| OBJECTIVEBy studying the effects of human umbilical cord mesenchymal stem cell-derived exosomes(hucMSC-Exos)on the proliferation and migration of heat-injured human fibroblasts,we provide a theoretical basis for hucMSC-Exos repair of burn wounds and new ideas and protocols for the clinical treatment of burn wounds.METHOD1.Umbilical cord mesenchymal stem cells(hucMSCs)were isolated from fresh umbilical cord tissue of healthy newborns and morphologically characterized as well as observed for the expression of surface markers CD34,CD45,CD29,CD90.2.hucMSC-Exos were obtained by PEG precipitation(polymer precipitation)combined with differential centrifugation.hucMSC-Exos morphology was observed by transmission electron microscopy.hucMSC-Exos size and concentration were analyzed by particle size analysis instrument.hucMSC-Exos surface specific markers CD63 and CD81 were detected by westernblot.3.The discarded scar tissues of patients who underwent scarring and skin grafting in Baogang Hospital,Inner Mongolia were obtained,and human dermal fibroblasts were isolated and cultured to observe their cell morphology and waveform protein expression.4.Fibroblasts in the logarithmic phase of growth were treated with heat damage,and a heat damage model was established,and CCK-8,cell scoring and Transwell assays were performed in turn to compare with normal fibroblasts and evaluate their proliferation and migration ability.5.The heat-damaged fibroblasts were divided into three groups,and different doses of hucMSC-Exos solution(0μg,50μg,100μg)were added,and CCK-8,cell scratch and Transwell experiments were performed sequentially to determine the effects of hucMSC-Exos on the proliferation and migration of heat-damaged fibroblasts.RESULTS1.After 14 d of hucMSCs tissue block culture,a large number of cells were seen growing from the tissue block,adhering to the bottle wall and growing;after successive passages to the 3rd generation,the cells were shuttle-shaped with uniform morphology and fish swarm-like arrangement.The extracted cell surface markers were positive for CD29 and CD90,while negative for CD34 and CD45,and the results were in accordance with the description of stem cell surface-specific marker characteristics by the International Society for Cellular Therapy.2.hucMSC-Exos vesicle-like structures with a bilayer-like circular morphology were visible under transmission electron microscopy.The diameters of the obtained hucMSC-Exos were mostly concentrated around 110 nm,which was consistent with the size of hucMSC-Exos;the concentration of hucMSC-Exos was 3.2×1010(Particles/m L).The extracted hucMSC-Exos were enriched with transmembrane proteins CD63 and CD81,which were consistent with the characteristics of hucMSC-Exos surface markers.3.Fibroblasts were subjected to passaging culture,and cells were seen to grow adhering to the culture flask wall with a shuttle-shaped cell morphology and swirling arrangement of cell clusters.Immunochemical staining of them showed a uniform brownish-yellow cytoplasm,i.e.positive expression of waveform proteins.4.After the fibroblasts were treated with thermal damage,their proliferative capacity was lower than that of normal fibroblasts at each observed time point(24h,48 h,72h),which was statistically different(P<0.05);the 24 h scratch healing rate of fibroblasts treated with thermal damage was(22.70±2.93)%,which was lower than that of normal fibroblasts(62.00±2.71)%,and there was a statistical difference between the two groups There was a statistical difference between the data(P<0.05);the number of normal fibroblasts passing through Transwell pores at 24 h was(70.60±5.96)and the number of fibroblasts passing through Transwell pores in the heat-damaged treated fibroblasts was(21.40±4.70),which showed that the migration ability of fibroblasts after heat-damaged treatment was significantly reduced(P<0.05).5.The effect of different doses of hucMSC-Exos(0 μg,50 μg,and 100 μg)on the proliferation of heat-injured fibroblasts was detected using the CCK-8 assay,and the results showed that at 24 h and 48 h,compared with 0 μg hucMSC-Exos,50 μg and 100 μg hucMSC-Exos could promote the proliferation of heat-injured fibroblasts,and the difference was statistically significant(P<0.05),while there was no significant difference between the two groups of 50 μg and 100 μg hucMSC-Exos;at 72 h,compared with 0 μg hucMSC-Exos,50 μg and 100 μg hucMSC-Exos could promote the proliferation of heat-injured fibroblasts,and the difference was statistically significant(P<0.05),and there was also a significant difference between the two groups of 50 μg and 100 μg hucMSC-Exos(P<0.05).The effect of different doses of hucMSC-Exos(0 μg,50 μg,100 μg)on the planar spatial migration of thermally injured fibroblasts was tested using a cell scratch assay,and the results showed that the scratch healing was(23.21±8.25)% in the 0 μg hucMSC-Exos group;(42.86±6.54)% in the 50 μg hucMSC-Exos group;and(55.42±8.53)% in the 100 μg hucMSC-Exos group.Both 50 μg and 100 μg hucMSC-Exos promoted the scratch healing ability of heat-injured fibroblasts,and the scratch healing ability of the 100 μg hucMSC-Exos group was stronger than that of the 50 μg hucMSC-Exos group,and the results were statistically different(P<0.05).The effect of different doses of hucMSC-Exos(0 μg,50 μg,and 100 μg)on the stereospatial migration of cells was examined using a transwell assay,and the results showed that the number of cell migration was(19.2±2.91)in the 0 μg hucMSC-Exos group;(31.47±4.31)in the 50 μg hucMSC-Exos group;and(39.53±3.11)in the 100 μg hucMSC-Exos group.The number of thermally injured fibroblast migration in the 50 μg and 100 μg hucMSC-Exos groups was significantly increased compared with the 0 μg hucMSC-Exos group,and the number of cell migration in the 100 μg hucMSC-Exos group was the highest,with a statistically significant difference(P<0.05).CONCLUSION1.hucMSC-Exos meeting the conditions of this experiment could be obtained by using PEG precipitation(polymer precipitation)combined with differential centrifugation.2.The thermal injury model established at 52℃ and 30 s is a better model for thermal injury of human fibroblasts in vitro.3.hucMSC-Exos can promote the proliferation and migration of heat-injured human fibroblasts. |
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