| Objective:To investigate the effect of IL-10-hAMSCs on the phenotype of macrophages in vitro and the effect of co-culture supernatants on the expression of fibroblast-related factors.In order to further study the use of IL-10-hAMSCs to regulate the wound inflammation and promote wound healing to provide preliminary data.Methods:(1)The primary hAMSCs were extracted by trypsin-collagenase two-step digestion method and cultured in DMEM-F12 medium containing 10% FBS.The hAMSCs were purified by differential adherence method.After flow cytometry,Vimentin immunofluorescence was used to identify hAMSCs.(2)Take P3-hAMSCs with good growth status,add the diluted virus liquid to the well plate according to the MOI = 30 measured in the pre-experiment,change the culture medium after 8-12 hours according to the cells state;Fluorescence was observed microscopically to assess transfection efficiency.The proliferation activity of hAMSCs in each group was tested by CCK8 kit and the growth curve was drawn.(3)The mouse skin fibroblasts were isolated and identified by vimentin immunofluorescence.Peritoneal lavage method was used to extract peritoneal macrophages of C57BL/6 wild mice and identified by the ink phagocytosis test.M1 macrophages were obtained after induction of IFN-γ and LPS transformation for 24-48 h.(4)The mouse peritoneal macrophages were co-cultured with hAMSCs.The experimental groups were as follows: group A: blank control group(medium supplemented alone)+ mouse peritoneal macrophage group;group B: hAMSC +mouse peritoneal macrophage group;group C: IL-10-hAMSCs + mouse peritoneal macrophages;group D: empty plasmid-hAMSCs + mouse peritoneal macrophages group;respectively cultured 0d,1d,3d supernatant was collected by ELISA Method to detect IL-12,Arg-1 and IL-10 content.At the same time,macrophages were collected at the corresponding time points,and the M1 / M2 polarization state of macrophages in each group was detected byusing CD16 / CD32(M1 macrophage surface molecule)and CD206(M2 macrophage surface molecule).(5)The culture supernatants of groups A,B,C and D were used to culture mouse skin fibroblasts.The proliferation of fibroblasts of groups A,B,C and D were detected by CCK8 kit and the growth curves were drawn.The cells were harvested at both time points of 3d and 5d respectively.The mRNA and protein expression of TGF-β1,type I collagen and type III collagen were detected by qPCR and Western blot.Results:(1)A large number of primary hAMSCs are obtained by trypsin-collagenase digesting method.A large number of high-purity hAMSCs are obtained by differential adherent method.The primary cells are diverse in morphology with many spindles,short fusiform or more Angular,disordered,fibrous growth.After passage subculture is spindle or spindle-shaped,spiral-shaped growth,with the directionality.Flow cytometry results of P3 generation amniotic mesenchymal stem cells(hAMSCs)indicate that CD90(96.88%),CD44(96.23%),CD73(99.54%)and CD105(98.58%)are positive.The results of flow cytometry analysis of IL-10-hAMSCs show that CD90(98.50%),CD44(95.20%),CD73(99.67%)and CD105(96.67%)are positive.The expression of CD34 + 11b+ 19 + 45 +HLA-DR of both cell lines is negative.Immunofluorescence indicated that Vimentin is highly expressed in hAMSCs but not CK19.(2)The lentivirus infect P3 generation of hAMSCs cells,observed by inverted fluorescence microscope after 72 h,IL-10 lentivirus and empty plasmid are infected with hAMSCs are expressed uniform green fluorescence,the virus infection efficiency can reach 90%.The growth curve of CCK8 method suggeste that the hAMSCs,empty plasmid-hAMSCs cells and IL-10-hAMSCs enter the logarithmic growth phase at 3~6days after a period of 1-2 days of slow growth,and enter the plateau phase on the 6th day.Curve showing "S" shape.After transfection,cell proliferation decrease slightly.(3)A large number of fibroblasts are successfully obtained by enzymatic digestion and grew rapidly in vitro.The primary cells showed short rod,long spindle,polygon and irregular shape.Vimentin immunofluorescence prompted the acquisition of higher purityfibroblasts.Macrophages are oval,short spindle and irregular morphology,ink phagocytosis test is positive phagocytosis percentage of up to 89%.(4)Flow cytometry analysis of hAMSCs and macrophages after co-culture showed that the M1-type surface markers are predominant in each group at day 0,while the percentages of M1-type cells in groups A,B,C and D are 98.20% and 99.20% 97.90%,97.20%.M1 is the main type of cells in each group.On the first day,the phenotype of each group starte to change.The percentage of M1 phenotype in groups A,B,C and D is95.20%,79%,64.20%,78.50%.Among them,the proportion of M1 cells in group C decrease most obviously.On the third day,the proportion of type M1 cells in group A,B,C and D were 90.70%,57.20%,11.20% and 55.80% respectively.The percentage of type M1 macrophages in group C is significantly lower than that on first day.88% transformed into M2 type macrophages,suggesting that IL-10-hAMSCs cells can effectively promote macrophage phenotype transformation and more efficient than simple hAMSCs and ordinary culture.(5)IL-12 levels in the co-culture supernatant of the control group at 0d,1d,and 3d are detected by ELISA assay: 474.840±2.978pg/mL,304.415±3.747pg/mL,325.938±12.229pg/ mL;The levels of IL-12 in the supernatant: 473.709±49.485 pg /mL,235.248±1.329pg/mL,183.395±11.268pg/mL;IL-12 in the IL-10 group:413.131±37.422pg/mL,199.429±0.319pg/m L and 138.084±1.491 pg/mL,respectively.The content of IL-12 in the empty plasmid group is 421.935±73.823 pg / mL,246.828±11.680pg/mL and 185.869±8.570pg/mL,IL-10content: 12.265±0.271 pg / m L,17.111±1.244 pg / mL,30.106±1.181 pg / mL;IL-10 content in the normal stem cell supernatant: 16.800±2.585 pg / m L,35.907±2.155 pg / mL.89.128±4.872 pg / m L;IL-10 content of IL-10 transfection group: 13.515± 0.716 pg / mL,77.906±1.849 pg / mL,258.012±5.784pg/mL;The contents of Arg-1 in the control group are0.730±0.060 ng / mL and 0.925±0.083 ng / mL,respectively,1.137±0.037 ng / mL;Arg-1contained in supernatant of normal stem cell group : 0.783±0.033 ng / mL,2.342±0.063 ng/ mL,4.671±0.094 ng / mL;Arg-1 contented of IL-10 transfecte group: 0.820±0.035 ng /mL,5.112±0.191 ng / mL,10.920±0.959 ng / mL.The contents of Arg-1 in supernatant ofempty plasmid group are 0.802±0.046ng/mL,2.588±0.372 ng / mL and 4.995±0.453 ng /mL,respectively.The content of Arg-1 and IL-10 in normal stem cell group,empty plasmid group and IL-10 transfection group is higher than that in control group on the 1 and 3 days,and the content of IL-10 Transfection group expression is particularly evident.The expression of IL-12 decreased gradually with the prolongation of co-culture time.Compared with control group,the content of IL-12 in normal stem cell group,empty plasmid group and IL-10 transfection group decrease significantly,and the decrease is more obvious in IL-10 transfection group Significance(P <0.05).(6)Fibroblasts with good growth status in each group are detected by CCK8 kit,the growth curve of four fibroblasts show "S" shape,and the proliferation rate of IL-10-hAMSCs cells is significantly faster than the other three groups,the proliferation rate of fibroblasts in stem cells and empty plasmid group is faster than that in control group,the difference is statistically significant(P <0.05).(7)qPCR detection of TGF-β1,type I collagen,type III collagen gene relativeβ-actin gene expression.Each group of supernatant cultured fibroblasts,with the extension of the culture time,TGF-β1,type I collagen,type III collagen mRNA expression increased gradually.At the same time compare with normal stem cells group,the empty plasmid group,IL-10 transfection group TGF-β1,type I collagen,type III collagen mRNA expression are higher than the control group,and IL-10 group compare with the normal stem cell group,empty plasmid group relative expression quantity is higher,the difference is statistically significant(P < 0.05).Through the Western blot test results show that: in the 3d and 5d,normal stem cells group,empty plasmid group,IL-10 transfection group TGF-β1,type I collagen,type III collagen expression increase with the prolongation of the culture time increased gradually,at the same time the normal stem cells group,empty plasmid group,IL-10 transfection group TGF-β1,I collagen,type III collagen expression are higher than the control group,IL-10 transfection group compare with normal stem cells group,empty plasmid group relative expression quantity is higher,the difference is statistically significant(P < 0.05).There is no significant difference between the common stem cell group and the empty plasmidgroup.Conclusion:1.IL-10-hAMSCs are capable of promoting the transformation of macrophages from pro-inflammatory phenotype M1 to tissue-repairing M2 in vitro as compared to hAMSCs.2.IL-10-hAMSCs and macrophages co-culture supernatant can promote the proliferation of fibroblasts and up-regulate the expression of TGF-β1,type I and type III collagen in fibroblasts,which is conducive to early wound healing. |
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