| ObjectiveMalignant tumor cells produce various bioactive molecules to inhibit host immune function, composing one of the most important mechanisms for tumor cell to avoide immune rejection. Blockage of the immunosuppressive effects of tumor cells is beneficial to control and cure malignant tumor. Ganoderma lucidum Polysaccharides (Gl-PS), a most important bioactive component of Ganoderma lucidum, have the antitumor and immunomodulation effects. However, it is waiting to be confirmed with direct convincing evidence that Gl-PS can antagnise the immunosuppression on immune cells caused by immunoinhibitor of malignant tumor. With the Gl-PS extracted and purified from the Ganoderma lucidum grown on basswood by Fuzhou Institute of Green Valley Bio-Pharm Technology, the antagonism of the Gl-PS against immunosuppressive effects of supernatants in tumor (mouse B16F10 melanoma cells) culture on lymphocytes was explored.Methods1. Cells strain: B16F10 cells, a high matetistic melanoma cell line of C57BL/6 mouse origin, and lymphocytes freshly prepared from splenocytes in BALB/C mouse and C57BL/6 mouse were used in this study.2. Preparation of supernatant of B16F10 cell culture: 2×104/ml B16F10 cells were seeded into the culture bottles for 10ml/bottle, and the media were replaced with fresh one when the cells grown to 80% of the bottom and followed by further culture for 8h. The supernatant of the cell culture were harvested, filted with 0.22μm, and stored at -20℃.3. Antagonism of the Gl-PS against suppressive effects of supernatants of tumor culture on lymphocytes proliferation induced by PHA: The C57BL/6 mouse spleen lymphocytes were cultured in the supernatant from tumor culture or the normal medium (1×106 cells /well). Gl-PS of different concentrations were added into the wells where the lymphocytes were cultured in the supernatant from tumor culture, and the normal medium instead of the Gl-PS was used as the control. The cells were cultured for 72h and the MTT assay was used to measured the OD value to evaluate the proliferate activity.4. Antagonism of the Gl-PS against suppressive effects of supernatants of tumor culture on mixed lymphocytes reactions: The double direction mixed lymphocytes reactions were performed. The equal amount of BALB/c mouse and C57BL/6 mouse spleen lymphocytes were cultured in the supernatant from tumor culture or the normal medium (1×106 cells /well respectively). Gl-PS of different concentration were added into the wells where the lymphocytes were cultured in the supernatant from tumor culture, and the normal medium instead of the Gl-PS was used as the control. The cells were cultured for 72h and the MTT assay was used to measure the OD value to evaluate the mixed lymphocytes reactions.5. Antagonism of the Gl-PS against suppressive effects of supernatants of tumor culture on lymphocytes for granzyme B and porforin expression displayed by immunocytochemistry: The C57BL/6 mouse spleen lymphocytes were cultured in the supernatant from tumor culture or the normal medium (1×10~6 cells /well). Gl-PS of different concentrations were added into the wells where the lymphocytes were cultured in the supernatant from tumor culture, and the normal medium instead of the Gl-PS was used as the control. The cells were cultured for 72h and the immunocytochemistry was used to display the granzyme B and porforin expression.6. Antagonism of the Gl-PS against suppressive effects of supernatants of tumor culture on lymphocytes for granzyme B and porforin expression detected by Western blot: The C57BL/6 mouse spleen lymphocytes were cultured in the supernatant from tumor culture or the normal medium (1×106 cells /well). Gl-PS of different concentrations were added into the wells where the lymphocytes were cultured in the supernatant from tumor culture, and the normal medium instead of the Gl-PS was used as the control. The cells were cultured for 72h and the Western blot was used to detect the granzyme B and porforin expression.7. Statistical analysis: The statistical software of SPSS15.0 was used and the data was analyzed with one way ANOVA. P values < 0.05 were considered significant.Result1.Effects of supernatants from tumor culture for suppression on lymphocytes proliferation induced by PHA: It was shown by MTT assay that with the presence of supernatants from tumor culture, 72h after induction by PHA, the OD value of syngenic mouse spleen lymphocytes was 1.132±0.086, obviously lower than that of RPMI 1640 control (1.797±0.075), and the difference between them was significant (t=16.447, p=000)2.Effects of supernatants from tumor culture for suppression on mixed lymphocytes reaction: It was shown by MTT assay that with the presence of supernatants from tumor culture, 72h after admixture of spleen lymphocytes from syngenic C57BL/6 mice and BALB/c mice, the OD value of mixed lymphocytes reaction was 0.825±0.157, obviously lower than that of RPMI 1640 control (1.334±0.083), and the difference between them was significant (t=8.097,p=000).3.Effects of supernatants from tumor culture for suppression on expression of porforin and granzyme B detected with immunocytochemistry in lymphocytes induced by PHA: It was shown by immunocytochemistry that with the presence of supernatants from tumor culture, 72h after induction by PHA, the expression of porforin and granzyme B in syngenic mouse spleen lymphocytes was obviously weaker than that of RPMI 1640 control.4.Effects of supernatants from tumor culture for suppression on expression of porforin and granzyme B detected with Western blot in lymphocytes induced by PHA: It was shown by Western blot that with the presence of supernatants from tumor culture, 72h after induction by PHA, the level of porforin and granzyme B expressed in syngenic mouse spleen lymphocytes were 0.7662±0.017 and 0.7857±0.056, obviously lower than that of RPMI 1640 control(both 1.000), and the difference between them was significant (t=23.698,p=0.002;t=6.658,p=0.003).5.Antagonism of the Gl-PS against suppressive effects of supernatants from tumor culture on lymphocytes proliferation induced by PHA: It was shown by MTT assay that with the presence of supernatants from tumor culture, different concerntration of Gl-PS acting on syngenic mouse spleen lymphocytes with the induction by PHA, and detected 72h after, the OD value of were 1.350±0.068, 1.400±0.053, 1.455±0.046 and 1.592±0.049 respectively, obviously higher than that of control without supernatants from tumor culture (1.132±0.086),with remarkable statistical significance (F=97.499,p=000). Except for 0.2μg/ml vs 0.8μg/ml,and 0.8μg/ml vs 3.2μg/ml, all defferences among other groups were statistically significant.6.Antagonism of the Gl-PS against suppressive effects of supernatants from tumor culture on mixed lymphocytes reaction: It was shown by MTT assay that with the presence of supernatants from tumor culture, different concerntration of Gl-PS acting on the mixture of mouse spleen lymphocytes, and detected 72h after,the OD value of was 1.170±0.205,1.209±0.12, 1.234±0.148 and 1.247±0.148, obviously higher than that of control without supernatants from tumor culture (0.825±0.157),with remarkable statistical significance (F=11.335, p=000). Except for all group with different concerntration of Gl-PS having statistically significant differences respectrvely compared with the control without supernatants from tumor culture (p=000), all defferences among other groups were not statistically significant (p>0.05).7.Antagonism of the Gl-PS against suppressive effects of supernatants from tumor culture on expression of porforin and granzyme B detected with immunocytochemistry in lymphocytes induced by PHA:It was shown by immunocytochemistry that with the presence of supernatants from tumor culture, different concerntration of Gl-PS acting on syngenic mouse spleen lymphocytes with the induction by PHA,and detected 72h after, the expression of porforin and granzyme B were obviously stronger than that of control without supernatants from tumor culture.8.Antagonism of the Gl-PS against suppressive effects of supernatants from tumor culture on expression of porforin and granzyme B detected with Western blot in lymphocytes induced by PHA: It was shown by Western blot that with the presence of supernatants from tumor culture, different concerntration of Gl-PS acting on syngenic mouse spleen lymphocytes with the induction by PHA, and detected 72h after, the level of porforin expression were 1.073±0.040, 1.127±0.037, 1.240±0.0143, 1.352±0.035 respectively, obviously higher than that of control without supernatants from tumor culture (1.000), with remarkable statistical significance (F=66.256, p=000). Except for 0.2μg/ml vs 0.8μg/ml, 3.2μg/ml and 12.8μg/ml vs RPMi 1640 control respectively without supernatants from tumor culture without statistal significance (p>0.05), all defferences among other groups were statistically significant(p<0.05). The level of granzyme B expression were 1.021±0.041,1.089±0.026,1.210±0.079和1.259±0.089 respectively, obviously higher than that of control without supernatants from tumor culture (1.000), with remarkable statistical significance (F=10.954, p=000). Except for 0.2μg/ml vs 0.8μg/ml, 0.8μg/m vs3.2μg/ml, 0.8μg/ml vs 12.8μg/ml, 3.2μg/ml vs 12.8μg/ml without statistal significance (p>0.05), all defferences among other groups were statistically significant(p<0.05).Conclusion1.Supernatant from B16F10 melanoma cell culture showed the immune-suppressive effects on lymphocytes of syngenic mouse.2.Gl-PS antagonised the immune-suppressive effects of Supernatant from B16F10 melanoma cell culture on lymphocytes of syngenic mouse. |
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