| Objective:Hepatocellular(HCC)is one of the major cancers in the world,and there are a large number of cancer patients in China.Because the early symptoms of liver cancer patients are not obvious,many of them have been found in the middle and late stages,and tumor metastasis has appeared in many parts of the body.It is not suitable for surgery.Chemotherapy is a common method of use,but the side effects of chemotherapy are relatively large and some patients are intolerant,which makes the treatment of patients with advanced liver cancer extremely limited.Despite the continuous improvement of treatment methods and the emergence of immunotherapy,the prognosis of liver cancer patients is still not optimistic.According to statistics,the five-year survival rate of liver cancer patients is only 10.8%,and most patients die from tumor metastasis and complications caused by tumor metastasis.The metastasis of liver cancer is a complex process that is affected by many factors.In addition,it has been reported in the literature that macrophages pass through the blood circulation of monocytes in the bone marrow to various tissues and organs of the body.It differentiates into mature macrophage M1 and macrophage M2 in different microenvironments.Different types of macrophages play different roles in the body’s immunity.M1 macrophages can kill tumors and reduce immunity.M2 macrophages can promote tumorigenesis and metastasis.When M2 macrophages increase after tumor patients,postoperative expectation is often poor.Early research by this group found that polyamines are closely related to the growth and metastasis of tumor cells.When the content of polyamines in tumor patients increases,patients are more likely to have tumor metastasis.So we wonder that spermidine may have an effect on the polarization of macrophages? Can it promote the polarization of macrophages into M2-type cells and promote the growth and metastasis of tumors? How does spermidine affect HCC metastasis through macrophages? The resolution of these problems may provide a new idea for tumor immunotherapy and new targets for the treatment of liver cancer patients.Methods:The promotion In vivo experimentsIn this study,human THP-1 cells were used to construct macrophage M1 and M2 models in vitro.The following experimental methods were used to verify the success of establishing the macrophage polarization model.1.Observe the morphological changes of induced macrophages M1 and M2 under the microscope2.The expression levels of CCR7,a protein marker of M1-type macrophages,and CD206,a protein marker of M2-type macrophages were detected by flow cytometry at the protein level.3.Real-time PCR was performed at the mRNA level to detect the relative expression levels of M1-type macrophage molecular markers iNOS,TNF-α,M2-type macrophage molecular markers MRC-1 and CCL-18.Spermidine was added in vivo when macrophages were induced to add cytokines,spermidine was added at the same time,and the polarization effect of spermidine on macrophages M1 and M2 was verified by using the established macrophage model experiment method.Then macrophages and liver cancer cells were co-cultured,and the effects of M1-type and M2-type macrophages on the migration of liver cancer cells were detected by wound healing experiments.Then,the effect of macrophages on the migration of HCC cells after the addition of spermidine was detected by using Trabswell to co-culture the macrophages with HCC cells.Then,the functional type of macrophages was studied,and the secretion factor IL-1β of M2-type macrophages was measured by ELisa.We hypothesized that IL-1β could promote the proliferation,migration and invasion of hepatocellular carcinoma cells and investigate the related mechanisms.In terms of proliferation,monoclonal assay and MTT assay were used to detect whether IL-1β promotes the proliferation of HCC cells.In terms of migration,we verified from the perspective of cell behavior,using the wound healing experiment to detect the effect of IL-1β on the transverse migration of liver cancer cells and the Trabswell experiment to detect the effect on the longitudinal migration of liver cancer cells.In terms of invasion,this study used a small matrix gel test to verify the effect of IL-1β on invasion of liver cancer cells.In terms of mechanism,we used western blot assay to explore the changes of proteins related to liver cancer cell metastasis.In vivoMouse lung metastasis model was established using mouse H22 liver cancer cells to evaluate the effect of spermidine on macrophage polarization and macrophage on tumor.Results:1.Cell morphology was observed under the microscope.The M1 type macrophages were long spindle shaped with significantly increased volume,while the M2 type macrophages were irregular oval shaped with significantly increased volume.2.flow cytometry experiments showed that CCR7,the protein marker of M1-type macrophages,increased significantly and the peak shifted to the right.CD206,a protein marker of M2-type macrophages,increased significantly and its peak shifted to the right.3.real-time fluorescence quantitative PCR detection showed that the expression of M1-type macrophage-related mRNA iNOS,TNF-α and M2-type macrophage-related mRNA CCL-18,MRC-1 mRNA were significantly increased..4.Flow cytometry and real-time fluorescence quantitative PCR experiments showed that spermidine inhibited the differentiation of macrophages into M1-type macrophages and promoted the differentiation of macrophages into M2-type macrophages.5.Macrophages and liver cancer cells were co-cultured.The wound healing experiment showed that M1-type macrophages inhibited the migration of liver cancer cells,while M2-type macrophages promoted the migration of liver cancer cells.6.We added spermidine and used small test to construct a co-culture microenvironment of macrophages,polyamines and liver cancer cells.The experimental results showed that M1-type macrophages inhibited the migration of liver cancer cells,while M2-type macrophages promoted the migration of liver cancer cells.7.The detection of IL-1β in macrophage exosomes by Elisa showed that spermidine could promote the secretion of IL-1β by M2-type macrophages.8.Monoclonal assay showed that IL-1β promoted the proliferation of HCC cells9.wound-healing assay and Transwell assay showed that IL-1β promoted the migration of HCC cells from both transverse and longitudinal migration,respectively.10.Transwell matrigid assay showed that IL-1β promoted the invasion of hepatocellular carcinoma cells.11.Immunoblotting experiments on 13 proteins showed that IL-1β could induce EMT transformation in HCC cells by AKT pathway,up-regulating p-AKT,Ecation protein and down-regulating n-cation protein.12.The lung metastasis model of H22 mouse liver cancer showed that spermidine up-regulate ARG-1 protein,a marker of M2-type macrophages,and down-regulate iNOS,a marker of M1-type macrophages,And therefore promote the metastasis of liver cancer cells.Conclusions:1.Spermidine can inhibit the polarization of macrophages toward M1-type,promote the polarization of macrophages toward M2-type,and reduce the immune function of the body.2.Spermidine can promote the secretion of cytokine IL-1β by M2-type macrophages,thus promoting the proliferation,migration and invasion of liver cancer cells.3.In vivo experiments on mice showed that Spermidine can promote the polarization of macrophages from M1-type to M2-type. |
PDF Full Text Request