M1/M2 Macrophages Play Different Roles In Adipogenic Differentiation Of PDGFR_?~+ Preadipocytes

Background:Free fat transplantation is frequently used in plastic surgery,but its application is limited by complications such as cysts,nodules and low volume retention rate.Adipocyte regeneration after fat transplantation is the key to maintain fat volume retention.In adipose tissue,the main source of mature adipocyte regeneration is PDGFR?+preadipocyte.Therefore,the effect of adipogenic differentiation of PDGFR?+preadipocyte is very important for fat regeneration.Adipose tissue after fat transplantation is confronted with complex inflammatory microenvironment.Macrophages are an important part of inflammatory microenvironment.Macrophages play an important role in regulating inflammatory microenvironment after fat transplantation,and then affect the adipogenic differentiation of PDGFR?+preadipocytes.However,does M1 and M2 macrophages,different subtypes of macrophages,promote or inhibit the adipogenic differentiation of PDGFR?+preadipocytes?Are there any differences in their roles?At present,there is no systematic research and direct evidence.ObjectiveIn this study,we designed co-culture experiment of macrophage supernatant and preadipocyte,macrophage clearance experiment of autologous fat transplantation in mice,and tested the adipogenesis of preadipocytes or tissues and the expression of PPAR? and C/EBPa,which are related to adipogenesis.We aimed to study on the effects of M1/M2 macrophages on the adipogenic differentiation of PDGFR?+preadipocytes and to verify M1 and M2 macrophages promoted or inhibited the adipogenic differentiation of PDGFR?+preadipocytes respectively.This study is mainly carried out in the following aspects:1.To investigate the difference of inflammatory factors secreted by M1 and M2 macrophages and their physiological functions.2.To investigate the infiltration timing and proportion of M1 and M2 macrophages in adipose tissue after fat transplantation.3.To verify the effect of M1 and M2 macrophages on the adipogenic differentiation of PDGFR?c+preadipocytes.MethodIn vitro experiment1.Primary bone marrow cells of mice were isolated and cultured in vitro.Bone marrow cells were stimulated to differentiate into macrophages by granulocyte-macrophage colony stimulating factor(GM-CSF)and polarized into M1 and M2 macrophages by interferon-gamma(IFN-gamma),lipopolysaccharide(LPS)and interleukin-4(IL-4),respectively.The supernatants of Ml and M2 macrophages were collected and tested by ELISA.2.Mouse primary adipose stem cells were isolated and cultured in vitro.PDGFR?+preadipocytes were separated and amplified with non-adipogenic medium or adipogenic medium.At the same time,M1 or M 2 macrophage supernatants were added for co-culture.3.The co-cultured PDGFR?+preadipocytes were stained with Oil red O staining.Real-time PCR was used to test PPARy and C/EBP? gene expression of PDGFRa+preadipocytes,western blot was used to detect PPARy and C/EBP? protein expression of PDGFR?+preadipocytes after co-culture.In vivo experiments1.Mouse inguinal fat was transplanted into the back of the same mouse to establish an autologous fat transplantation model.2.Flow cytometry was used to test the proportion of M1 and M2 macrophages in grafts tissue at 0,4,11,13,16,25 and 30 days after operation.3.The autologous fat model of mice was established again.Gadolinium chloride(GdCl3)was injected into fat grafts on the back of mice to selectively clear M1 macrophages;Mannosylated clodronate liposome(m-Clodrosome(?))was injected into fat grafts on the back of mice to selectively clear M2 macrophages;the control groups were set up in both groups.The grafts were injected twice a week for 8 weeks.4.Perilipin immunohistochemical staining was used to evaluate the effect of fat formation.Real-time PCR was used to test the expression of PPARy and C/EBPo?genes in adipose tissue,western blot was used to test the expression of PPARy and C/EBP? proteins in adipose tissue.ResultsIn vitro experiment1.The contents of inflammatory factors in supernatants of Ml and M2 macrophages were significantly different.The contents of TNF-a,iNOS and IL-12 in supernatants of Ml macrophages were significantly higher than those of M2 macrophages,while the contents of anti-inflammatory and pro-growth factors(IL-10,TGF-beta 1)in supernatants of M2 macrophages were significantly higher than those of M1 macrophages.2.In non-adipogenic group,PDGFR?+preadipocytes' adipogenic differentiation were not affected by macrophages,while in adipogenic group,M1 macrophages could significantly inhibit the adipogenic differentiation of PDGFR?+preadipocytes.M2 macrophages did not show inhibition or promotion effect.In vivo experiments1.After autologous fat transplantation in mice,the proportion of M1 macrophages increased in the early stage and decreased in the late stage,while the proportion of M2 macrophages was at a low level in the early stage and increased gradually in the late stage.2.After selective removal of M1 macrophages in fat grafts,the adipogenesis of the grafts increased significantly,but there was no significant change after selective removal of M 2 macrophages in the grafts.Conclusion1.M1 and M2 macrophages secrete different inflammatory factors and play different physiological functions.M1 macrophages mainly secrete pro-inflammatory factors,while M2 macrophages mainly secrete anti-inflammatory and pro-growth factors.2.In the early stage after fat transplantation,M1-type macrophages predominated,while M2-type macrophages gradually increased in the later stage.3.Macrophages can directly affect the adipogenic differentiation of preadipocytes,and M1/M2 macrophages play different roles:M1 macrophages can significantly inhibit the adipogenic differentiation of preadipocytes,while M2 macrophages have no significant effect on the adipogenic differentiation of preadipocytes.