| Objective: To better study tongue cancer and esophageal cancer in mice, we induced tongue cancer and esophageal squamous cell carcinoma on C57BL / 6 mice by using chemical carcinogen 4NQO through drinking water. At the same time, we detected the number and proportion of regulatory T cells (Treg) and myeloid-derived suppressive cells (MDSC) in pathological mice,s spleen and peripheral blood .Method: (1) The carcinogen 4-NQO stock solution was prepared in 1,2-propylene glycol at 200mg/ml(2%), and then was diluted in the drinking water at 100μg/ml, 50μg/ml, 10μg/ml.105 eight-weeks-old and female C57BL / 6 mice were randomly divided into four groups A, B, C and D, which contained 30, 25, 25, 25 mice in each. A group drank 100μg/ml 4NQO water to 16 weeks, then drank normal water. B group drank 50μg/ml 4NQO water to 16 weeks, then drank normal water. C group drank 10ug/ml 4NQO water to 16 weeks, then drank normal water. D group always drank normal water which contain the same concentration of 1,2- propylene glycol. When the mice (all groups of mice) were observed to 16, 20, 24, 28, 32 weeks , five mice were sacrificed in each group respectively. Then tongue, esophagus and spleen were taken to observation by eyes and histological assessment. (2) During the process of model constuction, we collected the A and D groups mice,s peripheral blood and spleen cells at 0, 1, 2, 4,12, 16, 20, 24, 28, 32 week, each time, we use 3 mice for this experiment. Flow cytometry was used to determine the number and proportion of CD4~+CD25~+Treg cells and CD11b~+Gr1~+MDSC cells. Results: (1) Compared to the control mice, 50μg/ml, 100μg/ml group yield the obvious pathological characteristics. But it have no difference between the control group and the 10ug/ml group until to 32 weeks. D group (the control group) mice had pink tongue mucosa, tongue papillae evenly distributed, had no white patches, ulcers, and their tongues could not be seen the formation of new objects. (2) Corresponded to the length of observation , A group and B group mice appeared white patch tongue mucosa, ulcers, erosions, granular and cauliflower-like new objects one after another. And the esophagus of A group and B group mice could be seen some swelling and redness symptoms. Pathological analysis showed that Group A and Group B mice,s tongue mucosa and esophageal wall undergone a classic pathologic changes: simple epithelial hyperplasia, dysplasia, carcinoma in situ , invasive cancer. The tongues of 16 weeks Group A mice mainly appeared mild and moderate dysplasia. At 20 weeks , appeared severe dysplasia, at 24 weeks carcinoma in situ could be seen and 28 weeks could be seen invasive cancer. At 32 weeks, 80% of Group A mice's tongues could be seen invasive carcinoma. Group A mice's esophagus can be seen mild dysplasia at 16 weeks, severe dysplasia at 20 weeks, invasive cancer at 28 weeks. The outward appearance of Group B mice's tongue and esophagus undergone similar changes with Group A mice, but its pathological changes was slow relatively. During the experiment period, Group C mice were similar to control group, it could not be observed any abnormal changes in outward symptoms and pathological analysis. (3) Group A and Group D mice's peripheral blood and spleen were detected by flow cytometry. From 0 weeks to 4 week, the number of CD4~+CD25~+Treg and CD11b~+Gr1~+MDSC was not significantly different between Group A and Group D . But at 8 weeks, the number of CD4~+CD25~+Treg and CD11b~+Gr1~+MDSC began to increased. At 16 weeks, it showed that the proportion of MDSC increased rapidly, at 32 weeks, the proportion was about 5 times more than control group. The Treg cells accounted for the proportion of CD4~+T cells was also significantly increased after 16 weeks.Conclusions: (1) We establish a C57BL / 6 mice model of tongue cancer and esophageal squamous cell carcinoma by 4NQO-drinking water. The process of carcinogenesis undergone the classic histopathological changes which contains simple epithelial hyperplasia, dysplasia ,carcinoma in situ, invasive cancer. This changes were similar to human oral cancer and esophagea cancer. In addition, this cancer model had the advantage of slow-growing, high incidence, induced cancer close to the spontaneous tumor, so it was an ideal animal model of tongue and esophagus cancer. (2) CD4~+CD25~+Treg and CD11b~+Gr1~+ MDSC was found significantly increased in cancer mice,s peripheral blood and spleen. It is suggested that the percentage of regulatory T cells and myeloid-derived suppressor cells significantly increased during the evolution of chemical-induced carcinogenesis, and this changes might be related to tumor immune escape.
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