| Objective: Radiotherapy is one of the most important methods againstcancer. In recent years, with the increasing deepened research on theradiobiology, high energy X-ray can inhibit or kill tumor cells by themechanism of which has an ionisation effect inside tumor cells and destructsthe structure of DNA in cancer cells. In addition, by means of modifying thetissue structure and activity of the tumor, X-ray can enhance the immun-ogenicity of tumor cells, which releases "danger signals", such as HMGB-1and hsp70, and up-regulates the expression of adhesion molecules, such asVCAM-1, MHC-I and ICAM-1."Danger signals" and adhesion molecules canfuther recruit immune cells and activate innate immune reponse and evenadaptive immune reponse. Thus, radiotherapy not only causes the destructionof signaling molecules, which is indispensable for tumor cells growth,but alsoplays more important role in immune regulation effect of anti-tumorimmunity.More and more basic and clinical researches have indicated that there is abetter therapeutic effectiveness against tumor by adopting combined treatment.Thus, it is accumulating acceptable that combination of radiotherapy andimmunotherapy is an available way against cancer.In our study, the aim is to observe the synergy effect between X-ray andDC vaccine against tumor, and to do some preliminary researches andanalyses related with the immune mechanism.Methods:1Observing the effect of apoptosis-inducing for4T1tumor cells treatedwith X-ray by FCM4T1tumor cells accepted X-ray at a dose of10Gy for three times intervalof24hours. Then, the rate of apoptosis cells was tested by FCM after24hours of the last radiation.2Detecting the expression of HMGB-1and TGF-β4T1tumor cells were cultured to logarithmic growth phase and weretreated with X-ray at a dose of10Gy for three times interval of24hours. Thenthe cells were collected and their total proteins were extracted for testing theexpression of HMGB-1and TGF-β after24hours of the last radiation.3Preparation, activity and presenting antigen function of DC vaccineBone marrow cells were collected from femoral bones of BABL/c micekilled by cervical dislocation. The differentiation of DCs were induced by IL-4and GM-CSF for5days. DCs cultured at5days were gathered again and thenumber of cells was distributed at1×106/ml in24-well plates. A finalconcentration of50μg/ml of the lysate of4T1tumor cells treated with X-raywas added to24-well plates, which were placed in5%CO2incubator with37℃water-saturated air for16hours. Then LPS(100ng/ml)or TNF-α(10ng/ml)was adopted to induce DC maturation. DCs treated with PBS were consideredas negative control. The expression of MHCⅡ,ICAM-1and CD86of CD11c+cells was analyzed by FCM.DCs treated with mitomycin C at final concentration of25μg/ml wereused as stimulating cells, while response cells were spleen cells obtained fromfemale C57BL/6mice. The respective proportion of response cells tostimulating cells at200：1,100:1,50:1, was added to U type of96-wells andwere incubated for96hours in5%CO2incubator with37℃water-saturatedair. Then20μl CellTiter96AQueous One Solution Reagent was added toevery well prior to4hours of terminating the incubation. The proliferation ofresponse cells was analyzed by MTS colorimetry.4Anti-tumor effect and mechanism of combination of X-ray and DCvaccineX-ray was adopted to treat the4T1tumor-bearing mice at a dose of10Gyfor sequential3times interval of24hours or at a dose of30Gy only one time.Subsequently, DC vaccines were injected intratumorally at1×106/mouse.Tumor growth curves were tested every day. The number of CD8+T cells and CD4+CD25+T-Reg cells related to the TILs (tumor-infiltrating lymphocytes)was analyzed. The cytotoxic activity and secretion of IFN-γ of tumor antigenspecific CTLs and the number of T-Reg cells, which are related with thespleens of4T1tumor-bearing mice were detected by the methods ofmicrocytotoxicity or FCM, respectively. Real-time PCR was used foranalyzing the expression of cytokines in the tumor cells and TILs, such as IL-6and IL-10; and the expression of chemokines such as CXCL16and CXCR6.Results:1X-ray at a dose of10Gy for sequential3times interval of24hourscould induce4T1cells apoptosis(apoptosis rate was about14.88%), as well asnecrosis. The expression level of HMGB-1indicated a increasing trend,whileTGF-β with a decreasing trend.2Activaty of DCs was promoted by using the method of combination ofX-ray and DC vaccine. Compared with the control group, DCs loaded withantigen and LPS could obviously induce the expression of ICAM-1, MHC IIand CD86, and could powerfully stimulate the proliferation of allogeneic Tlymphocytes,which was enhanced with the increasing number of DCs.3X-ray combined with DC vaccine significantly inhibit tumor cellsgrowth in vivo. In the group of exposure to10Gy for sequential3timesinterval of24hours combined with DC vaccine, tumor growth was the slowest;the production of IFN-γ of spleen lymphocytes and CTL activity were thestrongest. The combination of X-ray and DC vaccine up-regulated the numberof CD8+T cells, and had no obvious regulatory role for the T-Reg cells, butraised the ratio of CD8+T/T-Reg.4Compared with the control group, in the group of exposure to10Gy forsequential3times interval of24hours combined with DC vaccine, theexpression of IL-6and IL-10significantly decreased in tumor cells; theCXCL16had a increase trend. Of TILs, the expression of CXCR6markedlyup-regulated; the expression of IL-6and IL-10had no significantly change.Stat3activation was inhibited in tumor cells treated with X-ray at at a dose of10Gy for sequential3times interval of24hours by compared with control group, while the activity of NF-κB had no obvious change.Conclusions:1X-ray promoted the4T1tumor cells expressing HMGB-1, whilereduced the expression of TGF-β.2The lysate of4T1cells exposed to X-ray combined with LPSsignificantly promoted DCs maturation, activation, and antigen-presentingability.3The combination of X-ray and DC vaccine had synergy effect againsttumor.4The combination of X-ray and DC vaccine up-regulated the number ofCD8+T cells in tumor tissue and spleens, and had no obvious effect forregulating T-Reg cells, but raised the ratio of CD8+T/T-Reg.5The combination of X-ray and DC vaccine can inhibit of stat3-IL-6signaling pathway, which was conducive to improving the microenvironmentof the tumor immune... |
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