Effect of rat adipose-derived mesenchymal stem cells on macrophages phenotype in skin wound / 中华创伤杂志

ABSTRACT

Objective To investigate the effect of local injection of rat adipose-derived mesenchymal stem cells (ADSCs)on the phenotype of macrophages in skin wound.Methods The cryopreserved primary SD rat ADSCs were resuscitated and then sub-cultured.ADSCs of the third generation were used in the experiments.Thirty six SD rats were divided into ADSCs group (n =18) and control group (n =18) by random numbers table method.The full thickness skin wounds were established on bothsides of the spine.After the model establishment 0.2 ml ADSCs suspension labeled by live cell stain Chloromethylbenzamido derivatives of 1,l'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (CM-Dil) with the concentration of 5 × 106/ml was subcutaneously annularly injected in the skin wound of SD rats in ADSCs group.The SD rats in control group were given 0.2 ml serum-free Dulbecco modified Eagle medium (DMEM).On 3,7,and 14 days after injury,six rats were selected from each group to measure the wound area and healing rate.The healed wound tissues were harvested to observe the morphology by HE staining.The expressions of interleukin (IL)-10 were detected by immunohistochemistry staining.The double-positive expressions of CD68 and inducible nitric oxide synthase (iNOS) (M1 type macrophages) and of CD68 and arginase-1 (Arg-1) (M2 type macrophages) were detected by immunofluorescent staining.The distribution of CM-Dil-labeled ADSCs in healed wound tissue 14 days after injury was observed by inverted fluorescence microscope.Results (1) At day 3 after injury,the wound areas in two groups were covered with crust and surrounding redness,and the wound healing rates were slightly different;at day 7 after injury,the wound area of ADSCs group was significantly smaller than that of control group,and the healing speed and rate of ADSCs group was significantly higher than that of control group (P ＜ 0.01);at day 14 after injury,the healing rate of ADSCs group was nearly 99％ (P ＜ 0.01),and the healing skin tissue texture of ADSCs group was better than that of control group.(2) At day 3 after injury,there were a large number of inflammatory cells and disorganized collagen fiber in the wound areas of two groups;at day 7 after injury,the inflammatory cells infiltration reduced in ADSCs group compared with control group,and the collagen fiber arrangement in control group was in disorder;at day 14 after injury,the inflammatory cells in both groups obviously decreased,and ADSCs group had more new vessels and more orderly arrangement of collagen fiber than control group.CM Dil labeled ADSCs were seen in the healing wound tissue in ADSCs group.(3) At day 3 after injury,there was little difference in M1 type macrophage distribution in the two groups;ADSCs group had more M2 type macrophage cells than control group significantly (P ＜0.01);the expression of IL 10 in ADSCs group was not high,which did not differ from that of control group;at days 7 and 14 after injury,ADSCs group has fewer M1 type macrophage cells,more M2 type macrophage cells,and higher expressions of IL-10 than control group (P ＜ 0.01).Conclusion The ADSCs trasplantation can promote the change from M1 type to M2 type macrophages,facilitating wound regeneration and healing.