| Objective:To explore the optimization for establishment of mouse models of orthotopic bladder tumors, to evaluate the effects of pseudomonas aeruginosa (PA-MSHA) on bladder cancer in mouse subcutaneous tumor model and on the proliferation of splenic lymphocytes, to explore the mechanism involved in, and to provide new treatment strategies for bladder cancer.Methods:After1or24hours the pretreatment of murine bladder mucosa with silver nitrate, HCl, trypsin and ethanol, the mice were killed to obtain bladder tissue samples. Pathological changes were observed using hematoxylin and eosin staining. The microenvironment modification was assessed by electron microscopy. Mast cells were counted with toluidine blue staining. The glycosaminoglycan (GAG) layer was observed by periodic acid-Schiff staining. Mice were intravesically pretreated with the above four reagents, respectively, and the rate of tumor formation was determined. Subcutaneous bladder tumor model was established, and divided into four groups:normal saline treatment group, low PA-MSHA dose treatment group, medium PA-MSHA dose treatment group, and high PA-MSHA dose treatment group. PA-MSHA or normal saline were injected around the tumors. Three mice were sacrificed in each group at14,21and28after implantation, serum was collected from each mice at3000×g for5minutes, then CD4+or CD8+were detected through flow cytometry analysis. Tumor tissue, and major organs were obtained to observe pathological changes, VEGF and MVD were also assessed. The spleens were removed aseptically from C57BL/6mice, then spleen cell suspensions were treated with low PA-MSHA dose, medium PA-MSHA dose, high PA-MSHA dose and RPMI-1640culture medium as control for24,48,72h. At the end point, the optical density (OD) values of the samples were measured on a microplate reader.Results:In the model mice, a part of umbrella cells were sloughed off, the submucosal layer was exposed and incontinuous in some areas at one hour after pretreatment with trypsin and ethanol. Mucosal damages were more serious in the HCl and silver nitrate groups. However, there was no significant difference in the inflammatory cell infiltration between the experiment and control groups. Mild edema and congestion in the mucosa, tightly arranged epithelial cells, and continuous mucosa were observed in the mice after trypsin and ethanol pretreatment for24hours. By contrast, the thickness of mucosa was uneven and incomplete mucosa in some areas were still observed in the mice24hours after silver nitrate and HCL and NaOH pretreatment. Tumor size were apparently smaller in PA-MSHA-treated groups than those in control group, and a dose-dependent effect on the inhibition of tumor volume was shown. Meanwhile, a longest survival rate was observed in high PA-MSHA dose treatment group as well as a shortest survival rate in control group. VEGF expression and MVD were statistically decreased in PA-MSHA groups. At14days after implantation, CD4+, CD8+were increased in all PA-MSHA-treated groups. With the progression of administration, CD4+, CD8+,were obviously increased in PA-MSHA-treated groups. In the proliferation assay of splenic lymphocytes, the findings showed that the proliferation of splenic lymphocytes was increased by PA-MSHA with a dose-dependent and time-dependent manner.Conclusion: Pretreatment with silver nitrate and HCl, NaOH cause more serious damage in the bladder mucosa and may be regarded as an optimal choice in establishing orthotopic bladder tumor models in mice. Meanwhile, PA-MSHA has a apparently effect in the promotion of proliferation of splenic lymphocytes in vitro, inhibited the growth of tumor via decreased VEGF expression and MVD, and increased the level of CD4+or CD8+. PA-MSHA provides a new strategies for bladder cancer. |
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