Effects Of Hypoxia On Tumor-associated Macrophages

Objective: To acquire M2-type macrophages, through stimulating Human monocyticleukemia THP-1cells by phorbol esters (Phorbol-12-13-acetate, PMA) and IL-4(Recombinant human interleukin-4). Co-culture M2-type macrophages and Human coloncancer HCT-116cells in the condition of hypoxia or normoxia, to observe the effects ofhypoxia on the autophage and the expression levels of cytokines of M2-type macropages.Methods:1. Stimulating THP-1cells differentiate into macrophages by PMA, andthen uses IL-4differentiate macrophages into M2-type macrophages.2. In the conditionof hypoxia or normoxia, cultured M2-type macrophages or co-cultured M2-typemaerophages and HCT-116cells for24h or48h.3. Experimental groups: control group:M2-type macrophages were cultured alone in normoxia condition for24h and48h.Experimental groups: M2-type macrophages were cultured alone in hypoxia condition,M2-type macrophages were co-cultured with HCT-116cells in hypoxia condition for24hand48h, M2-type macrophages were co-cultured with HCT-116cells in normoixacondition for24h and48h.4. Collecting the M2-type macrophages which under differentculture conditions or cultured different hours. Extracted proteins, detected the expressionof each specimens’ beclin-1and microtubule-associated protein1light chain3(LC-3) bywestern-blot. Collecting each groups’ supernatant to measured the expressions of cytokineInterleukin-1β(IL-1β), Interleukin-12(IL-12) and Tumor necrotic factor-α(TNF-α) byusing enzyme-linked immunoabsorbent assay(ELISA).Results:1. successfully induced in THP-1cells differentiate into M2-typemacrophages. The differentiation ratio was86.35%.2. through ELISA text, it was nostatistically significant of the expression of IL-1βin the groups of M2-type macrophagesco-cultured with HCT-116at nomoxia condition (24h or48h) and the groups of M2-typemacrophages cultured alone in hypoxia condition (24h or48h) compared with controlgroup(24h or48h)(p＞0.05). It was statistically significant that the co-cultured groups inhypoxia condition(24h or48h)24h:0.7900±2.65%and48h:0.7433±4.16%compared with control group (24h or48h)24h:0.8717±5.11%and48h:0.8733±3.21%（P＜0.05）.The expression of IL-1βwere all reduce in each hypoxia groups(p＞0.05). IL-12andTNF-α was no significant difference between the experimental group and control group.The ratio of LC-3Ⅱ/LC-3Ⅰof M2-type macrophage which cultured at nomoxia condition(24h or48h) were no difference (p＞0.05). It was significant difference of the LC-3Ⅱ/LC-3Ⅰratio about M2-type macrophages cultured (24h or48h) or co-cultured(24h,48h) inhypoxia condition were0.3714±5.49%,1.0706±8.00%,0.4203±5.07%and1.1377±6.18compared with control group(24h or48h) was0.2590±0.78%,0.2617±0.82%（P＜0.05）,and positively correlated with the hypoxia time. Beclin1protein expression was nosignificant ratio differences of gray values in the normoxic group (P＞0.05). The grayvalue ratio of Beclin1/β-actin were difference between M2-type macrophage cultured aloneat hypoxia condition (24h or48h) and co-cultured (24h or48h) in hypoxia condition were0.4385±2.04%、0.6451±3.96%、0.6871±2.21%、0.8235±7.70%compared with controlgroup(24h or48h) was0.2876±0.67%、0.2856±1.42%（P＜0.05）and positively correlatedwith the hypoxia time.Conclusion:1. THP-1cells can successfully induced differentiate into M2-typemacrophages.2. M2macrophages’ autophagic level was significantly upregulated underhypoxic conditions.3. IL-1β secretion significantly down when M2macrophagesco-cultured with colon cancer cells under hypoxic conditions. There’s no significant effecton the secreting of IL-12and TNF-α.