一氧化氮对体外培养人表皮干细胞生物学行为的影响  

Biologic effect of nitric oxide on human epidermal stem cells in vitro

作　　者：詹日兴[1] 孙薇[1] 姚志慧[1] 崔艳艳[1] 杨思思[1] 谭江琳[1] 周俊峄[1] 王颖[1] 杨俊杰[1] 张小容[1] 胡晓红[1] 吴军[1] 罗高兴[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,疾病蛋白质组学重庆市重点实验室,重庆400038

出　　处：《中华烧伤杂志》2012年第2期125-129,共5页Chinese Journal of Burns

基　　金：基金项目：国家自然科学基金面上项目（30973116、81171809）;创伤、烧伤与复合伤国家重点实验室自主研究课题（SKLZZ200808、SKLZZ200820）

摘　　要：目的观察外源性NO对人表皮干细胞（ESC）脱黏附、增殖以及迁移功能的影响。方法采用改良Ⅳ型胶原快速黏附法分离、培养人ESC，倒置相差显微镜下观察其形态；蛋白质印迹法和免疫荧光法观察ESC标志物整合素p，和细胞角蛋H 19（ CK19）的表达。对贴壁生长的ESC行划痕处理，用O（对照）、1、10、100、500 μmolL NO供体S-亚硝基-N-乙酰-DL-青霉胺（SNAP）作用细胞12、24 h，检测细胞迁移率；Transwe11实验检测SNAP（浓度同上）对ESC趋化能力的影响（计数转膜细胞）。采用0、10、100、500 μmolL SNAP作用细胞1 h，黏附实验检测SNAP对ESC黏附功能的影响，计算黏附抑制率；于上述4种浓度SNAP作用O（即刻）、12、24、48 h，采用酶标仪检测ESC增殖水平， 数据以吸光度值表示。对数据进行单凶素方差分析和Dunnett z检验。 结果 （1）倒置相差显微镜下可见，细胞培养5-9 d形成小克隆；培养10 -14 d，细胞增殖明显加快。蛋白质印迹法和免疫荧光法检测结果显示，所分离的细胞均能表达CK19和整合素p．。由此鉴定该细胞为ESC。（2）与O μmolL SNAP怍用12、24 h后细胞迁移率[（35.7±0.3）%、（45.7 ＋5.0）%]比较，1-100 μmolLSNAP均能促进ESC迁移，其中100 μmolL SNAP作用后细胞迁移率达峰值[（48.8±2.7）%、（82.1±15.8）%，P值分别为8. 34、5.10，P值均小于0.01]。100 y,mo1/L SNAP作用后转膜细胞数显著多于O μmolL SNAP作用后（t=9. 24，P=0.00）。100、500 μmolL SNAP作用后，ESC黏附能力明显低于OμmolL SNAP作用后（t值分别为4.30、4.67，P值均为0.00）。100、500 μmolL SNAP作用24、48 h，细胞增殖能力明显强于OμmolL SNAP作用后（t值为2.84-8.17，P值均小于0.05）。 结论适宜浓度的外源性NO对体外培养人ESC的迁移功能有促进作用。外源性NO对体外培养人ESC具有脱黏附、促增殖作用。Objective To observe the effec：t of nitric oxide （NO） on adhesion, proliferation, and migration of human epidermal stem cells （ESC） in vitro. Methods ESC were isolated and cultured by the modified method of rapid actac,hment to type lV collagen. （1） Morphology of cells was observed under inverted phase-contrast microsc：ope. Expression levels of integrin [3, and cytokeratin 19 （ CK19） of cells were determined by Western blotting ancl immunofluorescenc：e staining. （2） After being creaced with sc：racching, ESC adhered to the wall was respectively treated with nitric oxide （ NO） donor S-nitroso-N-acetylpenicilla- mine （SNAP） in the concentration of l, 10, 100, 500 y.mol/L. ESC without treatment of SNAP was used as control. The migration rate of ESC was detected at post scratching hour （ PSH） 12 and 24. The chemotax-is of ESC （ treated with SNAP in above-mentioned concentration） was cested by Transwell assay, and che transferred c：ell number was c：ounted. （3） ESC was respec：tively treated with SNAP in the concentration of 10, 100, 500 y,mol/L for l h. ESC without treatment of SNAP was used as control. The adhesion of ESC was detected with adhesion test, and the inhibition rate of adhesion was c：alculated. The proliferation of ESC （ denoted as absorbance value） was determined by mic：roplate reader at post-treatment hour （ PTH） 0, 12, 24, 48. Data were processed with one-way analysis of variance and Dunnett t test. Results （1） Small clone f＇ormed on post culture days （PCD） 5 t0 9. On PCD 10 t0 14, cell proliferation sped up. CK19 and integrin [31were detected to be expressed in the isolated cells. The cells were identified as ESC. （2） Com- parecl with that of ESC without treatment of SNAP [ （ 35. 7 ＋0. 3） % , （ 45. 7 ＋5. 0） % ] , migration of ESC treated with SNAP in the concentration from l c0 100 y,mol/L was promoted at PSH 12 and 24. Migration rates of ESC treated with 100 pdmol/L SNAP were

关 键 词：一氧化氮 细胞运动 表皮干细胞 创面愈合 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]