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Antagonism of Ganoderma Luncidum Polysaccharides on Immune Suppression of Lymphocytes Caused by Supernatants of Melanoma Cells

Author: JiaJing
Tutor: SunLiXin；LiuKeXia
School: Chengde Medical College
Course: Oncology
Keywords: Ganoderma lucidum polysaccharides Melanoma Lymphocytes PHA Granzyme Perforin
CLC: R739.5
Type: Master's thesis
Year: 2010
Downloads: 98
Quote: 0
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Abstract

Objective: malignant cells produce a variety of biologically active molecules inhibit the body's immune function, constitute an important mechanism of tumor immune escape. Blocking this immunosuppressive effects of malignant cells, contribute to the control and treatment of malignant tumors. Ganoderma lucidum polysaccharides (Ganoderma lucidum Polysaccharides Gl-PS) is one of the main active ingredient of Ganoderma has anti-tumor and immunomodulatory effects. But whether it can antagonize the inhibition of malignant immune inhibitor of immune cells, be direct evidence has yet to be confirmed. Ganoderma lucidum polysaccharides of the experiment Fuzhou Green Valley Institute of Bio-Pharmaceutical Technology, separated from the wood cultivation of Ganoderma extract purified, highly metastatic mouse melanoma B16F10 cells, to explore Ganoderma polysaccharides antagonize tumor cells culture supernatant on the inhibition of lymphocyte. Method: 1. Cell sources: B16F10 cells used in this experiment is a derived from C57BL / 6 mice highly metastatic melanoma cell lines, lymphocytes freshly prepared in BALB / C mice and C57BL / 6 mice spleen lymphocytes. 2 Preparation B16F10 cell culture supernatant (referred tumor supernatant): 2 x 104/ml B16F10 cells were inoculated in 50ml of culture bottles, each bottle 10ml, culture medium was changed to 80% confluence, the culture was continued 8H, cultures were collected The supernatant, 0.22μm filter paper filter bacteria, repackaging, -20 ℃ cryopreservation spare. Ganoderma lucidum polysaccharides antagonize tumor supernatant of PHA-induced lymphocyte proliferation inhibition experiment: Take a 96-well plate C57BL / 6 mouse spleen lymphocytes cultured in tumor supernatant or normal medium, 1 × 106 cells / well, Ganoderma lucidum polysaccharides simultaneously with different concentrations of to cultured tumor supernatant spleen lymphocytes, without Ganoderma lucidum polysaccharides control and cultured for 72 hours, the OD value of MTT assay to determine cell proliferation activity. Ganoderma lucidum polysaccharides antagonize tumor supernatant mixed lymphocyte reaction inhibition experiments: the use of two-way mixed lymphocyte reaction. Take a 96-well flat-bottom cell culture plates, of the equivalent amount of 1 × 106 cells / well of BALB / c mice and C57BL / 6 mice lymphocytes cultured tumor supernatant or normal medium, while to the culture in the tumor supernatant Ganoderma lucidum polysaccharides in spleen lymphocytes with different concentrations of control without Ganoderma lucidum polysaccharides were cultured for 72 hours, the OD value of MTT assay to determine the mixed lymphocyte reaction. Immunocytochemistry detection of Ganoderma lucidum polysaccharides antagonize tumor supernatant lymphocyte granzyme B and perforin expression inhibition experiments: C57BL / 6 mouse spleen lymphocytes cultured tumor supernatant or normal medium, 1 × 106 cells / well, at the same time to train in tumor supernatant of spleen lymphocytes with different concentrations of Ganoderma lucidum polysaccharides without Ganoderma lucidum polysaccharides as a control, were cultured for 72 hours, collect nonadherent lymphocytes, smear Immunocytochemistry as positive staining, microscopy observations to cytoplasm stained brown. 6.Western blot detection of Ganoderma lucidum polysaccharides antagonize tumor supernatant on lymphocyte granzyme B and perforin expression inhibition experiment: mouse spleen lymphocytes cultured tumor supernatant or normal medium, 1 × 106 cells / well, while Ganoderma lucidum polysaccharides with different concentrations of spleen lymphocytes to cultured tumor supernatant without Ganoderma lucidum polysaccharides controls were cultured for 72 hours to collect nonadherent lymphocytes extracted protein determination of granzyme B, Western blot was used and perforin expression. The 7. Statistical analysis: Application SPSS15.0 statistical software, on the results of single-factor analysis of variance, p lt; 0.05 discriminative significant. Results: 1. Tumor supernatant of PHA-induced inhibition of lymphocyte proliferation: MTT results showed that the tumor supernatant role, 72h syngeneic mouse spleen lymphocytes of PHA-induced OD value of 1.132 ± 0.086, far lower in RPMI 1640 culture the liquid control group 1.797 ± 0.075, the difference was highly significant (t = 16.447, p = 000). Tumor supernatant inhibition of the mixed lymphocyte reaction: MTT results showed that the tumor supernatant role, the mixed lymphocyte reaction of spleen lymphocytes OD value of 0.825 ± 0.157, far lower than in RPMI 1640 culture The liquid control group, 1.334 ± 0.083, the difference was highly significant (t = 8.097, p = 000). Immunocytochemical staining tumor supernatant of lymphocyte granzyme B and perforin expression inhibition: the tumor supernatant role, PHA-induced 72h syngeneic mouse spleen lymphocyte perforin and granzyme B immune The cytochemical staining positive is far lower than the control group of RPMI 1640 medium. 4.Western blot detection of tumor supernatant 72h syngeneic mouse spleen lymphocytes granzyme B and perforin expression inhibition: In the the tumor supernatant role, PHA-induced lymphocyte perforin and granzyme B relative expression significantly lower than the levels were 0.7662 ± 0.017 and 0.7857 ± 0.056 RPMI 1640 culture fluid 1.000 of the control group, the difference was highly significant (t = 23.698, p = 0.002; t = 6.658, p = 0.003). Ganoderma lucidum polysaccharides antagonize tumor supernatant of PHA-induced inhibition of lymphocyte proliferation: MTT results showed, Ganoderma lucidum polysaccharides under different concentrations, the the tumor supernatant role the PHA induced the 72h after syngeneic mouse spleen lymphocytes OD values 1.350 ± 0.068,1.400 ± 0.053,1.455 ± 0.046 and 1.592 ± 0.049, significantly higher than the control group unused Ganoderma lucidum polysaccharides role 1.132 ± 0.086, and showed a dose-dependent relationship. But also failed to achieve despite inhibition of tumor supernatant RPMI 1640 medium control 1.796 ± 0.075, analysis of variance, the difference was highly significant (F = 97.499, p = 000); pairwise comparisons in addition to 0.2μg/ml with 0.8μg/ml, 0.8μg/ml and 3.2μg/ml group, no significant difference between the groups, the difference was highly statistically significant (p = 000). Ganoderma lucidum polysaccharides antagonize tumor supernatant inhibition of the mixed lymphocyte reaction: MTT results showed that in tumor supernatant action, mixed culture 72h mice with the gene mouse spleen lymphocytes with allogeneic mouse spleen lymphocytes OD values ??were 1.170 ± 0.205,1.209 ± 0.125,1.234 ± 0.148 and 1.247 ± 0.148, significantly higher than the control group not with Ganoderma lucidum polysaccharides role 0.825 ± 0.157 spleen lymphocytes, the difference was highly significant (F = 11.335, p = 000). Although showing a dose-dependent, but pairwise comparison analysis between each dose group in addition to the differences in the control group with Ganoderma lucidum polysaccharides role was highly significant (p = 000), the difference was not significant sex (p gt; 0.05 RPMI 1640 medium); each dose group results are nearly unaffected tumor supernatant inhibited conventional contrast 1.335 ± 0.083, analysis of variance, no significant difference in sex (p gt; 0.05). 7. Immunocytochemical staining Ganoderma lucidum polysaccharides antagonize tumor supernatant of lymphocyte granzyme B and perforin expression inhibition: Ganoderma lucidum polysaccharides under different concentrations 72h after PHA-induced syngeneic mouse spleen lymphoid tumor supernatant inhibition of cell perforin and granzyme B expression was significantly reduced or disappeared, the immunocytochemical staining positive was significantly higher than control group without the effect of Ganoderma lucidum polysaccharides. 8.Western blot detection of Ganoderma lucidum polysaccharides antagonize tumor supernatant inhibition of lymphocyte granzyme B and perforin expression: in different concentrations of Ganoderma lucidum polysaccharides under tumor supernatant of PHA-induced 72h after syngeneic mouse spleen lymphocytes perforation and granzyme B expression inhibition was significantly reduced or disappeared, perforin protein expression levels were 1.073 ± 0.040,1.127 ± 0.037,1.240 ± 0.0143 and 1.352 ± 0.035, significantly higher than the control group, not with the role of Ganoderma lucidum polysaccharides 1.000 , and showed a dose-dependent trend, the difference was highly significant (F = 66.256, p = 000). No significant differences (p gt; 0.05) in the high dose group (3.2μg/ml 12.8μg/ml) has been close to unaffected inhibit tumor supernatant control group 1.306 ± 0.029, Pairwise comparison analysis of each dose group and the control group is not the role of Ganoderma lucidum polysaccharides were significant differences between sex (p lt; 0.05) or highly significant (p lt; 0.01), in addition to 0.2μg/ml and 0.8μg/ml differences between no significant resistance p-gt; 0.05) between each dose group differences were highly significant significant (p lt; 0.01). Granzyme B protein expression levels were 1.021 ± 0.041,1.089 ± 0.026,1.210 ± 0.079 and 1.259 ± 0.089, significantly higher than the control group of 1.000 unused Ganoderma lucidum polysaccharides role, and a dose-dependent trend, the difference was highly significant ( F = 10.954, p = 000). The high-dose group (3.2μg/ml 12.8μg/ml) has been close to unaffected inhibit tumor supernatant control group 1.277 ± 0.090, no significant differences (p gt; 0.05). Pairwise comparison analysis of the low-dose group (0.2μg/ml, 0.8μg/ml) with Ganoderma lucidum polysaccharides role between the control group the difference was not significant sex (p gt; 0.05) high-dose group (3.2μg/ml, 12.8μg / ml) was highly significant (p lt; 0.01) between each dose group except between 0.2μg/ml and 0.8μg/ml, 0.8μg/ml and 3.2μg/ml, 0.8μg/ml 12.8 μg / ml between, 3.2μg/ml and 12.8μg/ml of the difference was not significant sex (p gt; 0.05) outside each dose group, the differences were significant sex (p lt; 0.05) or highly significant (p lt; 0.01). Conclusion: 1. B16F10 melanoma cell culture supernatants of mouse spleen lymphocytes with immunosuppressive effects. Ganoderma lucidum polysaccharides B16F10 melanoma cell culture supernatant of mouse spleen lymphocytes immune inhibition could be antagonized.

Antagonism of Ganoderma Luncidum Polysaccharides on Immune Suppression of Lymphocytes Caused by Supernatants of Melanoma Cells

Abstract

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