Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (19): 2993-2998.doi: 10.3969/j.issn.2095-4344.2014.19.007
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Cao Jing, Jiang Nan, Xu Yang-yang, Zhu Meng-lin, Yang Liu
Revised:2014-03-01
Online:2014-05-07
Published:2014-05-07
Contact:
Jiang Nan, Professor, Master’s supervisor, Department of Plastic and Cosmetic Surgery, Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
About author:Cao Jing, Studying for master’s degree, Department of Plastic and Cosmetic Surgery, Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
CLC Number:
Cao Jing, Jiang Nan, Xu Yang-yang, Zhu Meng-lin, Yang Liu. Human adipose-derived stem cells promote angiogenic capability of random pattern flaps in mice[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(19): 2993-2998.
2.1 实验动物数量分析 小鼠30只均进入结果分析,无脱落。 2.2 体外脂肪干细胞分离、培养、鉴定和标记结果:提取的脂肪干细胞培养至48 h绝大部分细胞贴壁生长,呈梭形成纤维细胞样,单层分布,均匀有序,见图1A。 流式细胞学检查显示,第3代脂肪干细胞表面抗原表达比例分别为CD105(93.30±1.24)%,CD90(97.25±2.33)%,CD31(0.20±0.04)%,CD34(3.82±0.86)%,CD45(3.12± 0.53)%,HLA-DR(0.81±0.09)%,同型对照均为阴性,说明本实验分离、培养所得到的细胞阳性表达多向分化潜能干细胞的标记CD105、CD90、不表达造血系统及内皮系统抗原标记CD31,CD34,CD45,不表达成纤维细胞抗原标记CD45、HLA-DR,与以往研究结果基本吻合[18]。 成脂诱导21 d后的脂肪干细胞经油红O染色,细胞内可见成片分布的亮红色圆泡,提示胞浆内有脂滴形成,见图1B。 成骨诱导21 d后的脂肪干细胞经茜素红染色,可见岛状分布的橙红色颗粒,提示有沉积的钙结节,见图1C。 CM-DiI标记后即刻观察,在荧光显微镜下见标记后的脂肪干细胞均显红色荧光,经消化、重悬后细胞呈球形,胞浆丰富,活性良好,CM-Dil标记阳性率大于90%,见图1D。"
2.3 皮瓣成活率比较情况 建模后7 d,成活皮瓣与坏死皮瓣间分界清晰,易被双盲观察者区别。实验组皮瓣成活面积为(3.78±0.16) cm2,成活率为(84.0±3.6)%;对照组皮瓣成活面积为(2.27±0.16) cm2,成活率为(72.2±3.4)%;两组皮瓣成活率比较,差异有显著性意义(P < 0.05)。 2.4 皮瓣组织中微血管密度的检测和CM-Dil荧光示踪 术后14 d,和对照组相比,实验组成活皮瓣内微血管的密度有明显的增加,对照组为每200倍镜下视野内(12.8±3.11)个,实验组为每200倍镜下视野内(32.2±2.59)个,两组比较,差异有显著性意义(P < 0.05)。实验组镜下可见少量红色荧光细胞分布于皮肤组织和皮下组织深层内,但未观察到典型的红色荧光与绿色荧光重叠现象,见图2,3。 2.5 皮瓣组织中细胞因子的表达 ELISA检测显示,实验组和对照组的皮瓣组织中均含有血管内皮生长因子,实验组血管内皮生长因子水平为(687.01±19.15) μg/L,对照组血管内皮生长因子水平为(453.60±26.37) μg/L,与对照组相比,实验组血管内皮生长因子分泌量显著增多(P < 0.05)。Western blot结果显示,实验组和对照组基质细胞衍生因子1表达均呈阳性,见图2。Image J软件对条带吸光度值的分析表明,实验组基质细胞衍生因子1蛋白与β-actin吸光度值的比值为0.78±0.04,对照组基质细胞衍生因子1蛋白与β-actin的比值为0.30±0.04,实验组基质细胞衍生因子1蛋白表达水平明显高于对照组(P < 0.05)。"
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