中国修复重建外科杂志

中国修复重建外科杂志

去铁胺促进BMSCs靶向归巢和血管新生的实验研究

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目的应用低氧模拟剂去铁胺(desferrioxamine,DFO)模拟组织缺氧环境,观察其是否能促进 BMSCs 在大鼠随意皮瓣中的归巢和血管新生。方法分离、培养荧光素酶转基因 Lewis 大鼠的 BMSCs 和成纤维细胞(fibroblast,FB)。选用 4 周龄 Lewis 雄性大鼠 40 只,在其背部形成 10 cm×3 cm 大小矩形皮瓣,然后随机分成 4 组,每组 10 只:A 组于大鼠球后静脉丛注射 200 μL PBS;B、C 组同上法分别注射浓度为 1×106个/mL 的 FB 和 BMSCs 200 μL;D 组同 C 组方法注射 BMSCs 后,腹腔注射 DFO[100 mg/(kg·d)],连续 7 d。术后 7 d,观察各组大鼠皮瓣成活情况并计算皮瓣成活率,采用激光散斑血流成像仪检测皮瓣血流情况;术后 30 min 及 1、4、7、14 d 采用生物发光成像检测移植细胞在大鼠体内分布情况;术后 7 d 行 CD31 免疫荧光染色计算毛细血管密度,免疫荧光检测基质细胞衍生因子 1(stromal cell derived factor 1,SDF-1)、EGF、FGF 及 Ki67 的表达情况;利用荧光素酶抗体标记移植的 BMSCs,免疫荧光染色观察其是否参与损伤组织修复。结果术后 7 d 各组缺血皮瓣坏死边界已明确,C、D 组皮瓣成活率明显高于 A、B 组,D 组高于 C 组(P<0.05)。激光散斑血流成像仪检测示,C、D 组皮瓣血流值显著高于 A、B 组,D 组高于 C 组(P<0.05)。生物发光成像示 BMSCs 随时间变化逐渐向缺血缺氧区迁移,最终分布到缺血组织中;术后 14 d D 组的光子信号明显强于其他组(P<0.05)。CD31 免疫荧光染色示,C、D 组毛细血管密度显著高于 A、B 组,D 组高于 C 组(P<0.05)。C、D 组 SDF-1、EGF、FGF 及 Ki67 的表达明显强于 A、B 组,D 组强于 C 组。术后 7 d 移植的荧光素酶标记 BMSCs 表达于组织的动脉弹力层、毛细血管处和毛囊处。结论DFO 可以加速 BMSCs 向随意皮瓣缺氧区域的迁移归巢,加速 BMSCs 在缺血组织中的分化,同时促进缺血组织血管新生。

ObjectiveTo investigate whether desferrioxamine (DFO) can enhance the homing of bone marrow mesenchymal stem cells (BMSCs) and improve neovascularization in random flaps of rats.MethodsBMSCs and fibroblasts (FB) of luciferase transgenic Lewis rats were isolated and cultured. Forty 4-week-old Lewis male rats were used to form a 10 cm×3 cm rectangular flap on their back. The experimental animals were randomly divided into 4 groups with 10 rats in each group: in group A, 200 μL PBS were injected through retrobulbar venous plexus; in group B, 200 μL FB with a concentration of 1×106 cells/mL were injected; in group C, 200 μL BMSCs with a concentration of 1×106 cells/mL were injected; in group D, cells transplantation was the same as that in group C, after cells transplantation, DFO [100 mg/(kg·d)] were injected intraperitoneally for 7 days. On the 7th day after operation, the survival rate of flaps in each group was observed and calculated; the blood perfusion was observed by laser speckle imaging. Bioluminescence imaging was used to detect the distribution of transplanted cells in rats at 30 minutes and 1, 4, 7, and 14 days after operation. Immunofluorescence staining was performed at 7 days after operation to observe CD31 staining and count capillary density under 200-fold visual field and to detect the expressions of stromal cell derived factor 1 (SDF-1), epidermal growth factor (EGF), fibroblast growth factor (FGF), and Ki67. Transplanted BMSCs were labeled with luciferase antibody and observed by immunofluorescence staining whether they participated in the repair of injured tissues.ResultsThe necrosis boundary of ischemic flaps in each group was clear at 7 days after operation. The survival rate of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Laser speckle imaging showed that the blood perfusion units of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Bioluminescence imaging showed that BMSCs gradually migrated to the ischemia and hypoxia area and eventually distributed to the ischemic tissues. The photon signal of group D was significantly stronger than that of other groups at 14 days after operation (P<0.05). CD31 immunofluorescence staining showed that capillary density in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). The expressions of SDF-1, EGF, FGF, and Ki67 in groups C and D were significantly stronger than those in groups A and B, and in group D than in group C. Luciferase-labeled BMSCs were expressed in the elastic layer of arteries, capillaries, and hair follicles at 7 days after transplantation.ConclusionDFO can enhance the migration and homing of BMSCs to the hypoxic area of random flap, accelerate the differentiation of BMSCs in ischemic tissue, and improve the neovascularization of ischemic tissue.

关键词: 低氧模拟剂; 去铁胺; BMSCs; 归巢; 血管新生

Key words: Hypoxia mimetic agent; desferrioxamine; bone marrow mesenchymal stem cells; homing; neovascularization

引用本文: 郑胜武, 杜子婧, 黄雄梅, 庄兢, 林根辉, 杨宇, 丁昕, 昝涛. 去铁胺促进BMSCs靶向归巢和血管新生的实验研究. 中国修复重建外科杂志, 2019, 33(1): 85-92. doi: 10.7507/1002-1892.201809065 复制

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