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ObjectiveTo investigate the effect of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway inhibitor PD98059 on the proliferation and killing function of γδT cells cultured in vitro. MethodsMononuclear cells were separated from peripheral blood in healthy subjects and placed in RPMI 1640 complete medium containing zoledronic acid and interleukin-2 to obtain γδT cells through induction and culture. The ERK1/2 specific inhibitor PD98059 was used to block the ERK1/2 signaling pathway in γδT cells, and the proliferation of γδT cells was measured by cell counting, and the killing function of γδT cells was measured by CCK-8 method. Flow cytometry was used to measure the expression of granzyme B and perforin in γδT cells. The t-test was used for comparison of continuous data between groups. ResultsAfter 10 days of culture, the purity of γδT cells reached 87.94%±2.36%. The number of γδT cells treated with PD98059 was significantly lower than that of control cells ［(6.74±0.36)×105/ml vs (9.42±0.31)×105/ml, t=-12.708, P＜0.001］. Compared with the control cells, those treated with PD98059 had significantly higher positive expression rates of granzyme B and perforin (granzyme B: 48.89%±1.31% vs 41.58%±1.58%, t=7.582, P＜0.001; perforin: 65.92%±3.29% vs 33.49%±2.83%, t=15.478, P＜0001) and a significantly higher killing rate of HepG2 cells (69.28%±4.96% vs 48.34%±3.01%, t=11.201, P＜0.001). ConclusionThe ERK1/2 specific inhibitor PD98059 can inhibit the proliferation of γδT cells, but it can enhance the in vitro killing function of γδT cells.
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Objective:To investigate the anti-tumor effect of Hyperoside.Methods: Human γδT cells were amplified by isopentenyl pyrophosphate from peripheral blood cells.The proliferation capacity of γδT cells was measured with CCK-8 assay after treated with different concentrations of Hyperoside.Cytotoxicity of γδT cells was detected with LDH assay , and the expression of granzyme,perforin CD107a and IFN-γonγδT cells were measured by flow cytometry before and after treatment.Results: Hyperoside could significantly stimulate the proliferation of γδT cells at the concentration of 3.13-12.5 μg/ml.Cytotoxicity and expression of granzyme,perforin and IFN-γofγδT cells were increased after treatment.Conclusion:Hyperoside could enhance cytotoxicity of humanγδT cells through up-regulation of granzyme ,perforin CD107 a and IFN-γexpression.
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Objective:To investigate the effect of 2-deoxy-D-glucose (2-DG) on hunmanγδT cells on the expression of CCR5 and killing function in vitro.Methods:UsingγδT medium to cultivate peripheral blood mononuclear cell( PBMCs) in vitro.After co-cultured with various concentrations of 2-DG for 48 h,the expression of CCR5 and killing activities of γδT cells for each group were detected by flow cytometry and CCK-8 methods.Results: 2-DG could not promote the growth of γδT cells with the increase in concentration from 0 μmol/L to 1.0 μmol/L and decreased thereafter.The certain concentration ( 0-2.0 μmol/L ) of 2-DG could upregulate the expression of CCR5 in dose dependent manner.Besides,at 0.5μmol/L and 1.0μmol/L of 2-DG could increase the ex-pression of CD107a and perforin and have no effect on the granzyme B.Conclusion: Human γδT cells isolated from peripheral blood treated with 2-DG could promote the expression of CCR5 and increase the killing activities at certain concentration in vitro.
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Objective:To investigate the mechanisms of TWS119 induced CCR5 expression in hunman γδT cells. Methods:After treatment with various concentrations of TWS119 for 48h, the expression of CCR5 in γδT cells were detected by flow cytometry. The p-STAT3 and GAPDH expression were examined by Western blot analysis. Results: TWS119 could upregulate the expression of CCR5 in dose dependent manner. Western blot analysis revealed that TWS119 inhibit phosphorylation of STAT3,but had no significant impact on GAPDH. In addition, pretreatment of γδT cells with 0. 5 μmol/L STAT3 specific phosphorylation inhibitor Stattic could upregulate the expression of CCR5 and enhance the TWS119 induced CCR5 expression. Conclusion: TWS119 could upregulate CCR5 expression of γδT cells by inhibiting STAT3 phosphorylation in vitro.
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Objective To investigate the effect of DHA on proliferation and killing activity of γδT cells against SW1990 cells in vitro.Methods γδT cells were generated in vitro by stimulating peripheral blood mononuclear cells of healthy donors in RPMI 1640 completed medium containing IPP and IL-2 for 8 d,and then co-cultured with different concentrations of DHA for 48 h.Proliferation rates of γδT cells for each group were detected by MTT method.The perforin,granzyme B and CD107a expression in γδT cells were verified by flow cytometer.The cytotoxic activity of γδT cells against SW1990 cells were analyzed by CCK-8 kit.Results The purity of γδT cells in each group reached 75.46％ ±5.32％ after 8 d of culture.Compared with the control group,the proliferative capability of γδT cells were enhanced significantly after treated with 50-100 μmol/ml DHA for 48 h,moreover,cytotoxicity against SW1990 cells and perforin,granzyme B and CD107a expression of the γδT cells treated with DHA were higher than the control group.Conclusion DHA could enhance the antitumor activity of γδT cells,which may be associated with the upregulation of perforin and granzyme B expression in γδT cells.
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Objective To observe the local immue response and changes of angiogenic factors of tumor cells in Heps-bearing mice after mesenchymal stem cell (MSC) are administrated. And to explore the feasibility and safety of MSC for liver tumors therapy. Methods MSC were obtained through adherent culture method. Phe-notypes of MSC were analyzed by flow cytometry. MSC were labeled with DAPI in vitro. 54 Mice of 8 weeks of age with subcutaneously transplanted liver carcinomas were developed randomly. When the maximal diameters of the tumor reached 0.5 - 0.8cm, they were divided into three groups randomly: MSC group, DAPI group and NS control group. 2 × 10~6 MSC and MSC marked by DAPI were administrated into the mice right rear back tumor tissue. The survival time of the tumor-bearing mice was recorded and the mean survival time was calculated. Immunohistochemical staining was performed to count CD4~+ T cells and CD8~+ T cells in the local tumor,as well as to examine the expression of vascular endothelial growth factor ( VEGF) in tumor cells. Results In the MSC group,the mean survival time was 45 d (95%CI;33 ～56 d) ,in the NS control group, the mean survival time was 33 d ( 95%CI : 28 ～ 37 d). There was a statistical significance in the difference between them ( P < 0.05). Immunohistochemical staining results showed as follow: the number of CD4~+ T cells and CD8~+ T cells in the MSC group decreased significantly in comparison with the NS control group at early stage. The expression of VEGF also decreased obviously in comparison with the NS control group and induced tumor cells necrosis at late stage. The survival time of MSC group was prolonged. Conclusion MSC can engraft in Heps-bearing tumor tissue, and inhibit T lymphocyte cellular immunity at early stage. It can reduce the number of CD4~+ T cells and CD8~+ T cells and promote tumor growth. MSC can down regulate VEGF expression and induce tumor cells necrosis at late stage. By this way,it can prolong the survival time of Heps-bearing mice.