more closely resembles that of the sheep lung than that of the mouse,which has facilitated the study of sheep lung adenomas as a model for human lung cancer.Therefore,in this experiment,the chemical ethyl carbamate and sheep lung adenoma virus capsid protein(JSRV Env)were used in carcinogenicity tests in mice and lakesheep to investigate the stable establishment of lung adenoma models in mice and sheep.Methods: 1.10 mice as well as 3 lake sheep were randomly selected and the drug induction test was carried out by applying ethyl carbamate to mice as well as lake sheep respectively;10 mice and 3 lake sheep were injected with 5% dextrose saline.Blood was collected from mice and lambs every 5 weeks and all animals were executed after 30 weeks of dosing and the lungs of mice and lambs were dissected and fixed in 4% formalin.Measurement of the T cell surface molecules CD4 molecules,CD8 molecules and the cytokines IL-4,TNF-α and IFN-γ on the collected blood.Dissected lung tissue was sectioned for observation under a light microscope;immunohistochemistry was used to detect the tumour markers CEA,NSE and TTF-1.2.The recombinant eukaryotic expression plasmid pc DNA3.1-env was produced and 20 mice were randomly selected to be divided into transfected and blank control groups,10 mice in each group;6 lake sheep were selected to be divided into transfected and blank control groups,3 mice in each group.In vivo transfection reagents were used for tracheal drip injection into mice and lake sheep.Expression of the eukaryotic expression plasmids in the lungs of mice and lakesheep was examined by RT-PCR 3 weeks after transfection.Blood was collected from mice and lake sheep every 5 weeks and all experimental animals were executed after 30 weeks of transfection.CD4 molecules,CD8 molecules and cytokines IL-4,TNF-αand IFN-γ were measured in the collected blood.Dissection was observed and sections of diseased lung tissue were made for observation under a light microscope;immunohistochemistry was used to detect the tumour markers CEA,NSE and TTF-1.Results: 1.The results of induction with ethyl carbamate showed that the serum immune indexes of mice were significantly lower in CD4 molecules,significantly higher in CD8 molecules and significantly lower in CD4/CD8 compared with the control group;the serum lymphokine TNF-α was significantly higher.Greyish-white adenoma nodules were seen on the surface of the lung tissue and microscopic examination revealed papillary hyperplasia of type II alveolar epithelial cells,infiltration of macrophages in the alveolar cavity and positive for tumour markers CEA and TTF-1.There were no significant changes in serum immune parameters in the lake sheep compared to the control group.Microscopic examination did not reveal papillary hyperplasia in alveolar and bronchial epithelial cells,and the tumour marker TTF-1showed positive cells in the bronchial epithelium,but CEA and NSE were negative.2.In vivo transfection of the recombinant eukaryotic expression plasmid pc DNA3.1-env showed that the eukaryotic expression plasmid was expressed in mouse lung tissue 3 weeks after transfection in tracheal drip mice,and immune indexes detected CD4 molecules,CD4/CD8 were significantly reduced and CD8 molecules,IFN-γ and TNF-α were significantly increased.The lungs show greyish-white nodules of varying shapes and sizes,with proliferation of alveolar wall epithelial cells and fine bronchial epithelial cells growing in a papillary pattern into the alveolar lumen or fine bronchial lumen,and positive for the tumour markers CEA and TTF-1.Env gene expression could be detected in the lungs after 3 weeks of transfection in lake sheep.No significant changes in lymphocytes and cytokines were observed.No characteristic pathological changes were found in the histological observation of pathology,and the results of tumour markers CEA and NSE were negative.Conclusions: 1.In the secondary carcinogen ethyl carbamate drug induction assay,ethyl carbamate800 mg/kg administered intraperitoneally twice a week for 5 weeks induced lung adenocarcinoma in C57BL/6 mice.In an in vivo transfection assay with sheep lung adenoma cystic membrane protein,a tracheal drip was applied to C57BL/6 mice that produced tumourigenic effects and eventually led to the development of lung adenomas,further demonstrating that expression of the lake sheep lung adenoma cystic membrane protein gene alone was sufficient to induce lung adenomas in the animals.2.Neither the carbamate drug induction test nor the sheep lung adenoma cystic membrane protein in vivo transfection test produced lung adenomas in lakesheep,but epithelial-derived cancer cells were found in both tumour marker TTF-1 assays.