| Part I The impact of Anti- TIM3 and MEK inhibitor on the immune function,proliferation and apoptosis of melanomaObjective: To evaluate the anti-tumor effect of MEK inhibitor and Anti-TIM3 in melanoma.Methods:The grouped Mouse melanoma model is treated with medicine(the first group: control; the second group: Anti- TIM3; the third group: MEK inhibitors GSK1120212; the fourth group: Anti- TIM3 + GSK1120212).PBMC extracted from mouse melanoma model, using flow cytometry to detect the PBMC lymphocyte subsets and IL- 17 level.at the same time using the CCK 8, wound healing to detect proliferation, migration in each experimental group of mouse melanoma cell lines B16F10, To evaluate the antitumor effect of MEK inhibitors in multiple dosing approach.Results:Flow cytometry shows that Anti- Tim3 can increase CD8 + T cells and IL-17 a level in mouse melanoma PBMC(Anti-Tim3 group CD8+(%)66±5(%);GSK1120212 group CD8+(%)31±4.5(%);Control group CD8+(%)35±4(%);GSK1120212+Anti-TIM3 group CD8+(%)44±4(%);Anti-Tim3 group IL-17a(%)14.5±2.8(%);GSK1120212 group IL-17a(%)3±1.1(%);Control group IL-17a(%)3.1±1.3(%);GSK1120212+Anti-TIM3 group IL-17a(%)8.9±2.6( %)).mice survival experiments show that GSK1120212 group had product inhibition rate reached 30%(* * * P < 0.01, n = 5), and GSK1120212 + Anti- TIM3 joint group had product inhibition rate reached 40.8%(* * * P < 0.01, n = 5). In contrast,also Anti- TIM3 group induced the inhibition effect on the size of tumor growth,it is not clear.CCK 8, wound healing experiments have shown that B16F10 proliferation and migration ability decreased significantly in GSK1120212 and joint group,there is no significant difference of cell proliferation and migration in Anti-TIM3 group and control group.In CCK 8 for 4 h,the inhibiting effect of GSK1120212 and joint group reached the highest peak of 21%, 24%(* * P < 0.01),With the passage of time,to 36 h B16F10 inhibition of proliferation activity almost entirely disappeared. In the evaluation of the antitumor effect of MEK inhibitors in multiple dosing approach.we find the tumor inhibition efficiency of the size is more than 30%(* * * P < 0.01)in lavage group, in contrast,the inhibition rate of the two groups of subcutaneous injection and intraperitoneal injection is about 15%.Conclusions:There is a synergistic effect of Anti-TIM3 and MEK inhibitors in the anti-tumor effect of melanoma. Among this, Anti-Tim3 can restore the activity of CD8+T cells. MEK inhibitor can promote the apoptosis of melanoma and inhibit the proliferation and migration of B16F10. It is suggested that the anti-tumor mechanism of Anti-TIM3 and MEK inhibitor is different.Part ⅡThe influence of TIM3 expression through MEK reducingObjective:To evaluate the level of TIM3 expression through MEK reducingMethods:The grouped Mouse melanoma model is treated with medicine(the first group: control; the second group: MEK inhibitors GSK1120212).PBMC extracted from mouse melanoma model, using flow cytometry to detect TIM3 level on lymphocyte subsets in PBMC.Observe the PMA stimulation effect on TIM3 expression in vitro TIM3 Jurkat T cell surface.At the same time through the Si-h-MEK2 plasmid transfecting to Jurkat T cells to compare TIM3 expression level compared to control.Results:GSK1120212 block MEK in the model of mouse melanoma,the result shows that TIM3 level in CD8 + T cells increased significantly( GSK1120212 group CD8+TIM3+17.6±2(%),control group CD8+TIM3+ 5.95±3.7(%),**P<0.01).In contrast,the TIM3 level in CD4, NK has no significant difference.Jurkat T cell was activited after stimulated with PMA and TIM3 level increased(*P<0.05). the results of Flow cytometry and real-time PCR shows that TIM3 m RNA and protein expression level of Si-h-MEK2 group was significantly higher than control( Flow cytometry :Si-h-MEK2 group Jurkat T cells TIM3+15.5±2.5(%),Control group TIM3+4.9±2.8(%),**P<0.01)Conclusions : The downregulation of MEK can promote the expression of TIM3,Ras/Raf/MEK/ERK signaling pathway plays an important role in the development of cancer. |
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