Difference Of The Expression Of DAMP In Tumor Released Autophagosome And Its Effect On IL-10~+B Cells Under Hypoxic And Starvation State

During the early tumorigenesis, tumor tissue obtained oxygen and nutrients from the blood circulation in the normal tissue surrounded. With the rapid tumor growth and new blood vessels formed, the supply of blood oxygen cannot satisfy the requirement of rapid tumor proliferation. Therefore, the tumor tissue will stay in ischemia and hypoxia conditions for a long time. In such a unsuitable environmental condition, thanatosis will occur in tumor tissue and the tumor cells will release Danger-associated molecular-pattern(Danger-associated molecular-pattern, DAMP)into the blood circulation or intercellular space, such as HMGB1, HSP90. Its advanced glycosylation end-products of receptor ligand (RAGE) and toll-like receptor 4 (TLR-4) mediated the pathway which can promote tumor cell autophagy under stress.It is now shown that, autophagy is a survival mechanism for cells responding to serious environment factors such as lack of nutrition, oxidative stress, hypoxia and infection. The cells could resistant to apoptosis by depredating and recycling intracellular protein molecules directly.Previous studies show that damaged organelles in cytoplasm and the cytoplasm components as metabolic product are packaged by double membrane-structured autophagy vesicles which become autophagosome. However, whether the DAMP molecules released from intracytoplasm are wrapped into autophagosome or bonded to the corresponding receptors on the surface of autophagic vacuole membrane, and the effect on the immune cells of DAMP in tumor released autophagosome(TRAP) with different content remain to be further research.OBJECTIVE:1. To confirm whether there are differences among the Danger-associated molecular-pattern and its contents;2. To define the effect of TRAP under hypoxic and nutrient-deprived state on IL-10+ cells and its function.METHODS:1. Detecting the existence of DAMP and its expression in the TRAP under hypoxia and starvation state1.1 Detecting the expression of DAMP in the TRAP from primary hepatocellular carcinoma cell line:HepG2, SMMC7721 and Huh7;1.2 Detecting the expression of DAMP in HepG2 cell lysate under hypoxia and starvation state;1.3 Detecting the expression of DAMP in HepG2-TRAP under hypoxia and starvarion state.2. Detecting the effect of TRAP on inducing B cells into IL-10+B cells and IL-10+B cell’s function under hypoxia and starvarion state2.1 HepG2-TRAP under hypoxia and starvarion state is co-cultured with healthy donor B cells for 72 hours. Then the IL-10+B cells proportion and the IL-10 levels of the culture supernatant will be detected;2.2 The B cells which are co-cultured with TRAP collected under hypoxia and starvation state for 72 hours will be co-cultured with PBMC for 96 hours. After co-culturing, the proliferation of T cells will be detected.RESULT:1. Detecting the existence of DAMP molecules and its expression in the TRAP under hypoxia and starvation state1.1 TRAP surface by flow cytometry HMGB1 content, SMMC7721-TRAP in HMGB1 mean fluorescence intensity (MFI) was significantly higher than Huh7-TRAP and HepG2-TRAP (102vs31vs27); Western blot detection of TRAP in HMGB1 and HSP90 content by comparing relatively gray values, we can see 7721-TRAP in HMGB1 were significantly higher than Huh7-TRAP and HepG2-TRAP (67%vs45%vs37%),7721-TRAP in HSP90 was higher than Huh7-TRAP and HepG2-TRAP (90%vs68%vs62%).1.2 Upregulation of hypoxia DAMP content, lack of nutrition increases autophagy. Western blot analysis of cells HMGB1, HSP90, LC3 content, by comparing the relative intensity, we can see the content of the cells under hypoxic conditions HMGB1 than normal conditions and lack of nutritional condition was significantly increased (95%vs50%vs68%), HSP90 content also higher than normal conditions and lack of nutrition conditions (98%vs38%vs47%); lack of under nutrition cells LC3-Ⅱ content was significantly increased compared to (94%vs 15%vs18%) in normal and hypoxic conditions.2. TRAP can induce more IL-10+B cells and the content of IL-10 in supernatant are higher under hypoxic state,and inhibit the proliferation of T cells.1.3 TRAP hypoxia upregulated in DAMP content. By mass spectrometry to hypoxic conditions than normal conditions TRAP and TRAP, we found HMGB1 and HSP90 levels were increased in hypoxic conditions;TRAP surface by flow cytometry HMGB1 content, we found that the average fluorescence of HMGB1 in hypoxic conditions TRAP intensity (MFI) was significantly higher than normal conditions TRAP (128vs29), no lack of nutrition HMGB1 TRAP in mean fluorescence intensity (MFI) significant difference compared to normal conditions (31 vs29); TRAP by Western blot analysis of HMGB1, HSP90 content, by comparing the relative intensity, known under hypoxic conditions TRAP HMGB1 in content than in normal conditions and lack of nutrition condition significantly increased (127%vs30%vs48%), HSP90 content is also higher than normal conditions and lack of nutrition conditions (135% vs76% vs120%).2. TRAP can induce more IL-10+B cells and the content of IL-10 in supernatant are higher under hypoxic state,and inhibit the proliferation of T cells.2.1 TRAP-induced B cell hypoxia become IL-10+B ratio is higher than the lack of nutritional conditions and normal conditions TRAP TRAP (7.4%vs4.6%vs3.6%); culture supernatant IL-10 content was also higher than lack of nutrition conditions and normal conditions TRAP TRAP (147vs45vs40) (pg/mL); it produces IL-10+B on the CD8+T cell proliferation inhibition is also higher than the lack of nutritional conditions and normal conditions TRAP TRAP (43.8% vs48.7% vs50.5%), were lower than the 61.7% in the control group; CD4+T cells also get similar results.2.2 Through statistical analysis of HMGBl and TRAP-induced HSP90 content with their IL-10+B cells, correlation, we can see its contents induced HMGBl TRAP in IL-10+B cells were highly correlated to 0.869,p<0.001; TRAP in HSP90 its contents induced IL-10+B cells also have a certain correlation, correlation coefficient of 0.589, p<0.05. TRAP content of HMGB1 highly correlated with IL-10 in the supernatant of correlation coefficient of 0.713, p<0.05, TRAP HSP90 content in the supernatant IL-10 content is not relevant, the correlation coefficient was 0.537,p= 0.072, no statistically significant.2.3 Block the HMGB1 in TRAP may reduce IL-10+B cells proportion (2% vs3.6%), the content of the supernatant IL-10 also decreased (27vs52) (pg/mL).CONCLUSION:TRAP under hypoxia and starvation state could induce B cells into IL-10+B cells and inhibit the proliferation of T cells.