中国修复重建外科杂志

中国修复重建外科杂志

人脂肪来源干细胞对小鼠压疮愈合影响的实验研究

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目的探讨人脂肪来源干细胞(human adipose-derived stem cells,hADSCs)对小鼠压疮创面愈合的影响。方法取自愿捐赠的人脂肪组织,采用机械分离联合胶原酶消化法获取 hADSCs 并传代。取第 3 代细胞行成骨、成脂、成软骨分化以及流式细胞仪鉴定。取健康自愿者捐赠的外周血,采用离心法制备富血小板血浆(platelet rich plasma,PRP),并行血小板计数。取 45 只 C57BL/6 小鼠采用磁铁夹压法于背部制备压疮模型,随机分为 3 组(n=15);hADSCs 组、PRP 组及对照组创面分别注射 100 μL 经携带绿色荧光蛋白(green fluorescent protein,GFP)标记后的 hADSCs(1×106个)、人 PRP、PBS。注射后观察压疮创面愈合情况,5、9、13 d 计算创面愈合率;5、11、21 d 取标本行 HE 染色、Masson 染色以及 CD31、S100 免疫组织化学染色,观察创面血管、神经再生情况。hADSCs 组于 11 d 时荧光示踪法观察细胞定植及成活情况。结果经诱导分化及流式细胞仪鉴定分离培养细胞为 hADSCs。PRP 血小板计数显著高于正常外周血(t=5.781,P=0.029)。大体观察,注射后 hADSCs 组创面愈合优于 PRP 组及对照组,5、9、13 d,hADSCs 组创面愈合率明显高于 PRP 组及对照组(P<0.05)。组织学观察示,与 PRP 组及对照组相比,hADSCs 组炎性细胞浸润、炎性反应明显减轻,胶原沉积明显增多,且 21 d 时可见皮肤附属器再生;注射后各时间点,hADSCs 组胶原表达量均显著高于 PRP 组及对照组(P<0.05)。免疫组织化学染色示,各时间点 hADSCs 组新生血管数、S100 阳性细胞百分比均明显多于 PRP 组和对照组(P<0.05)。荧光示踪法显示注射 11 d 后 hADSCs 可定植于创面并成活。结论局部移植的 hADSCs 通过促进血管新生和神经再生,进而促进小鼠压疮创面愈合、提高愈合质量。

ObjectiveTo investigate the effect of human adipose-derived stem cells (hADSCs) on pressure ulcers in mouse.MethodsThe subcutaneous adipose tissue from voluntary donation was harvested. Then the hADSCs were isolated and cultured by mechanical isolation combined with typeⅠcollagenase digestion. The 3rd generation cells were identified by osteogenic, adipogenic, chondrogenic differentiations and flow cytometry. The platelet rich plasma (PRP) from peripheral blood donated by healthy volunteers was prepared by centrifugation. The pressure ulcer model was established in 45 C57BL/6 mice by two magnets pressurized the back skin, and randomly divided into 3 groups (n=15). The wounds were injected with 100 μL of hADSCs (1×106 cells) transfected with a green fluorescent protein (GFP)-carrying virus, 100 μL human PRP, and 100 μL PBS in hADSCs group, PRP group, and control group, respectively. The wound healing was observed after injection. The wound healing rate was calculated on the 5th, 9th, and 13th days. On the 5th, 11th, and 21st day, the specimens were stained with HE staing, Masson staining, and CD31 and S100 immunohistochemical staining to observe the vascular and nerve regeneration of the wound. In hADSCs group, fluorescence tracer method was used to observe the colonization and survival of the cells on the 11th day.ResultsThe cultured cells were identified as hADSCs by induced differentiation and flow cytometry. The platelet counting was significantly higher in PRP group than in normal peripheral blood group (t=5.781, P=0.029). General observation showed that the wound healing in hADSCs group was superior to those in PRP group and control group after injection. On the 5th, 9th, and 13th days, the wound healing rate in hADSCs group was significantly higher than those in PRP group and control group (P<0.05). Histological observation showed that compared with PRP group and control group, inflammatory cell infiltration and inflammatory reaction were significantly reduced in hADSCs group, collagen deposition was significantly increased, and skin appendage regeneration was seen on the 21st day; at each time point, the expression of collagen was significantly higher in hADSCs group than in PRP group and control group (P<0.05). Immunohistochemical staining showed that the number of neovascularization and the percentage of S100-positive cells in hADSCs group were significantly better than those in PRP group and control group on the 5th, 9th, and 13th days (P<0.05). Fluorescent tracer method showed that the hADSCs could colonize the wound and survive during 11 days after injection.ConclusionLocal transplantation of hADSCs can accelerate healing of pressure ulcer wounds in mice and improve healing quality by promoting revascularization and nerve regeneration.

关键词: 压疮; 脂肪来源干细胞; 富血小板血浆; 血管新生; 神经再生; 创面愈合; 小鼠

Key words: Pressure ulcer; adipose-derived stem cells; platelet-rich plasma; revascularization; nerve regeneration; wound healing; mouse

引用本文: 邓呈亮, 刘志远, 姚远镇, 刘睿池, 魏在荣, 王达利. 人脂肪来源干细胞对小鼠压疮愈合影响的实验研究. 中国修复重建外科杂志, 2018, 32(6): 726-735. doi: 10.7507/1002-1892.201801031 复制

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