| Mesenchymal stromal/stem cells(MSCs)are a type of pluripotent stem cells existed in different tissues such as placenta,bone marrow,umbilical cords and adipose tissue,which having the characteristic of vitro amplification,strong proliferation ability,low immunogenicity.Recent years,they have been widely used in the basic and clinical experimental study of various diseases including hepatocellular carcinoma.Tumor microenvironment(TME),mainly composed of immune cell subsets,cancer cells,cytokines,contributes to the development of hepatocellular carcinoma.Tumor cells suppress the activation of immune cells through a variety of mechanisms to evade the host autoimmune response.Therefore,improving the immune microenvironment of hepatocellular carcinoma(HCC)will provide a new breakthrough for clinical treatment of HCC.Myeloid-derived suppressor cells(MDSCs),a myeloid-derived cell subgroup with immunosuppressive function,are closely associated with the development of tumors.However,it is whether that MSCs regulating MDSCs in hepatocellular carcinoma?And the related mechanisms are unclear.The above issues need to be further characterized.PurposeBased on the above issues,this study aims to investigate the effects of MSCs treatment on MDSCs,lymphocyte subpopulations,macrophage polarization,and plasma related cytokine levels in liver cancer bearing mice,and explore the immune mechanisms of MSCs treatment for HCC.MethodsThe mouse hepatocellular carcinoma model was constructed,and the mouse bone marrow MSCs were isolated,cultured and identified.Then the MSCs were injected into the caudal vein to the hepatocellular carcinoma bearing mouse model.The model mice were randomly divided into high-dose MSCs group,low-dose MSCs group and control group.The changes of MDSCs in liver cancer were detected by immunohistochemistry.The changes of M1/M2 ratio of CD11b~+MDSCs,CD3~+T cells,CD19~+B cell,NKp46~+NK cell,CD4~+CD25~+Foxp3~+T cell subsets,CD86~+and CD206~+macrophages were examined by flow cytometry.The changes of IL-1,IL-4,IL-6 and IFN-γin plasma were detected by liquid chip instrument cytokine content.Results(1)After isolation,culture and purification,mouse bone marrow mesenchymal stem cells were identified by flow cytometry and induced differentiation.The surface markers CD34,CD11b and CD45 of mouse P3 bone marrow mesenchymal stem cells were negative;CD90,CD54 and CD44 were positive,and had the ability of adipogenic,osteogenic and chondrogenic differentiation after induction.(2)After inoculation of H22 tumor,the mass can be touched in vitro.As time continuation of the mass,there was no abdominal water formation,and the maximum diameter of the tumor was 0.79±0.12 cm,and the tumor rate was 100%,the in situ model of mouse liver cancer was successfully established.Mouse bone marrow mesenchymal stem cells were cultured to the 5th generation of cells,and evenly distributed,and the state was good.(3)The brown photographic value of CD11b~+MDSCs was 6157.80±758.79 in theliver of the control group,1926.68±1152.53 in the low dose MSCs group,and1548.20±865.12 in the high dose MSCs group.Among them,the optical density value of negative control group was obviously higher than that of the low dose and the high dose MSCs group(P<0.01),and there was no obviously significant difference between the low dose and the high dose MSCs group.(4)The mouse whole blood CD11b~+MDSCs of control group,low dose MSCs group,high dose MSCs group detected by flow cytometry respectively accounted for23.14±2.06,16.28±2.67 and 13.76±2.13.The serum CD11b~+MDSCs levels of the high doses MSCs group was obviously reduced compared with that of control group and low dose MSCs group(P<0.01).The serum CD4~+CD25~+FOXP3~+-Treg cells respectively accounted for 2.28±0.50,1.36±0.34,0.93±0.28,in whole blood of control group,low dose and high dose MSCs group mice.Compared with that of the control group,the serum Tregs levels of high-dose MSCs group were significantly decreased(P<0.01).After MSCs treatment,the MDSCs of the peripheral blood in the hepatocellular carcinoma mice consistent with Tregs lymphocyte proportional trend.(5)The proportion of CD3e~+cells in peripheral blood lymphocytes of the control group,low-dose MSCs group and high-dose MSCs group was 67.39±4.94,56.79±4.07 and 76.36±6.77 respectively.The significant difference existed in the control and the high-dose MSCs group(P<0.05).The ratio of CD19~+cells accounted as 17.16±3.13,6.77±2.94,13.15±5.61,and there were no obviously significant differences in the above three groups.The ratio of NKp46~+cells accounted as4.31±2.01,5.56±0.91,10.49±4.35 respectively,in which the difference between control and high doses MSCs group was significant(P<0.05).(6)The proportion of CD86,CD11b and CD206 cells in macrophages was examined by flow cytometry.And the results showed that the proportion of M1 in high-dose MSCs group was obviously higher than that in the control group(P<0.05).(7)The serum IL-1,IL-4,IL-6 and IFN-γwere detected by Luminex liquid chip instrument.And the results displayed that the serum IFN-γlevel of the high-dose MSCs group was higher than that of the control and low-dose MSCs group(P<0.05),while there was no significant difference among other groups.ConclusionsMSCs treatment improve the immunity of tumor mouse bodies,which may be related to inhibiting MDSCs and Treg cells in lymphocytes.And different doses of MSCs may have a certain difference on the improvement of immune function in tumor bearing mice. |
PDF Full Text Request