Experimental Study On The Therapeutic Effect Of Daphnetin Combined With Dendritic Cells Vaccine On H22-bearing Mice

Objective:To observe the therapeutic effect of daphnetin combined dendritic cells(DCs) vaccine on H22-bearing mice and preliminarily explore its possible mechanism of anti-tumor. Methods:1、Use MTT methods to exam the surpress effect of inhibition daphnetin of different concentrations to human hepatoma Hep G2 cells and mice hepatoma proliferation of H22;2 、 Culture DC in vitro and observe cell morphology under an inverted microscope, use repeated freezing and thawing prepared tumor cells whole cell antigen pulse DC to prepare DC tumor vaccine;3、The anti-tumor effect of daphnetin combined with DC vaccines in vivo: establish a mouse hepatoma H22 tumor-bearing model, random Ly divide the tumor-bearing animals into a control group, 5-Fu group, daphnetin group, DC vaccine group, daphnetin combined with DC vaccine group, set another healthy control groups, each group with eight mice. Inject saline 0.2m L intraperitoneally to both control group and tumor-bearing mice every day for 14 days;As for 5-Fu group, inject 0.2m L 5-Fu to each mouse intraperitoneally by 20 mg / kg·d dose everyday for 14 days; For daphnetin group, inject 0.2m L Daphnetin by 4mg / kg·d dose per mouse intraperitoneally everyday for 14 days; for DC vaccine group, subcutaneously inject DC vaccine around the inoculation peritumoral area after the first 1d and 7d of tumor cell inoculation(5×106/mouse). For daphnetin combined with DC vaccine group: inject 0.2m L daphnetin by 4mg/kg·d dose per mouse intraperitoneally everyday for 14 days; At the same time, subcutaneously inject DC vaccine around the inoculation peritumoral area after the first 1d and 7d of tumor cell inoculation(5×106/mouse). Sacrifice all the Mice under treatment after 15 days, count the tumor inhibition rate, spleen index and thymus index;4、Use MTT method to assay the activity of mice spleen natural killer(NK) cell and the proliferation index of Con A-induced T lymphocytes;5、HE staining, observe the pathological changes of liver cancer H22 under light microscope;6、Use ELISA to detect the levels of IFN-gamma、IL-12、IL-4 levels of cytokines in serum;7、Use real-time quantitative PCR method to detect the expression of Bcl-2、Bax m RNA in the tumor tissue of mice in each group. Results:1、①Inhibition rate(%) were from 63.71±2.20 to 10.10±1.71,when Hep G2 cells were treated by the daphnetin with the concentration from 250 to 0.48(μg/m L) lasts for 48 hours.IC50 value(μg/m L) was 21.89±0.70;②Inhibition rate(%) were from 85.52±5.15 to 19.79±5.47,when H22 cells were treated by the daphnetin with the concentration from 250 to 0.48(μg/m L) lasts for 48 hours. IC50 value(μg/m L) was 16.89±9.64.2、Treated groups have inhibition effects on the xenografts growth of mice of which the 5-Fu group is the most obvious. The tumor weight of tumor-bearing control group, 5-Fu group, daphnetin group, DC vaccine group, daphnetin combined with DC vaccine group respeictively(1.817±0.388) g,(0.424±0.309) g,(0.833±0.314) g,(1.206±0.523) g,(0.493±0.306) g, the tumor weight of each group is lower than that of the tumor-bearing control group(P<0.05).The inhibition rate of daphnetin combined with DC vaccine group is the highest, the significance level of differences when compared to the daphnetin group was less than 0.5, and the significance level of differences when compared to the DC vaccine group was less than 0.5; The significance level of differences when DC vaccine group compared to the daphnetin group was less than 0.05.3、①The spleen index of daphnetin group, DC vaccine group, daphnetin combined with DC vaccine group separately was 8.504±0.508,10.878±3.916 and 12.902±2.927, higher than the 7.075±0.379 of tumor-bearing control group(P<0.05). The significance level of the differences when combination therapy group compared to the daphnetin group and DC vaccine group was less than 0.5, but the difference between daphnetin group and DC vaccine group showing no statistically significant(P>0.05).The spleen index of 5-Fu group is 5.638±1.567, lower than that of the tumor-bearing control group(P<0.05). ② The thymus index of daphnetin group,daphnetin combined with DC vaccine group is higher than that of tumor-bearing control group, indicating that it can promote the thymus development of mice, but the difference is no statistically significant(P>0.05). But the thymus index of 5-Fu group is lower than that of tumor-bearing control group and the healthy control group, the significance level of differences was less than 0.5.4、Tumoricidal rate of NK cells of Daphnetin group, daphnetin combined with DC vaccine group is higher than that of tumor-bearing control group and 5-Fu group(P<0.05), the tumoricidal rate of daphnetin combined with DC vaccine group is up to(64.40±9.99)%, followed by daphnetin group of(59.50 ±10.90)%,while the 5-Fu group greatly reduces the NK cell killing activity, lower than the control group and tumor-bearing control group(P<0.05).5、T lymphocyte proliferation stimulation index induced by Con A of daphnetin group, daphnetin combined with DC vaccine is higher than tumor-bearing control group and 5-Fu group(P<0.05); T lymphocyte proliferation stimulation index induced by Con A of 5-Fu group is lower than that of the tumor-bearing control group and the healthy control group(P<0.05).6、Pathological changes of tumor tissues for each group:HE staining showes that daphnetin combined with DC vaccine group presenting large areaof necrosis,more tumor cells showing nuclear fragmentation,morphological changes of apoptosis and necrosis like nuclear condensation and so on; less necrotic area presents in tumor-bearing control group, and the necrotic area is less,vigorous growth of tumor cells, necrosis and apoptosis cells are less; daphnetin group and the DC vaccine group also present significant necrosis, but the degree of necrosis significantly reduces compared to daphnetin combined with DC vaccine group.7、Peripheral cytokines of the mice of the treated groups: ①The content of serum IL-12 of three treatment groups is higher than that of tumor-bearing control group(P<0.05), among which, daphnetin combined with DC vaccine group> daphnetin group>DC vaccine group(P<0.05). ②the content of serum IFN-gamma of three treatment groups is higher than that of the tumor-bearing control group and 5-Fu group, among which,the daphnetin combined with DC vaccine group is higher than that of daphnetin group and DC vaccine group(P<0.05), but there is no significant difference when daphnetin group compared to DC vaccine group(P>0.05), daphnetin combined with DC vaccine group is also higher than that of the healthy control group(P<0.05). ③the content of serum IL-4 of three treatment groups is lower than that of the tumor-bearing control group(P<0.05),among which the daphnetin combined with DC vaccine group is lower than that of daphnetin group and DC vaccine group(P<0.05), there is no statistical significance when daphnetin group compared to DC vaccine group(P>0.05).8 、 The RT-q PCR results show that Daphnetin group, DC vaccine group, daphnetin combined with DC vaccine group can reduce the expression of Bcl-2 m RNA in tumor tissue and increase the expression of Bax m RNA in tumor tissues, the significance level of the diffierences when compared to the tumor-bearing control group was less than 0.5; wherein the Bcl-2 m RNA and Bax m RNA ratio of the daphnetin combined with DC tumor vaccine group is the smallest. Conclusion:1、Daphnetin has inhibition effect of different degrees on Hep G2 cell growth, showing a good dose-dependent in the concentration range of 0.48~250μg/m L; Daphnetin also has inhibition effect on H22 cell growth in the concentration range of 0.48~250μg/m L, also showing a good dose-dependent;2、Daphnetin combined with DC vaccine can improve the immune function in tumor-bearing mice,has inhibitory effect on mouse H22 transplanted tumor, and the effect is better than single use of daphnetin or DC vaccine.