关键词: HepLPCs, BMDM, M1型巨噬细胞, M2型巨噬细胞, 极化

Abstract: Objective To investigate the effects of human hepatocellular derived liver precursor like cells (HEPLPCS) supernatant on macrophage subsets M1 and M2, respectively.Methods The primary liver cell line was purchased from Ziride liver, and the hepatocyte amplification and culture system (TEM) developed by the previous team was used to amplify and transdifferentiate the primary liver cells into liver precursor like cells (HEPLPCS) in vitro. During the culture process, the supernatant of the HEPLPCS was collected, and after centrifugation, the cells were frozen and stored for later use. Macrophages were derived from bone marrow of C57 mice (BMDM). After erythrocyte lysis treatment, mice M-CSF (40 ng/mL) was added for culture, and stimulated and induced for 7 days. Adherent macrophages were obtained for the experiment. Macrophages in the resting state were stimulated by LPS (100 ng/mL) to produce m1-type macrophages in the inflammatory state and IL-4(40 ng/mL) to produce M2-type macrophages with anti-inflammatory and tissue repair functions. On the basis of the directional induced macrophage polarization, the culture supernatant HepLPCs conditions with different polarization states of macrophages to jointly develop, collect the culture supernatant of different time points, cell RNA and protein, by flow cytometry quantitative PCR ELISA methods such as comprehensive evaluation HepLPCs culture supernatant on macrophages, the influence of different subgroup.Results M0 macrophages were obtained by inducing BMDM adherent by GM-CSF stimulation in mice. The purity of macrophages was identified by flow cytometry, and F4/80+ (total macrophage marker) expression accounted for 92.8%. Directionally induced macrophage polarization: LPS and IL-4 were used to stimulate the cells for six h, respectively, and the cells were harvested for flow test and qPCR test. F4/80+,CD11c+ expression accounted for 88.1%. F4/80+ , CD206+ expression accounted for 97.0%. After 6 hours of LPS stimulation, the expression of M1-related inflammatory genes were upregulated: IL6, IL1β, and iNOS were upregulated by 823.200±174.500 times, 8.389±0.029 times, and 24.650±1.196 times. After 6 hours of IL-4 stimulation, M2-related gene expression increased: CD206 expression was upregulated 114.000± 3.579 times, IL10 expression was upregulated 2.634±0.028 times, and Arg1 expression was upregulated 53.260±8.083 times.M1 type and M2 type macrophages were successfully induced. On this basis, the expression of polarization related genes in M1-type macrophages was down-regulated after co-culture with HEPLPCS-CM: IL6 expression was 346.300±20.810, IL1β expression was 11.290±0.10, iNOS expression was 169.800±9.711.The levels of IL6, IL1β and iNOS were 138.700±32.130pg/ (mL×10⁵ cell), 0.710±0.019pg/ (mL×10⁵ cell) and 0.095±0.001pg/ (mL×10⁵ cell) respectively.The expression of polarization-related genes in M2 macrophages was upregulated: CD206 expression was 40.88±0.1768, IL10 expression was 22.2±0.1414, and Arg1 expression was 32.25±0.2121.IL10 secretion increased to 108.052±0.472 pg/ (mL×10⁵ cell).Conclusion The conditional medium of HEPLPCS can inhibit the LPS-induced inflammatory activation of macrophages, significantly reduce the expression of genes and secreted proteins of inflammation-related factors, and promote the expression of repair type M2 macrophages induced by IL4 and increase the secretion of anti-inflammatory factor IL-10.

Key words: HeplPCs, BMDM, M1 macrophages, M2 macrophages, Polarization