Human Amniotic Epithelial Cells Prevent RAW264.7Cells Converting Into M1Status Induced By LPS And Its Preliminary Mechanism Research

Objective To study the modulation effect of human amniotic epithelial cells (H-AEC), human amniotic mesenchymal cell (HA-MSC), umbilical mesenchymal cells (UC-MSC) on the M1status of RAW264.7cells induced by LPS, to discuss the difference of their role in preventing the inflammation status differentiation of the RAW264.7cells induced by LPS.MethodsIsolate and culture cells:Use T/E digestion process to get H-AEC from amniotic on human placenta. Use hyaluronidase, dispase and collagenase digestion process to get HA-MSC and UC-MSC.Group of experiments:(1) Groups of H-AEC prevent the M1status of RAW264.7cells induced by LPS:①Blank control group: RAW264.7cells were normal cultured in48hours;②Positive control group:After RAW264.7cells were normal cultured in48hours,100ng/ml LPS were used to induce RAW264.7cells4hours;③Interferential control group:2.5×105RAW264.7cells were co-cultured with5×105H-AEC48hours;④Experimental group:After2.5×105RAW264.7cells were co-cultured with5×105H-AEC48hours,100ng/ml LPS were used to induce RAW264.7cells4hours.(2) Groups of comparing H-AEC HA-MSC and UC-MSC preventing the M1status of RAW264.7cells induced by LPS:①Positive control group:After RAW264.7cells were normal cultured in48hours,100ng/ml LPS were used to induce RAW264.7cells4hours;②H-AEC group:After2.5×105RAW264.7cells were co-cultured with5×105H-AEC48hours,100ng/ml LPS were used to induce RAW264.7cells4hours;③HA-MSC group:After2.5×105RAW264.7cells were co-cultured with5×105HA-MSC48hours,100ng/ml LPS were used to induce RAW264.7cells4hours;④UC-MSC group:After2.5×115RAW264.7cells were co-cultured with5×105UC-MSC48hours,100ng/ml LPS were used to induce RAW264.7cells4hours.Detection methods:Cell wound scratch assay detects RAW264.7cells’ migration ability; NO assay kit detects the nitric oxide concentration in each group; RT-PCR detects M1genes and M2genes of RAW264.7; Western blotting detects cytoplasmic protein p-IκBa and nuclear protein NFκB in (1) groups; ELISA detects the secretion of TGF-β1.Results:(1) Experimental group’migration rate was14.7%±4.5%. Comparing to the positive control group (31.1%±11.0%), the difference was obvious (p<0.05).(2) H-AEC group nitric oxide concentration was24.26±0.72μM/L. Positive control group was45.65±1.78μM/L. The difference was obvious (p<0.05). HA-MSC and UC-MSC nitric oxide concentration were44.52±2.51μM/L and42.25±0.76μM/L (p>0.05vs positive control group).(3) The expression of M1genes and M2genes:(A) Group H-AEC M1genes such as IL-1β TNFa NOS-2and INFβwere down-regulated expression. Group H-AEC M2genes such as CD206CD36and Arg-1were up-regulated expression. The difference was obvious;(B) HA-MSC and UC-MSC groups M1gene INFβwas down-regulated expression and M2genes Arg-1CD206and CD36up-regulated expression. The difference was obvious.(4) Experimental group the expression of p-IκBa and NFκB were lower than Positive control group.(5) Experimental group lower chamber’TGF-β1secretion was more than up chamber and both were more than H-AEC TGF-β1secretion.Conclusions:H-AEC can deeply inhibit M1genes expression and deeply induce M2genes expression. The possible mechanism is H-AEC’ inhibiting effect on passage way NFκB. HAEC, HAMC, UMSC have the effect of preventing the RAW264.7differentiate into inflammatory status but the role and the mechanism might be different from one another.