人羊膜上皮干细胞来源外泌体对大鼠全层皮肤缺损创面愈合的影响  

Effects of human amniotic epithelial stem cells-derived exosomes on healing of wound with full-thick- ness skin defect in rats

作　　者：赵彬[1] 吴高峰[1] 张熠杰[1] 张伟[1] 杨芳芳[1] 肖丹[1] 曾凯旋 石继红[1] 苏琳琳[1] 胡大海[1] 

机构地区：[1]第四军医大学西京医院全军烧伤中心,烧伤与皮肤外科,西安710032

出　　处：《中华烧伤杂志》2017年第1期18-23,共6页Chinese Journal of Burns

摘　　要：目的探讨人羊膜上皮干细胞来源外泌体对大鼠全层皮肤缺损创面愈合的影响。方法（1）取笔者单位妇产科5名足月分娩孕妇的羊膜组织，采用胰蛋白酶消化法分离人羊膜上皮干细胞并观察细胞形态；取第3代细胞，用罗丹明标记的鬼笔环肽染色，观察细胞骨架；另取第3代细胞，采用流式细胞仪检测细胞表面标志物CD29、CD31、CD34、CD90、CD105、SSEA3、SSEA4和免疫相关标志物人白细胞抗原DR位点（HLA－DR）的表达；另取第3代细胞，进行成脂、成骨诱导分化鉴定。（2）取第3代人羊膜上皮干细胞，用含体积分数10%无外泌体FBS的DMEM培养基培养，超速离心法从细胞培养上清液中提取人羊膜上皮干细胞来源外泌体，用扫描电镜观察其形态。（3）取6只成年SD大鼠，于每只大鼠背部制备4个面积为1 cm×1 cm的全层皮肤缺损创面，按随机数字表法将每只大鼠创面分为对照组及25、50、100 μg/mL外泌体组（每组6个创面），分别于创面四周皮下多点均匀注射总体积100 μL的PBS及25、50、100 μg/mL人羊膜上皮干细胞来源外泌体。伤后7、14、21 d，观察创面愈合情况计算创面愈合率。伤后21 d，取各组创面愈合组织，HE染色，观察计数皮肤附件；Masson染色，观察胶原纤维排列。对数据行重复测量方差分析、随机区组方差分析、单因素方差分析及Bonferroni检验。结果（1）分离培养的细胞呈典型的鹅卵石样，细胞表面分布较多微绒毛；细胞CD29、CD90、SSEA3、SSEA4阳性表达率高于50.0%，CD105阳性表达率为8.0%，CD31、CD34和HLA－DR阳性表达率几乎为0；细胞经诱导后可分化为脂肪细胞和成骨细胞。综上，培养细胞鉴定为人羊膜上皮干细胞。（2）人羊膜上皮干细胞来源外泌体为直径50～150 nm的圆形或椭圆形囊泡。（3）伤后7、21 d，各组创面愈合率相近（P值均大于0.05）。伤后14 d，50、100 μg/mLObjectiveTo investigate the effects of human amniotic epithelial stem cells-derived exosomes on healing of wound with full-thickness skin defect in rats.Methods（1） Human amniotic epithelial stem cells were isolated from the amnion tissue of 5 full-term pregnant women in Department of Obstetrics of our hospital by the method of trypsin digestion, and their morphology was observed. The third passage of cells were stained with rhodamine-phalloidin for cytoskeleton observation. The third passage of cells were identified with flow cytometry through the detection of expressions of cell surface markers CD29, CD31, CD34, CD90, CD105, SSEA3, SSEA4 and immunity-related marker human leukocyte antigen-D related site （HLA-DR）. The third passage of cells were also assessed the ability of adipogenic and osteogenic differentiation. （2） The third passage of human amniotic epithelial stem cells were cultured in DMEM medium supplemented with 10% exosome-free fetal bovine serum. Exosomes were isolated from culture supernatant by the method of ultracentrifugation and represented with scanning electron microscope for morphologic observation. （3） Six adult SD rats were anesthetized, and four 1 cm×1 cm sized wounds with full-thickness skin defect were made on the back of each rat. The wounds on the back of each rat were divided into control group, 25 μg/mL exosomes group, 50 μg/mL exosomes group, and 100 μg/mL exosomes group according to the random number table （with 6 wounds in each group）, and a total volume of 100 μL phosphate buffered saline, 25 μg/mL exosomes, 50 μg/mL exosomes, and 100 μg/mL exosomes were evenly injected around the wound through multiple subcutaneous sites, respectively. The wound healing rate was calculated based on measurement on post injury day （PID） 7, 14, and 21. On PID 21, the healed wound tissue of each group was collected and stained with HE to observe and count skin accessories, and the arrangement of collagen fibers was observed with Masson staining. Data were processed wi

关 键 词：创伤和损伤 羊膜 干细胞 外泌体 伤口愈合 

分 类 号：R641[医药卫生—外科学]