| BackgroundMacrophages are widely distributed throughout various tissues and organs. They playan important role in keeping the internal environment stable, body defense, inflammationregulation, promoting wound repair, etc. Macrophage is considered terminal differentiationcell, but recent researchs show that it has certain self proliferation ability and differentiationpotential, which can be converted into lymphatic endothelial cells in the body. Furthermorein vitro they can be converted into vascular endothelial cells, smooth muscle cells andmuscle cells. In addition, under different microenvironment macrophages can be activatedinto different functional phenotypes. Interestingly, redifferentiation in appropriateconditions can also happen. So, we think macrophages have strong plasticity. Macrophagespresent different phenotypic characteristics under different microenvironment, making themin the body under different physiological and pathological conditions can play differentfunctions. These characteristics make macrophages closely related to a variety of diseasessuch as tumors, metabolic syndrome occurrence and development.Resting state of macrophages under different microenvironment can be activated intodifferent phenotypes of two types: inflammatory macrophage and anti-inflammatorymacrophages, also called M1and M2macrophages. M1cells induced by gamma-IFN orgamma–IFN-LPS can trigger type Th1immune response, playing a role in defensingbacteria infection, aggravating the inflammatory response, and eventually leading to tissuedamage. M2cells induced by IL4or IL4-IL13, although, due to the different activationconditions can also be subdivided into M2a, M2b, M2c three subgroup, main trigger Th2immune response, playing a role in defensing against parasitic infections, promoting tissuerepair and reducing or regulating the inflammatoryimmune response. Studies have found that the M1and M2macrophages under certainmicroenvironment can be respectively redifferentiation into M2-like or M1-like macrophages, having great plasticity. In atherosclerosis and Ⅱ diabetes animal model, M2macrophages can translate into M1-like macrophages in situ sample. While in tumor animalmodel, observed under the effect of exogenous IL-12M2-like phenotype of tumor cell canbe transformed into M1-lik.. In vitro experiments have confirmed that the M1and M2macrophages in respectively suitable for M2and M1differentiation under the condition ofincubation, the cells presented M1-like or M2-like characteristics.The redifferentiation of macrophage is closely related to many diseases occurrence anddevelopment. In Ⅱ type diabetes, normal adipose tissue macrophages is M2-like andsecrete arginase-1.But with the increase of the body obesity, they from M2-like to theM1-like phenotype transformation and secreted TNF alpha or other inflammatory factors,such as il-6, iNOS, etc, which interfere with the normal adipocyte insulin signaling pathway,and lead to insulin resistance and Ⅱdiabetes. Similarly, in the sclerosis of arterial congeeappearance, macrophage transform from M2to the M1-like characteristics which is closelyrelated to the progress of disease.In the occurrence and progress of tumor, influenced bytumor microenvironment the tumor associated macrophages transform from M1-like toM2-like phenotypes. which inhibits the body’s immune response, promotes tumorangiogenesis and tumor new matrix remodeling, eventually to promote tumor growth andmetastasis. Therefore, the research has the vital significance in polarization plasticitymechanism of macrophage. At the same time, this study can deepen our understanding ofthe mechanism of immune cells to adapt to different microenvironments, providing theimportant theoretical basis for the variety of the treatment of diseases.Method1. Mice bone-marrow derived macrophages were induced in vitro. Flow cytometry wasused to detect cell purity.2. RAW264.7macrophages differentiate into to M1, M2cells induced by LPS and IL-4respectively. Real time PCR was used to measure the two types of macrophages markergene and key transcription factors mRNA levels,such as TNF-α,IL-6,Arginase-1,Fizz-1,IL-1β,MCP-1,MR,CEBPδ,PPARγ,IRF4,IRF5.3. Mice primary bone-marrow derived macrophages were cultured in vitro, anddifferentiate into to M1, M2induced by LPS and IL-4respectively under the optimizedconditions. Real time PCR was used to measure the genes mRNA levels which were mentioned above.4. CCK-8kits was used to detect the effects of histone acetylation enzyme inhibitorsTSA and DNA methylation transferase inhibitors Aza on macrophage proliferation ability.Real time PCR was used to measure TNF-α,IL-6,Arginase,Fizz-1mRNA levels after TSAand Aza were added to the process of macrophages polarization induced by LPS and IL-4respectively.Results1. In this study, we employ the optimized condition to generat mice bonemarrow-derive macrophages.The FITC-F4/80positive rate of bone marrow-derivemacrophages reach98.6percent, in addition,the APC-CD11b positive rate reach98.2percent, double positive rate reached98.1percent.2. RAW264.7macrophage lineages are functionally polarized into M1and M2cells bystimulating macrophages with lpopolysaccharide and interleukin-4respectively.The mRNAexpression levels of TNF-α,IL-6reached the peak in M1macrophages after induction bylpopolysaccharide for8hours. Meanwhile, The mRNA expression levels of arginase,fizz-1reached its peak in M2macrophages after induction by interleukin-4for24hours.3. Bone marrow macrophages are fuctinally polarized into M1and M2under theoptimized conditions respectively, and show the significant function phenotypes of M1,M2;M1macrophage was able to redifferentiate to M2-like macrophage while M1macrophage cloud redifferentiate to M2-like macrophage when induced conditions in vitroenvironment changed.The mRNA levels of TNF-α,IL-6,arginase-1,fizz-1changedcorresponding to the redifferentiation.4.50nmol/L Trichostatin,1μmol/L5-Aza-CdR have the weakest effect on theproliferation of bone marrow-derive macrophage. Trichostatin A,5-Aza-CdR could enhancthe mRNA expression levels of TNF-α,IL-6,arginase-1and fizz-1in M1,M2primarymacrophages.Conclusions1. Stimulation of macrophage with lpopolysaccharide and interleukin-4make themacrophage to obtain diffirent functional phenotype under the condition of different timeinterval. Lpopolysaccharide induces the most significant M1-type macrophage generationby stimulating macrophages8h in vitro,in addition, interleukin-4is kown to generate the most significant M2macrophages by stimulating macrophages24h in vitro. Thepolarization of macrophage is a continuous process, M1and M2were only two extremestate.2. M1macrophage was able to redifferentiate to M2-like macrophage while M1macrophage cloud redifferentiate to M2-like macrophage when induced conditions in vitrochanged.The functional differntiation of macrophage showed the strong plasticity.3. Trichostatin A,5-Aza-CdR could enhanc the mRNA expression levels of M1/M2specific marker genes in macrophages respectively,including TNF-α,IL-6,arginase-1andfizz-1.Histone acetylation and DNA methylation may participate in the polarization of themacrophages. |
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