AMD3100骨髓动员剂及单核细胞亚型变化对小鼠皮肤创面愈合模型影响的实验研究

发布时间：2018-07-28 07:21

【摘要】：目的建立小鼠背部皮肤全层缺损模型,应用AMD3100进行骨髓动员,研究在骨髓动员情况下单核巨噬细胞的亚型变化及其对创面愈合的影响。方法将小鼠分为实验组和对照组,单剂量实验组造模前40分钟给于腹腔注射5mg/kg AMD3100,对照组注射等量PBS。同时于小鼠背部造出5mm的创面。确定6小时、12小时、1天、3天、7天、14天为取材的时间点,通过大体观察、组织切片、免疫组化、流式细胞术等手段来观察愈合情况及单核细胞动力学变化。另外,采用每隔24小时腹腔重复注射AMD31005mg/Kg,对照组则注射等剂量PBS。术后1天、2天、3天观察伤后愈合情况。结果除1只单次注射实验组小鼠术后约3小时死亡外,其他的小鼠均存活良好。实验组创面较对照组愈合明显,创伤后7天时,单次注射组和对照组创面面积分别为1.28±0.95mm2、3.14±2.84mm2,p0.05.连续注射组实验组创面愈合较对照组较慢,创伤后3天时,连续注射组和对照组创面面积分别是17.65±2.60mm2、15.12±2.74mm2,p0.05.组织切片HE染色显示创伤后早期(6小时-24小时)单次注射实验组中性粒细胞、单核细胞等炎症浸润较对照组提前出现,以血管周围明显,伴毛细血管扩张、水肿等。创伤后3天,单次注射实验组微血管生成较对照组多,细胞数量增多,以核大深染的类上皮细胞为多。创伤后7-14天,单次注射实验组纤维母细胞增多,微血管增生。内皮细胞及成纤维母细胞增多。免疫组化显示单次注射实验组的α-SMA阳性增高,微血管密度增加。单次注射实验组CD68表达较对照组高。流式细胞术显示单次注射实验组Gr-1highCD11b+细胞群于创伤后6小时、12小时、1天、3天、7天占门内细胞总数分别为46.78±5.70%,19.09±9.25%,14.53±4.19%,4.15±1.81%,11.51±8.78%。对照组分别为23.58±5.85%,27.63±1.42%,15.72±4.97%,9.90±0.60%,9.33±0.91%。在6小时、3天时二者有显著性差异。在皮肤组织中,Gr-1highCD11b+细胞群在6小时、1天和3天时占比例分别为：1.35±0.06%,3.72±1.82%,2.71±1.68%,而对照组为0.86±0.18%,3.00±0.59%和1.66±0.15%。在6小时二者有显著性差异。结论通过单剂量AMD3100阻断SDF-1/CXCR4轴,能够从骨髓中动员出大量的单核细胞等造血干细胞,以Gr-1highCD11b+单核细胞群较多,而Gr-1lowCD11b+细胞群较少。单核细胞在伤后早期大量迅速招募到皮肤受损组织,清除伤口内的组织细胞碎片异物等,以尽快结束炎性反应期尽早启动抗炎修复期。在炎症中后期,组织中单核巨噬细胞增多,通过发挥巨噬细胞启动组织修复再生能力等,从而促进创面的愈合。
[Abstract]:Objective to establish a full-thickness skin defect model in mice and to investigate the changes of mononuclear macrophage subtypes and their effects on wound healing by bone marrow mobilization (AMD3100). Methods mice were divided into experimental group and control group. The mice were injected intraperitoneally with 5mg/kg AMD3100 40 minutes before the model was made in the single dose group, and the control group was injected with the same amount of 5mg/kg. At the same time, the wounds of 5mm were made on the back of mice. The time points of 6 hours, 12 hours, 1 day, 3 days, 7 days and 14 days were determined to observe the healing and the dynamic changes of monocyte by gross observation, tissue section, immunohistochemistry, flow cytometry and so on. In addition, AMD31005 mg / kg was injected intraperitoneally every 24 hours, while the control group was injected with PBSat the same dose. Wound healing was observed 1 day 2 days and 3 days after operation. Results except for one single injection, the mice in the experimental group died about 3 hours after operation, the other mice survived well. The wound healing in the experimental group was more obvious than that in the control group, and the wound area in the single injection group and the control group was 1.28 卤0.95mm2and 3.14 卤2.84mm2p0.05at 7 days after trauma, respectively. Wound healing in the continuous injection group was slower than that in the control group, and the wound area in the continuous injection group and the control group was 17.65 卤2.60 mm ~ (2) and 15.12 卤2.74 mm ~ (2) (p 0.05) respectively at 3 days after trauma. The HE staining showed that neutrophils were injected into the experimental group at the early stage (6 hours to 24 hours) after trauma. The inflammatory infiltration of monocytes appeared earlier than that of the control group, especially around the blood vessels, accompanied by capillary dilatation, edema and so on. On the 3rd day after trauma, the microvascular formation and the number of cells in the experimental group were more than those in the control group, especially in the large and deep stained epithelioid cells. At 7-14 days after trauma, fibroblasts and microvascular hyperplasia were increased in the experimental group. Endothelial cells and fibroblasts increased. Immunohistochemical staining showed that 伪 -SMA positive and microvessel density increased in single injection group. The expression of CD68 in the experimental group was higher than that in the control group. Flow cytometry showed that the total number of Gr-1highCD11b cells in the door was 46.78 卤5.70 卤19.09 卤9.25 卤4.19 卤4.15 卤1.81 卤11.51 卤8.78, respectively. The control group (23.58 卤5.85) was 27.63 卤1.42 (15.72 卤4.97) and 9.90 卤0.60 (9.33 卤0.91). There was a significant difference between them at 6 hours and 3 days. The percentage of Gr-1high CD11b cells in skin tissue at 6 hours and 3 days was 1: 1.35 卤0.06 and 3.72 卤1.82 and 2.71 卤1.68, respectively, compared with 0.86 卤0.18 卤0.59% and 1.66 卤0.15 in the control group. There was a significant difference between the two groups at 6 hours. Conclusion A large number of hematopoietic stem cells, such as monocytes, can be mobilized from bone marrow by blocking SDF-1/CXCR4 axis by single dose AMD3100. The monocytes of Gr-1highCD11b are more than those of Gr-1lowCD11b. Mononuclear cells were rapidly recruited into damaged skin tissue in the early stage of injury and removed tissue cell fragments from the wound so as to start the anti-inflammatory repair phase as soon as possible after the end of the inflammatory reaction period. In the middle and late stage of inflammation, the mononuclear macrophages in the tissues increased, and the ability of tissue repair and regeneration was activated by the macrophages, so as to promote the wound healing.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R641

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