The Experimental Study On The Role Of Calreticulin -expression In The Immune Environment Of Model Mice And The Use In The Transplanted Immune Therapy

Objective: To investigate the impact of the calreticulin (CRT) expresssion on the B16 cells membrane by the mitoxantrone (MIT), and the immune response of mice generated by the change; To observe the impact of B16 membrane antigen with high expression of CRT as a vaccine on model mice; also the effect of DCs -CTLs loaded with the B16 cell membrane antigen with high expression of CRT on model mice.Methods: To observe the expression of CRT on the surface of B16 cells treated by MIT by immunofluorescence. To observe model mice injected by different doses of MIT and vaccinated by membrane antigen of B16 cells and by membrane antigen of B16 cells treated by MIT, and to observe the effect of DCs-CTLs loaded with membrane antigen of B16 cells and membrane antigen of B16 cells treated by MIT by Immunohistochemistry and flow cytometry.Results:1. Four ex-vivo groups of B16 cells treated by different concentration of MIT : 0.0μg/ml, 0.25μg/ml, 1.0μg/ml, 4.0μg/ml. The percentage of CRT expression on B16 cells of four groups are (29.40±3.57)%, (42.80±4.59)%, (56.20±5.55)%, (72.20±2.94)%, the expressions of CRT are increased and dose depended (P<0.05).The result of Western blotting shows that the expression of B16 cells treated by 4.0μg/ml MIT is 472±23,and that treated by 0.0μg/ml MIT is 117±18 (P<0.01).2. Compared with the control group , the expressions of CRT in tumor tissue of tumor-bearing mice injected by 2.0, 4.0, 8.0 mg / kg of MIT increased in varying degrees. CRT relative expressions of different groups detected by flow cytometry are: 5.13±0.95; 7.40±0.86; 9.17±1.06, and compared with 3.21±1.37 of control group. there are significant differences between them (P<0.05).3. Immunohistochemistry experiments shows that: the number of CD11c~+ DCs within the tumor tissues of 2.0, 4.0, 8.0 mg / kg MIT groups and the MIT-B16 protein vaccine group are significantly higher than the control group of tumor-bearing mice (P <0.05);CD3~+ T-cells within the tumor tissues of 2.0, 4.0 mg / kg MIT and the MIT-B16 protein vaccine groups were significantly higher than the number of tumor-bearing control mice (P <0.01)4. Flow cytometry detection of T lymphocytes of mice spleen shows that the rates CD4~+ T lymphocytes of 4.0 mg / kg MIT group, MIT-B16 protein vaccine group, B16-DCs-CTLs Group, MIT-B16-DCs-CTLs Group increase significantly compared with tumor-bearing mice (P <0.01);the rates of CD8 ~+ T lymphocytes in mice spleen of 2.0,4.0,8.0 mg / kg MIT group, MIT-B16 protein group, B16-DCs-CTLs group and MIT-B16-DCs-CTLs Group significantly increased compared with tumor-bearing mice (P <0.05)5. The mice were killed at the 14th day, after that the volumes (cm3) of tumor were measured, tumor volumes of 2.0,4.0,8.0 mg / kg MIT groups are 4.07±0.77,2.35±0.54,1.21±0.39,and they are significantly smaller than 6.94±0.81cm3 of control group (P <0.01). And also the differences of tumor volumes between experimental groups are significant (P <0.05);The tumor volumes of B16 protein vaccine group, MIT-B16 protein vaccine group are 4.15±0.57,1.67±0.33,and the differences of tumor volumes between two groups are significant (P <0.05); The tumor volumes of B16-DCs-CTLs group, MIT-B16-DCs-CTLs group are 1.09±0.25,0.59±0.21,and there is a significant difference between them (P < 0.05);the tumor volume of MIT-B16-DCs-CTLs group was less than the other groups (P <0.05).Conclusions:1. MIT were able to increase the expression of CRT on the surface of B16 tumor cells, similarly, MIT can increase the expression of CRT in melanoma tissue of mice, and in the extent of mice can bear, CRT expression was dose-dependent; appropriate doses of MIT can uplift the number of T cells and DCs infiltrated in tumor tissue and the rates of CD4 ~+, CD8 ~+ T lymphocyte in the spleen of mice, and form a specific anti-tumor immune therapy.2. The membrane protein vaccine of MIT-B16 with high expression of CRT can increase the number of T cells and DCs infiltrated in tumor tissue and the rate of CD4 ~+, CD8 ~+ T lymphocytes in the spleen of mice, also improves immune environment of anti-tumor ,and protect the mice from being attacked by the tumor.3. To treat the tumor with the DCs-CTLs stimulated by membrane protein of MIT-B16 with high expression of CRT, the rates of CD4 ~+, CD8 ~+ T lymphocytes in the spleen of mice increase significantly. The immune environment advances greatly, and the treatment achieves a desired efficacy.