The Changes Of Immune Cells In Different Microenvironments At Different Stages And The Culture Of Primary Oral Cancer Cells Of Mouse Model

Objective:1.To explore the effects of immune cells in different microenvironments on targeting lymphatic metastasis of oral cancer and their mechanisms,by detecting the number of CD8+ T cells,CD28+ T cells and myeloid derived suppressor cells(MDSC cells)in different stages and microenvironments(lymph node and bone marrow)of mice oral cancer lymphatic metastasis model.2.To construct the primary oral squamous cell carcinoma(OSCC)cell line of Balb/c mice oral cancer lymphatic metastasis model.Methods:The 4NQO drinking method was used to construct the lymphatic metastasis model of oral cancer in Balb/c mice.a.Normal control group(Normal)(n=10):drinking tap water;b.Negative control group(8 Weeks,the tongue mucosa has not shown pathological changes,but there is already 4NQO drug effect)(n=10):4NQO feeding After 8 weeks.c.Dysplasia group(n=10): 20 weeks after 4NQO feeding;d.Primary oral cancer group(OSCC)(n=10): 24 weeks after 4NQO feeding;e.primary Oral cancer group(OSCC)(n=10): 28 weeks after 4NQO feeding f.submandibular lymph node metastasis group(Metastasis)(n=10):4NQO feeding 32 weeks g.submandibular lymph node metastasis group(Metastasis)(N=10): Week 36 after 4NQO feeding.Metastasis group(n=10):Week 40 after 4NQO feeding.Collect the lymph nodes and bone marrow tissues of mice in the above groups.The number of immune cells(CD8+T cells,CD28+T cells,MDSC cells)in different stages and microenvironments(lymph nodes,bone marrow)was detected by flow cytometry.2.The tongue tissue of squamous cell carcinoma of Balb/c mice oral cancer model was collected and cells were cultured by Dispase Ⅱ enzyme digestion method.The morphology and ultrastructure of the cells were observed by phase contrast microscope and transmission electron microscope.The surface markers(pan-ck,S100,p75,etc.),cell growth curve,tumorigenesis of naked mice and karyotype of cells were detected.Results:1.The results of flow cytometry are as follows,(1)in lymph nodes and bone marrow,the number of CD8+ T,CD28+ T,CD11b+ and Gr-1+ cells was significantly higher than that in normal control group from the 8th week to the40 th week,except that the number of CD28+ T cells was slightly lower in the20 th and 24 th week.The differences were statistically significant.(2)The number of CD8+ T,CD28+ T,CD11b+ and Gr-1+ cells in lymph nodes was significantly higher than that in bone marrow at the corresponding groups,except that the number of CD28+ T cells in lymph nodes was lower than that in bone marrow at the 24 th week.There were statistically differences.(3)In bone marrow,the number of CD8+ T and CD28+ T cells increased from the 8th week to the 24 th week,peaked at the 24 th week,and began to decline slowly from the28 th week to the 40 th week,but still in the plateau stage.In lymph nodes,the number of CD8+ T and CD28+ T cells decreased from the 8th week to the 24 th week,then CD8+ T increased from the 24 th to the 40 th week,while CD28+ T increased from the 24 th to the 32 nd week,then decreased.The difference was statistically significant.(4)In lymph nodes and bone marrow,CD11b+ and Gr-1+ cells showed an increasing trend with time from the 8th to the 40 th week,and the differences were statistically significant at different time periods.2.Under the condition of dispase Ⅱ pancreatin digestion combined with serum-free culture,the survival rate of Balb/c mice OSCC primary cells can reach 90%,which has been stably transmitted to 40 generations.Pan-CK immunohistochemical staining was positive in cytoplasm culture cells.Conclusion:1.The number of CD8+T,CD28+T and MDSC cells(CD11b+ and Gr-1+)in lymph nodes and bone marrow increased significantly as 4NOQ induced tumor progression and metastasis in Balb/c mice.It suggests that the immune function has changed in microenvironment,which may be related to the occurrence of DTC and the formation of metastasis.2.The expression of CD8+ T,CD28+ T and MDSC cells(CD11b+ and Gr-1+)of Balb/c mice in lymph nodes was higher than that in bone marrow.The difference of expression may be the reason of lymphatic metastasis targeting in oral cancer.3.In lymph nodes,with the occurrence and development of cancer until metastasis,the increasing immunosuppression caused by the increasing number of MDSC cells,and the decreasing of immune function caused by the decreasing number of CD28+ T cells in the metastasis period play an important role in the occurrence of lymphatic metastasis.On the other hand,the number of CD8+ T cells increased during the process of cancer formation and metastasis,which may not be related to lymphatic metastasis.Therefore,MDSC cells may be more effective as a target to inhibit lymphatic metastasis of oral cancer in the future.4.The Balb/c mice OSCC cells can be cultured successfully by dispase Ⅱ pancreatin digestion combined with serum-free culture condition,which has reached 40 generations.