| Objective: To investigate the effect of human amniotic mesenchymal stem cells( human amniotic mesenchymal stem cells, h AMSCs) on HA and collagen expression in ECM mice and improve the mechanism of h AMSCs in promoting wound healing, and provide basic research for the transformation of h AMSCs into clinical application..Methods: HAMSCs was extracted from the three to five cells, and the cell surface molecular markers were analyzed by flow cytometry and the differentiation and Induction of differentiation of osteoblasts and osteoblasts in order to identify h AMSCs. 96 C57BL/6 mice were divided into three groups, the normal group(normal mice), the control group(DMEM) and the experimental group(h AMSCs), 32 Animals in each group. Control group mice back on both sides of the spine to establish full-thickness skin defect model(each 2, 64). DMEM culture medium 100 ul was used in 64 wounds around the wound surface. Experimental mice back on both sides of the spine full-thickness skin defect model was established with the method of wound after subcutaneous ring around h AMSCs(1 x 106 cell / ml) 100 u L, not making the skin wounds in normal mice as normal controls. In modeling after one day,three days, seven days and fourteen days the wound model skin specimens were cut off. Records in the control group and experimental group was the gross appearance of a wound healing process, and through the cell tracer experiment observation h AMSCs survival after transplantation, collagen accumulation Masson staining observation,immunohistochemical staining after observing wound skin tissue positive expression in high molecular weight HA, and q PCR technology high molecular weight HA wound skin tissue, gene expression in I and III. Result Human amniotic mesenchymal stem cell growth curve showed that the cell growth of Qu Xiancheng "S" shape. FCM results showed that: the fourth generation h AMSCs were highly expressed CD105,CD90, CD73, the positive rate was 96.6%, 95.1%, 98.1%, low expression or not to express CD34, CD11 b, CD19, CD45, HLA-DR, the negative rate was 0.3%. Induced to differentiate into adipose cells culture for 21 days seasonal oil red O staining,visible lipid droplets at a red; osteogenic induction culture 21 d Alizarin Red S staining,visible red calcified nodules. Mice of the experimental group in the dorsal wound 3, 7,14 d three time points healing rates were 33.93±2.71, 75.17±3.69, 98.1±1.66 is obviously better than control group 27.29±3.41,62.37±5.24,95.46±2.56(P < 0.05)and scar healing than other groups are smaller and softer. Through cell tracer prove local transplantation h AMSCs after 14 d modeling is still alive. Immunohistochemical staining detection experimental high molecular weight HA expression quantity, high molecular weight HA respectively at 1, 3, 7, 14 d expression was 40.321±2.348,49.463±1.976,45.463±2.498,40.463±2.341, the expression quantity showed a trend of increasing before reducing, amount to 3 d maximum expression, then reduce the changes. The expression of high molecular weight HA in the control group respectively at 1, 3, 7, 14 d expression was37.463±2.243, 43.463±2.082,36.463±1.932,30.463±2.987. The expression level of the control group was the same as that of the experimental group. The normal group of high molecular weight HA expression volume in the range of 27.463±2.671 in order to spend extra time, each time point were less than the experimental group and control group, and experimental group per time point were greater than in the control group(P< 0.05), the differences were statistically significant.. q PCR results from the study showed that the high molecular weight HAm RNA expression in 1, 3, 7, 14 d were 0.587±0.046,0.854±0.176, 0.679±0.033, 0.524±0.066, the control group was 0.277±0.033,0.376±0.034, 0.261±0.046, 0.234±0.024, the normal group was 0.176±0.076,Experimental group of type III collagen m RNA expression in 1, 3, 7, 14 d respectively were 0.215±0.014,0.218±0.026,0.225±0.006,0.216±0.048 the control group were0.215±0.014, 0.218±0.026, 0.225±0.006, 0.216±0.048, the normal group was0.151±0.012, The experimental group of type I collagen m RNA expression in 1, 3, 7,14 d were 0.251±0.028,0.316±0.045,0.322±0.011,0.298±0.021, the control grouwere 0.505±0.007,0.547±0.079,0.682±0.195,0.719±0.133, the normal group was0.146±0.003, the expression pattern and form echoing with the immunohistochemical results of phase(P< 0.05), and type I/III collagen ratio in 1, 3, 7 and 14 d in the experimental group were 1.171±0.046,1.469±0.176,1.434±0.033,1.430±0.066, the control group were 2.926±0.333, 2.800±0.034, 3.481±0.046, 3.602±0.024. The normal group were 0.971±0.076, the ratio of control group was higher than the experimental group and the normal group, the experimental group was higher han the normal group(P< 0.05), the differences were statistically significant. Masson staining of collagen stained blue. The normal group as normal skin tissue, the collagen reticulation, collagen gaps larger; 1d, 3d experimental group compared with the control group collagen closely spaced, collagen relatively thicker, interstitial collagen are relatively small, stained darker; control group collagen slender, arranged in disorder, interstitial collagen increases, with pale staining; 7d, 14 d in experimental group compared with the control groups of collagen are arranged in a reticular structure, gap between the collagen is increased, collagen relatively thinner, control group disorder of collagen fibers was swirling tight collagen was thick, interstitial collagen arrangement.Conclusion: 1.HAMSCs can regulate the extracellular matrix components, raised high molecular weight HA, type III collagen, cut type I collagen, reduce the proportion of type I/III collagen.2. Local injections of h AMSCs in mice back skin defect can promote healing of biological effects. |
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