Feasibility of adipose-derived mesenchymal stem cell sheet transplantation for wound healing

doi:10.3969/j.issn.2095-4344.1581

Abstract

Abstract:

BACKGROUND: Adipose-derived mesenchymal stem cells are often used for tissue repair and functional reconstruction due to their paracrine actions of multiple growth factors, easy access and immunomodulatory effects. The prevention and treatment of esophageal stenosis after esophageal endoscopic submucosal dissection is a challenge for doctors and patients. Adipose-derived mesenchymal stem cell transplantation combined with the cell sheet technology has the potential to promoting wound repair and preventing esophageal stenosis.
OBJECTIVE: To investigate the biological characteristics of adipose-derived mesenchymal stem cell sheet, and to analyze the feasibility of preventing esophageal stenosis after esophageal endoscopic submucosal dissection.
METHODS: The green fluorescent protein-labeled adipose-derived mesenchymal stem cell sheets were prepared using temperature-sensitive culture dishes and were set as the experimental group. Conventionally adherent adipose-derived mesenchymal stem cells were used as the control group. Levels of vascular endothelial growth factor, transforming growth factor β1 and hepatocyte growth factor in the two groups were compared by ELISA, RT-qPCR and western blot. The skin ulcers of nude mice were prepared, and randomized into cell sheet group and model group. The green fluorescent protein-labeled adipose-derived mesenchymal stem cell sheets were then implanted into the skin wound in the cell sheet group.
RESULTS AND CONCLUSION: The number of cells in the cell sheet was high with enriched extracellular matrix, and there was an interconnection between cells. The expression levels of vascular endothelial growth factor, transforming growth factor β1 and hepatocyte growth factor in the experimental group were all higher than those in the control group. The adipose-derived mesenchymal stem cell sheet on the back of nude mice was successfully transplanted, and the wound healing in the cell sheet group was faster than that of the model group. Experimental findings indicate that adipose-derived mesenchymal stem cell transplantation combined with the cell sheet technology is feasible to prevent stenosis after esophageal endoscopic submucosal dissection, but a large number of basic and clinical studies are warranted.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Intercellular Signaling Peptides and Proteins, Ulcer, Tissue Engineering

CLC Number:

R456
R318

Chen Meihong, Dang Yini, Zhu Xudong, Li Xuan, Peng Lei, Yang Jiajia, Zhang Guoxin. Feasibility of adipose-derived mesenchymal stem cell sheet transplantation for wound healing[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(5): 790-796.

Figures/Tables 2

2.1 脂肪间充质干细胞的生长特性及膜片形态第3代脂肪间充质干细胞接种于温度敏感培养皿24 h后，即可见细胞贴壁生长，呈纤维状或长梭形，见图1A；第3代细胞的生长曲线呈S形，前2 d细胞生长缓慢，至第3天进入对数生长期，第5天进入生长平台期，见图1B；荧光显微镜下可见约80%的脂肪间充质干细胞标记绿色荧光蛋白，见图1C；脂肪间充质干细胞连续培养1周后，培养皿底部出现半透明乳白色膜样物质，细胞膜片呈皱缩状，倒置显微镜及苏木精-伊红染色可见膜片中细胞含量较高，细胞外基质丰富，细胞间连接紧密，膜片结构致密且厚度均匀，见图1D-F。 2.2 脂肪间充质干细胞的干细胞特性流式细胞仪检测第3代脂肪间充质干细胞有99.30%的细胞表面表达CD73、93.91%的细胞表达CD90、99.35%的细胞表达CD105，而仅有0.35%的细胞表达CD34、0.17%的细胞表达CD45、0.08%的细胞表达HLA-DR，这与原代干细胞高表达CD73、CD90、CD105，低表达CD34、CD45、HLA-DR特性一致[20-23]，见图2；成脂诱导培养基诱导培养第18天，油红O染色可见细胞胞浆内存在大小不一的橘黄色脂滴，表明成脂分化阳性，见图3；成骨诱导培养基诱导培养第16天，茜素红染色显示细胞中后出现深色钙结节，表明成骨分化阳性，见图4。 2.3 脂肪间充质干细胞膜片中血管内皮生长因子、转化生长因子β1、肝细胞生长因子基因及蛋白的表达水平实时定量PCR结果显示：细胞膜片3种基因的表达水平均高于对照组(P < 0.05)，见图5A-C；进一步Western blot验证了膜片中基因所对应的蛋白表达情况，与PCR检测结果一致，膜片血管内皮生长因子、转化生长因子β1、肝细胞生长因子3种蛋白的表达水平均高于对照组，见图5D。 2.4 脂肪间充质干细胞膜片培养上清液中血管内皮生长因子、转化生长因子β1、肝细胞生长因子的表达水平 ELISA结果显示，膜片移植前培养液生长因子含量显著高于干细胞专用完全培养基和基础培养基(P < 0.000)，见图6，而基础培养基和干细胞专用完全培养基中3种因子的含量接近。 2.5 裸鼠皮肤溃疡愈合模型的验证将脂肪间充质干细胞膜片移植至裸鼠人工皮肤溃疡创面，图7A-C显示贴膜组裸鼠皮肤溃疡愈合速度快于模型组，贴膜组裸鼠皮肤溃疡约于造模后第8天全部愈合，而模型组为造模后12 d，造模后1周时，贴膜组溃疡表面血痂基本脱落，而模型组稍晚于贴膜组，于第10天全部脱落；造模后第2天和第6天小动物活体成像及造模后1周组织切片荧光显微镜观察可见绿色荧光蛋白标记的脂肪间充质干细胞成功贴附并整合于创面组织，见图7D，E；待溃疡完全愈合后取其瘢痕组织行免疫组化，发现贴膜组的血管内皮生长因子、转化生长因子β1、肝细胞生长因子表达水平高于模型组，见图7F。"

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