| Objective:There are many bioactive molecules produced by malignant tumor cells which can inhibit the immune function,as an important mechanism for tumor to escape from immune attack.It has been proved by many experiments that Ganoderma lucidum polysaccharides(Gl-PS)have the antitumor effects.It can improve the function of cellular immunity and humoral immunity.In this study,the spleen lymphocytes of syngenetic mouse were cultured in the supernatant of B16F10 cells with G1-PS of different concentrations(0.2,0.8,3.2,12.8μg/ml),and those in RPMI-1640 culture solution instead of supernatant of B16F10 cells and G1-PS were used as control.FasL on lymphocytes were tested by immunocytochemistry and Western blot,and CD71 by immunofluorescence technique and flow cytometry.The research was to investigate the antagonism of the Gl-PS against immunosuppressive effects of supernatants in tumor cell on lymphocytes.Methods:1.Source of the cells:B16F10 cell was a high matetistic melanoma cell line derived from C57BL/6 mouse,and lymphocytes were prepared from splenocytes of C57BL/6 mouse.2.Preparation of supernatant of B16F10 cell:2×105/ml B16F10 cells were seeded into the culture bottles(10ml/bottle),and the culture solution were changed by 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.Detection of the antagonism of the Gl-PS against suppressive effects of culture supernatant of B16F10 melanoma cells on FasL in lymphocytes by immunocytochemistry:The C57BL/6 mouse spleen lymphocytes were cultured in the supernatant of tumor culture or the normal medium(1×106 cells/well).G1-PS of different concentrations were added into the wells where the lymphocytes were cultured in the supernatant of tumor culture,and the normal medium instead of the Gl-PS and supernatant of tumor culture was used as the control.The cells were cultured for 72h,then harvested and smeared,and immunocytochemistry was used to display the FasL.The cells stained with brown color were determined positive.4.Detection of the antagonism of the Gl-PS against suppressive effects of B16F10 melanoma cell supernatant on FasL in lymphocytes by Western blot:The C57BL/6 mouse spleen lymphocytes were cultured in the supernatant of tumor culture or the normal medium(1×106 cells/well),and Gl-PS of different concentrations were added into the wells where the lymphocytes were cultured in the supernatant of tumor culture,and the normal medium instead of the Gl-PS and supernatant of tumor culture was used as the control.The cells were cultured for 72h,and were harvested for Western blot to detect FasL.5 Detection of the antagonism of the Gl-PS against suppressive effects of B16F10 melanoma cell supernatant on CD71 in lymphocytes by Immunofluorescence technique:The C57BL/6 mouse spleen lymphocytes were cultured in the supernatant of tumor culture or the normal medium(1×106 cells/well),and 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 and supernatant of tumor culture was used as the control.The cells were cultured for 48h,and were harvested for Immunofluorescence technique to display the CD71,The cells stained with red fluorescence were determined positive.6.Detection of the antagonism of G1-PS against suppressive effects of B16F10 melanoma cell supernatant on CD71 in lymphocytes by Flow cytometry:The C57BL/6 mouse spleen lymphocytes were cultured in the supernatant of 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 of tumor culture,and the normal medium instead of the Gl-PS and the supernatant of tumor culture was used as the control.The cells were cultured for 48h,and were harvested for Flow cytometry to detect the CD71,the data was obtained and analyzed by FACSCalibur flow cytometer and Cell Questsoftware.7.Statistical analysis:The statistical software of SPSS 15.0 was used and the data was analyzed with one way ANOVA.P values<0.05 were considered significant.Result:1 Effects of suppression of supernatants of tumor culture on the expression of FasL detected by immunocytochemistry in lymphocytes activated by PHA:It was shown that with the presence of supernatants of tumor culture,72h after activation by PHA,the expression of FasL in syngenic mouse spleen lymphocytes was obviously weaker than that of RPMI 1640 control group.2 Effects of suppression of supernatants of tumor culture on expression of FasL detected by Western blot in lymphocytes activated by PHA:It was shown that with the presence of supernatants of tumor culture,72h after activation by PHA,the level of FasL expressed in syngenic mouse spleen lymphocytes(0.524±0.208)was obviously lower than that(1.054±0.171)of RPMI 1640 control group with statistical significance(t=3.409,p<0.05).3 Effects of suppression of supernatants of tumor culture on expression of CD71 detected by Immunofluorescence technique in lymphocytes activated by PHA:It was shown that with the presence of supernatants of tumor culture,48h after activation by PHA,the expression of CD71 in syngenic mouse spleen lymphocytes was obviously weaker than that of RPMI 1640 control group.4 Effects of suppression of supernatants of tumor culture on expression of CD71 detected by Flow cytometry in lymphocytes activated by PHA:It was shown that with the presence of supernatants of tumor culture,48h after activation by PHA,the expression of CD71 in syngenic mouse spleen lymphocytes(53.645 ± 1.280)was obviously weaker than that(60.108 ± 1.469)of RPMI 1640 control group with statistical significance(t=5.755,p<0.05).5 Antagonism of the G1-PS against the suppressive effects of supernatants of tumor culture on expression of FasL detected by immunocytochemistry in lymphocytes activated by PHA:It was shown that with the presence of supernatants of tumor culture and Gl-PS with different concerntration acting on syngenic mouse spleen lymphocytes,72h after activation by PHA,the expression of FasL was obviously stronger than that of control without the G1-PS.6 Antagonism of the G1-PS against suppressive effects of supernatants of tumor culture on expression of FasL detected by Western blot in lymphocytes activated by PHA:It was shown that with the presence of supernatants of tumor culture and G1-PS with different concerntration(0.2,0.8,3.2 and 12.8μg/ml)acting on syngenic mouse spleen lymphocytes,72h after activation by PHA,the levels of FasL were respectively 0.799±0.168、1.233±0.176、1.261±0.298 and 1.581±0.226,higher than that of control group without G1-PS(0.524±0.208),with statistical significance(p<0.05)except for 0.2μg/ml group.The levels of FasL in 0.8μg/ml,3.2μg/ml and 12.8μg/ml groups were higher than RPMi 1640 control group without supernatants of tumor culture without statistal significance(p>0.05).7 Antagonism of the G1-PS against suppressive effects of supernatants of tumor culture on expression of CD71 detected by Immunofluorescence technique in lymphocytes activated by PHA:It was shown that with the presence of supernatants of tumor culture and G1-PS with different concerntration acting on syngenic mouse spleen lymphocytes,48h after by PHA,the expression of CD71 was obviously stronger than that of control without the Gl-PS.8 Antagonism of the G1-PS against suppressive effects of supernatants of tumor culture on expression of CD71 detected by Flow cytometry in lymphocytes activated by PHA:It was shown that with the presence of supernatants of tumor culture and Gl-PS with different concerntration(0.2μg/ml,0.8μg/ml,3.2μg/ml and 12.8μg/ml)acting on syngenic mouse spleen lymphocytes,48h after activation by PHA,,different concerntration of Gl-PS acting on syngenic mouse spleen lymphocytes with the activation by PHA,and detected 48h after,the levels of CD71(58.812±1.161.60.533±0.476.61.458±2.493 and 62.705±2.880)were respectively higher than that(53.645±1.280)in control group without Gl-PS,with statistical significance(p<0.05).CD71 in 0.8μg/ml,3.2μg/ml and 12.8μg/ml groups were even higher than that(60.108 ± 1.469)in RPMi 1640 control group without supernatants of tumor culture,without statistal significance(p>0.05).Conclusion:1 Supernatant of B16F10 melanoma cell culture showed the suppressive effects on CD71 and FasL expression on lymphocytes of syngenic mouse.2 Gl-PS antagonised the suppressive effects of supernatant of B16F10 melanoma cell culture on lymphocytes of syngenic mouse. |
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