Objective: To explore the effects of macrophages with high expression of TL1A on the activation and proliferation of HSCs in vitro. Methods: The Bone marrow-derived macrophages (BMMs) and peritoneal macrophages (PMs) from wild type (WT) and myeloid-overexpressed TL1A transgenic mice were isolated, differentiated and activated. HSCs were harvested from activated macrophages culture supernatant (CM). HSCs were detected by immunofluorescence and real-time Q-PCR. And the proliferation was detected by CCK-8 and BrdU assay kit. The levels of IL-1β and PDGF-BB in macrophage culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Results: BMMs-derived CM-intervention HSCs were used to detect the expression of α-smooth muscle actin (α-SMA) on the 2nd, 4th and 6th day respectively by immunofluorescence method. There was no significant difference between the two groups on the 2 nd and the 6th day, P > 0.05; On day 4, the CM/Tg group was significantly higher than that of CM/WT group, P < 0.01; the results of CMs derived from PMs were consistent with the above trend. The expression of α-SMA mRNA on the 2nd, 4th and 6th day was detected by real-time Q-PCR method using BM-derived CMs. No significant difference was found between the groups on the 2nd day (P > 0.05).α-SMA mRNA increased further on the 4th and 6th day, and the level of CM/Tg in CM/Tg group was significantly higher than that in CM/WT group (P < 0.05). The detection results of CMs derived from PMs were consistent with the above trend. The results of CCK-8 assay and BrdU assay showed that the proliferation rate of HSCs in CM Tg group was significantly higher than that in CM/WT group (P < 0.01). The CMs derived from PMs were used to interfere with HSCs. And the results were consistent with the above trend. For BMMs, the levels of IL-1β and PDGF-BB in the lipopolysaccharide (LPS) + IFNγ/Tg culture supernatant were significantly higher than those in the LPS+IFNγ/WT group (P < 0.01). For the culture supernatants of PMs Liquid test results consistent with the above trend. Conclusion: Macrophages with high expression of TL1A could enhance the activation and proliferation of HSCs by increasing the secretion of IL-1β and PDGF-BB.
目的： 探讨体外高表达肿瘤坏死样因子配体1A（TL1A）的巨噬细胞对肝星状细胞（HSCs）的活化、增殖的影响。 方法： 分离、分化并活化C57BL/6野生型（WT）与髓系高表达TL1A的转基因（M-Tg）小鼠骨髓来源的巨噬细胞（BMMs）和腹腔巨噬细胞（PMs）；并分离WT小鼠原代HSCs；收取活化后的巨噬细胞培养上清液为条件培养基（CM）干预HSCs，应用免疫荧光法及real-time Q-PCR方法检测HSCs的活化、CCK-8法及BrdU法检测HSCs增殖情况，酶联免疫吸附试验法测定各组巨噬细胞培养上清液中白细胞介素1β和血小板衍生生长因子（PDGF）-BB的含量。 结果： 应用BMMs来源的CM干预HSCs，免疫荧光法分别检测第2、4、6天α-平滑肌肌动蛋白(α-SMA)表达量，第2天和第6天时各组间差异无统计学意义，P > 0.05；第4天时CM/Tg组明显高于CM/WT组，P < 0.01；PMs来源的CM干预HSCs的检测结果与上述趋势一致。应用BMMs来源的CM干预HSCs，real-time Q-PCR法分别检测第2、4、6天α-SMA mRNA表达量，第2天时各组差异无统计学意义，P > 0.05；第4、6天时α-SMA mRNA进一步升高，且CM/Tg组明显高于CM/WT组，P < 0.05；PMs来源的CM培养HSCs的检测结果与上述趋势一致。CCK-8法和BrdU法检测HSCs增殖速度结果显示CM/Tg组明显高于CM/WT组，P < 0.01；应用PMs来源的CM干预HSCs，检测结果与上述趋势一致。对于BMMs、脂多糖+干扰素γ/Tg组培养上清液中白细胞介素1β和PDGF-BB表达水平显著高于脂多糖+干扰素γ/WT组，P < 0.01；PMs的培养上清液检测结果与上述趋势一致。 结论： 高表达TL1A的巨噬细胞可能通过IL-1β和PDGF-BB分泌增加促进HSCs的活化与增殖。.
Keywords: Hepatic stellate cells; Interleukin-1β; Macrophages; Platelet-derived growth factor-BB; Tumor necrosis factor-like ligand 1 aberrance.