| BackgroundHepatocellular Carcinoma(HCC)is one of the most common and highly malignant tumors that seriously endanger the health of all human.Radiotherapy is one of the most important methods for the treatment of HCC.Radiotherapy is closely related to tumor microenvironment.The distant effect during radiotherapy is one of the products of this complex relationship.Distant effect was first proposed by MoE.Distant effect of radiotherapy refers to the phenomenon that distant metastases outside the irradiation path also respond to the irradiation of the primary tumor.Irradiation-induced bystander effect(RIBE)or distance effect describes a set of biological effects that occur in non-target cells while receiving signals or active factors from target cells exposed to ionizing radiation.RIBE changed the pattern of our knowledge of radiobiological effects and clearly showed that the damaging effects of ionizing radiation are not only tumor nuclear DNA damage,but also from cytoplasmic or extracellular signaling events,i.e.off-target effects.Some studies have reported that the distancing effect is caused by the systemic anti-tumor immune response,but the mechanism of how tumor cells act on immune cells to exert anti-tumor effect is still unclear.Exosomes(Exos)are substances with double vesicle-like structure,in which the main components of Exosomes are proteins,nucleic acids and lipids.Some researchers have found that exosomes are an important signal transmission medium between cells and play an essential role in the physiological and pathological processes of the body.Dendritic cells(DCs)play an important role in the initiation of anti-tumor specific immune response in humans.DC first recognizes,absorbs and processes antigen,and then delivers the processed antigen peptide fragment and major histocompatibility complex(MHC)to the surface of T cells in the form of complex and activates the initial T lymphocytes,thus activating the immune response of T lymphocytes.This study aims to study whether irradiated HEPA1-6 cells can affect the function of dendritic cells through the release of exosomes,induce the anti-tumor immune response of the body,and then mediate the distant effect of radiotherapy in liver cancer patients.Objective(1)To observe the effects of exosomes on DCs maturation and function in irradiated HEPA1-6 cells.(2)To observe the anti-tumor immune responses in vivo mediated by exosomes treated DCs of irradiated HEPA1-6 cells.Methods(1)HEPA1-6 cells were cultured and irradiated with 8Gy×3 times during the logarithmic growth phase.Exosomes in the supernatant of irradiated and unirradiated cells were extracted by the extraction method of exosome kit.(2)Exosomes extracted were identified(labeled as Exo-IR and Exo-Con),and the expressions of CD63,TSG101 and other signature proteins on exosomes were detected by Western-blot method;The morphology and size of exosomes were observed by transmission electron microscopy.Protein content was determined by BCA kit,and the samples were stored in-80℃ refrigerator.(3)DC2.4 cells were cultured,and PBS,ExO(Exo-IR and Exo-Con)and LPS were co-incubated with DCS for 48h,respectively,and the surface molecules of DCS were determined by flow cytometry.(4)The MACS sorting scheme was adopted to classify mouse T lymphocytes.The lymphocytes prestained with fluorescent dye CFSE and DCS were co-incubated with a cell ratio of 5:1 for 5 days,and the division and proliferation of T lymphocytes were measured and analyzed by flow meter.(5)DCs from each treatment group were injected into normal immune-bearing mice inoculated with ectopic model through tail vein.The tumor size of mice was recorded and the tumor volume of mice was calculated on the next day.(6)The tumor-bearing mice were sacrificed one week after the end of 3 injections,and their spleens,tumor tissues and lymph nodes were isolated.The division and proliferation of T cells in different tissues were determined by flow cytometry.Results(1)The exosomes extracted from the supernatant of tumor cell culture were related to the mode and dose of irradiation.When the cell supernatant was irradiated at 8Gy×3,the amount of protein exosomes secreted from the cell supernatant and the stimulating effect on immune cells were the most significant.(2)In the extracts of HEPA1-6 cell culture medium,a group of bilayer like structures with a diameter of 30-100nm were observed by transmission electron microscopy.Western-blot analysis confirmed that it was rich in CD63 and TSG101,and lacked Cytochrome C,which was consistent with the characteristics of"exosomes" reported in literature.(3)Microscopically,Exo-IR and Exo-Con treated DCS,respectively.DCS stimulated by Exo-IR had slightly longer dendrites.Flow test results,and exobiology-con compared with DCs group,exobiology-MHC-on the surface of the ir can raise DC2.4 Ⅱ CD80,CD83,CD86 and CD40 molecules expression(p<0.05).(4)The proliferation induction effect of DC-Exo-IR on mouse T lymphocytes was stronger than that of DC-Exo-Con;DC-Exo-IR could promote the proliferation of CD8+T cells(P<0.05).(5)Mice were inoculated with ectopic tumors,and DCS of different treatment groups were injected into mice through the tail vein.The results showed that compared with the DC-Exo-Con and IDC groups,the tumor volume and growth rate of the DC-Exo-IR group were the smallest and the slowest(P<0.05).The number of CD8+T lymphocytes was significantly increased in the tumor tissues and spleen tissues of mice(P<0.05).ConclusionThe irradiated hepatoma cells of mice could induce the increase of exosomes secretion.Exos can induce the maturation of dendritic cells and stimulate the proliferation of T lymphocytes in irradiated mice.Dendritic cells treated with Exos of irradiated hepatocellular carcinoma of mice can mediate a strong anti-tumor immune effect in tumor-bearing mice. |
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