The Effects Of HAMSCs On The Phenotype Of Mice Macrophage And IL-10 Expression In Vitro And In Vivo

Objective: To investigate the effects of human amniotic mesenchymal stem cells on the phenotype of mice macrophage and IL-10 expression in vitro and in vivo, Prove about whether h AMSC has the function of control mice macrophage phenotype transformation and promote the IL-10 expression, complete the research on the mechanism of h AMSCs improving skin wound healing,provide the basic information for further clinical application.Methods: 1、 h AMSCs were separated with two steps of trypsin-collagenase digestion and differential sticking wall, cultivation with DMEM medium, extend to fourth generation, the OD values was measured by MTT assay and draw the cells growth curves of 1-7 days, using flow cytometry instrument identification and after h AMSCs were induced to differentiation of adipogenic and osteogenic cells the identification of them was made. 2、 C57BL/6 wild mouse peritoneal macrophages were extracted by peritoneal lavage. The cells were cultured in DMEM medium which containing INF-γ. 3、 The medium which containing INF –γ was removed after 5 days,Set up h AMSCs co-culture group and individual culture group,the h AMSCs co-culture group macrophages cultured in 2m L DMEM medium with 1 × 104 fourth generation of h AMSCs,individual culture group cultured in same medium alone. The content of IL-12(M1 macrophages secretion), Arg-I(M2 macrophages secreted) and IL-10 in supernatant were detected by ELISA on 1 day and 7 days. 4、 The skin on both sides of the spine in 56 healthy male C57BL/6 wild mouse was used to establish the model of full-layer skin defect, removed the hair and designed 10 mm x 10 mm size square.Set up experimental group and the control group, each group of 28. experimental group were injected with 100μL h AMSCs(1×106 cells/100μL) in the subcutaneous layer of the skin surrounding the each wound, control group mice injected with 100 μL DMEM medium surrounding the each wound.5、 The skin on both sides of the spine in 2 healthy male C57BL/6 wild mouse was used to establish the model of full-layer skin defect. The moment establishments were done, injected with 100μL h AMSCs(1×106 cells/100μL) which were CM-Dil marked annularly in the subcutaneous layer of the skin surrounding the wound. sliced the samples of skin wound model in 14 th days, and Survival of ADSCs after immunofluorescent staining were observed. 6、 Then we records of general appearances and calculation of healing rate of skin wound in the first、3rd、7th and 14 th days in both experimental and control group,while the changes in tissue structure after HE staining, the expression of IL-10 in the skin wound after q PCR, double-positive expression of CD68 and i NOS(M1 macrophage) and of CD68 and Arg-1(M2 macrophages) after immunofluorescent staining were observed.Results: 1、 A large number of stable proliferation of h AMSCs can be obtained,48 hours after primary vaccination h AMSCs were seen growing adherently. Growth curve of the fourth generation appeared to be S-shaped, which was accorded with Logistic growth curve. During the period of 3-5 days, it was in the exponential growth stage, after which it went into the platform stage and then cells stopped proliferating. Flow cytometry appraisal: CD73(98.1%), CD90(96.7%), CD105(95.1%), CD45、CD34、 CD11b、CD19、HLA- DR(0.5%).After 21 days since adipogenic induced cultivation oil red O staining was done and the lipid droplets were shown red. After 21 days since osteogenic induced cultivation and Alizarin red S staining, red calcified nodules were visible. 2、 Mice peritoneal macrophages were roundness 、oval、spindle or irregular in shape, many thorn-like protrusions extend on cell surface, and the ink particles with phagocytosis and phagocytic percentage was 85%. 3、 Detected by ELISA,The concentration of IL-12 in supernatant on fisrst day and 7th days were 43.36±1.93ng/L、22.22±1.47ng/L, The concentration of Arg-I in supernatant on fisrst day and 7th days were 5.48±1.38 U/L、12.52±0.46 U/L, the concentration of IL-10 in supernatant on fisrst day and 7th days were 65.35±5.99 ng/L 、220.69±7.11ng/L in the h AMSCs co-culture group;The concentration of IL-12 insupernatant on fisrst day and 7th days were 42.74±2.42ng/L、33.08±1.10ng/L,The concentration of Arg-I in supernatant on fisrst day and 7th days were 5.13±0.48 U/L、6.07±0.13 U/L,The concentration of IL-10 in supernatant on fisrst day and 7th days were 62.48±2.17 ng/L、75.54±7.50 ng/L in individual culture group. we observed that the concentration of IL-12 in the h AMSCs co-culture group supernatant were much lower than that of individual culture group on 7thday while the concentration of Arg-I 、IL-10 in the h AMSCs co-culture group supernatant were lower at the time point(P <0.05). 4、 The healing rate of skin defect on first、3th、7th、14th day were(1.23±0.39)%、(33.93±2.71)%、(75.17±3.69)%、(98.14±1.66)% in experimental group; and the healing rate of skin defect on first、3th、7th、14th day were(1.11±0.11)%、(27.29±3.41)%、(62.37±5.24)%(95.46±2.56)% in control group.Obviously, the healing rate of skin defect in experimental group was higher than that of control group(P <0.05). 5、 local transplantation of h AMSCs were still alive after 14 days since modeling. 6、 Images of tissue HE stained demonstrated that in the experimental group less inflammatory cell infiltration at the 3th、7th day than that in the control group. 7、 Immunofluorescence staining showed that double-positive expression of CD68 and i NOS on first、3th、7th、14th day were(36.84±5.52)%、(32.73±3.56)%、(18.17±1.33)%、(5.41±2.19)%,double-positive expression of CD68 and Arg-i double on first、3th、7th、14th day were(2.08±1.04)%、(30.18±4.13)%、(38.74±3.71)%、(21.01±1.75)% in experimental group. double-positive expression of CD68 and i NOS on first、3th、7th、14th day were(37.29±6.78)%、(43.97±2.98)%、(23.31±2.51)%、(6.51±1.28)%, double-positive expression of CD68 and Arg-i double on first、3th、7th、14th day were(1.59±1.54)%、(23.58±1.66)%、(31.06±2.61)%、(18.71±1.58)%in control group.In the images of tissue immunofluorescence staining we observed that the positive rate of M2 macrophage in the experimental group were much higher than that of control group on 3thday, 7thday while the positive rate of M1 macrophage expressed were lower at the different time point(P <0.05).8、 expression of IL-10 in the skin wound on first 、 3th 、 7th 、 14 th day were 0.0152±0.0025、0.2827±0.0133、0.0535±0.0054、0.0209±0.0055 in experimental group; expression of IL-10 in the skin wound on first 、 3th 、 7th 、 14 th day were 0.0127±0.0021、0.1208±0.0043、0.0326±0.0046 、0.0148±0.0021 in control group. expression of IL-10 in experimental group on 3thday, 7thday were distinctly higher than that of control group at each time(P<0.05).Conclusion: 1、 HAMSCs has the function of promoting the transformation of mice M1 macrophage to M2; 2、 HAMSCs transplantation for skin wound could improve the full-layer skin defect healing in mice.The mechanism might partly be about the adjustment of phenotypes in macrophages、increase expression of IL – 10 and inhibit inflammation in the wound by HAMSCs.