| Objective:1.Observe the influence of β-elemene on proliferation and apoptosis in mice hepatoma cell line BNL;2. To unveil the immune mechanisms underlying the anti-tumor effect of P-elemene, we observed the immune response in vivo and in vitro and the therapeutic effect of P-elemene combining with DC/Dribble vaccine in mice hepatic cancer model.Methods:1.Mice hepatoma cells BNL were cultivated in vitro and then treated with various concentration of β-elemene.The effect of β-elemene on the growth of mice hepatoma cell line BNL was detected by CCK-8.The effect of β-elemene on the apoptosis of BNL cells was determined by fluorescence microscope and FCM with Annexin V FITC/PI staining;2. BNL hepatoma cells were used to establish subcutaneous model in Balb/c mouse.Dentritic cells were induced in vivo through combined injection of GM-CSF and Flt3-L expression plasmid and splenocyte from injected mice were harvested and counted for identification by FCM. DRibbles derived from BNL cells were extracted through "step by step" high-speed centrifugation;3. The splenocyte obtained from health mice were incubated with different stimulations for72hours, which divided into the control group, the DRibble group, the DC group and the DC/Dribble vaccine group, and each group was divided into different concentrations of β-elemene0,4,10,40(μg/ml) stimulation.The proliferation effect of splenocyte was detected by CCK-8,the IFN-γ and IL-4concentration in culture medium was detedted by ELISA;4.The mice immunized previously were divided into4groups,the control group, the β-elemene group, the vaccine group and the united group.PBS was given to the control group by subcutaneous and intraperitoneal injection. At the dose of50mg/kg of the β-elemene was given to the β-elemene group and united group by intraperitoneal injection once a day for7successive days. DC/Dribble vaccine was injected into the lymph node to the vaccine group and united group on the1st day, and DC/Dribble vaccine was given by subcutaneous injection on the3rd day and5th day. All the mice were killed on the10th day, the spleen cells obtained sterilely were incubated with Dribble or cell culture for72hours, the supernatant of cultured cells were collected and the contents of IFN-γ were measured by ELISA. In addition, the spleen cells obtained from the united group were incubated with different stimulations for72hours,which divided into the control group, the DRibble group, the DC group and the DC/Dribble vaccine group, the supernatant of cultured cells were collected and the contents of IFN-y were measured by ELISA. Establish liver tumor-burdened mice model, then the BNL bearing mice were randomly divided into4groups, the control group, the β-elemene group, the vaccine group and the united group, the treatment were the same as the immune ways.The mice were observed in tumor size and the survival period. On the23rd, the mice were killed, stripping the tumor tissue by HE staining, the pathology morphological characteristics of tumor tissue were observed by light microscopes.Results:1.The result showed that the P-elemene obviously inhibited the proliferation of BNL cells in both time and dose dependment anners. In early stage, β-elemene can induce apoptosis and the apoptpsis rate increased with the increase of the concentration ofp-elemene.In later stage, necrotic cells increased significantly;2.BNL cells were succeed in constructing subcutaneous HCC model in Balb/c mouse and the dose of2x106cells was selected to establish tumor-bearing mouse model.The size of spleen increased a lot and the number of splenocytes amplified much more after injected of the two plasmids. According to the identification of FAC,20%of the splenocytes were CD11c+cells. The determination of total protein of DRibble was2μg/ul;3. In vitro, we found that both the vaccine group and the Dribble alone group can promote the proliferation of splenocytes, and the vaccine group is better than other groups (P<0.001). The best β-elemene’s concentration is10μg/ml in which the effect of promoting cell proliferation is strongest. The spleen alone group and the DC alone group cannot stimulate the proliferation of splenocytes with various concentration of β-elemene (P>0.05).The trend of the secretion of IFN-y was the same as the proliferation of splenocytes,but level of IL-4is too low to statistical significance;4. In vitro,we found that when the mice were immunized previously by different ways, compared with other three groups, the secretion of IFN-y in the united group was significantly higher (P<0.01),the β-elemene group and the vaccine group was higher than the control group (P<0.01);when the spleen cells obtained from the united group were incubated with different stimulations, the secretion of IFN-γ in the DC/Dribble vaccine group and the Dribble group were higher than the control group (P<0.01), when the dose of the β-elemene was10μg/ml, the secretion was significantly higher (P<0.01). In vivo, under the combination therapy, tumors grew more slowly, the tumor size and the survival period compared with the other groups had statistically significant (P<0.01).In HE staining, the surrounding connective tissue of the tumor was wrapped tightly and completely, which had a large number of inflammatory cell infiltration,such as neutrophils, monocyte macrophages and lymphocytes.Conclusion:Inhibiting liver cancer cell proliferation and promoting apoptosis is an important mechanism of P-elemene’s antitumor effect. β-elemene associated with DC/DRibble Vaccine can induce strong specific immune response in vivo and in vitro in mice and mainly induce cellular immune response.β-elemene may exert its antitumor effect via enhancing the DC antigen presenting function. |
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