中国组织工程研究

中国组织工程研究 ›› 2013, Vol. 17 ›› Issue (10): 1766-1772.doi: 10.3969/j.issn.2095-4344.2013.10.010

• 干细胞移植 stem cell transplantation • 上一篇    下一篇

皮下移植骨髓间充质干细胞修复大鼠糖尿病足溃疡

万江波1，蔡 黔2，刘 毅2   

  1. 1 兰州大学第二医院，甘肃省兰州市 730000
    2 解放军兰州军区兰州总医院全军烧伤整形外科中心，甘肃省兰州市 730050
  • 收稿日期:2012-07-29 修回日期:2012-09-10 出版日期:2013-03-05 发布日期:2013-03-05
  • 通讯作者: 蔡黔，博士，副教授，主任医师，硕士研究生导师，解放军兰州军区兰州总医院全军烧伤整形外科中心，甘肃省兰州市 730050 caiqianmd@163.com
  • 作者简介:万江波★，男，1987年生，江西省进贤县人，汉族，兰州大学在读硕士，主要从事干细胞治疗糖尿病足溃疡研究。 maidibo@163.com
  • 基金资助:

    兰州军区面上项目(CLZ11JB07)。

Subcutaneous transplantation of bone marrow mesenchymal stem cells for repairing diabetic foot ulcers in rats

Wan Jiang-bo1, Cai Qian2, Liu Yi2   

  1. 1 The Second Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China
    2 Center of Burns and Plastic Surgery, Lanzhou General Hospital, Lanzhou Command, Lanzhou 730050, Gansu Province, China
  • Received:2012-07-29 Revised:2012-09-10 Online:2013-03-05 Published:2013-03-05
  • Contact: Cai Qian, Doctor, Chief physician, Associate professor, Master’s supervisor, Center of Burns and Plastic Surgery, Lanzhou General Hospital of Lanzhou Command, Lanzhou 730050, Gansu Province, China caiqianmd@163.com
  • About author:Wan Jiang-bo★, Studying for master’s degree, the Second Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China qiaodanbo@163.com

PDF

421

被引次数

8

摘要：

背景：糖尿病足溃疡严重威胁患者的健康甚至生命，目前缺乏良好的治疗手段，而干细胞疗法在组织再生和创面修复方面展现出独特的优势及潜力。
目的：评价骨髓间充质干细胞经创缘皮下移植对大鼠糖尿病足溃疡的治疗效果。
方法：全骨髓贴壁法体外培养Wistar大鼠骨髓间充质干细胞至第3代，经DAPI标记。36只雄性Wistar大鼠随机分成骨髓间充质干细胞移植组、正常足溃疡对照组、糖尿病足溃疡对照组，大鼠糖尿病经腹腔注射链脲佐菌素诱导，所有大鼠在双后足背上切取一3 mm×7 mm矩形全层皮肤制成足溃疡模型。于骨髓间充质干细胞移植后第2，5，8，11天评估各组大鼠创面愈合情况及相关蛋白和基因水平的表达情况。
结果与结论：正常足溃疡对照组的创面愈合率大于骨髓间充质干细胞移植组和糖尿病足溃疡对照组，但后2组之间无差异。冰冻切片发现骨髓间充质干细胞移植组荧光强度和区域在第5天达到高峰。苏木精-伊红染色显示第5天正常足溃疡对照组肉芽组织最厚，骨髓间充质干细胞移植组比糖尿病足溃疡对照组厚。免疫组化发现骨髓间充质干细胞移植组CD31平均小血管数目在第8天和第11天比糖尿病足溃疡对照组多(P < 0.05)；骨髓间充质干细胞移植组平均Ki-67阳性细胞数在各时间点均比糖尿病足溃疡对照组多(P < 0.01)。ELISA和RT-PCR结果均显示，正常足溃疡对照组血管内皮细胞生长因子表达水平较骨髓间充质干细胞移植组、糖尿病足溃疡对照组高。第5天和第8天骨髓间充质干细胞移植组表达水平显著高于糖尿病足溃疡对照组(P < 0.01)。说明骨髓间充质干细胞经创缘皮下移植明显促进大鼠糖尿病足溃疡的愈合，此疗效可能是通过促进创伤中血管内皮细胞生长因子的表达实现的。

关键词: 干细胞, 干细胞移植, 骨髓间充质干细胞, 糖尿病足, 创面修复, 溃疡, 肉芽组织, 血管内皮细胞生长因子, 细胞增殖, 血管化, 其他基金, 干细胞图片文章

Abstract:

BACKGROUND: Diabetic foot ulcers threaten the patients’ health even survival. It lacks of an effective treatment. But stem cell therapy possesses special advantages and potential in tissue regeneration and wound healing.
OBJECTIVE: To assess the therapeutic effect of subcutaneous transplantation of bone marrow mesenchymal stem cells around the wounds of diabetic foot ulcers in rats.
METHODS: Bone marrow mesenchymal stem cells were separated from male Wistar rats and cultured by the whole bone marrow adherence method until the third generation. Then the bone marrow mesenchymal stem cells were labeled by 4,6-diamino-2-phenylindole in vitro. Thirty-six male Wistar rats were randomly divided into three groups: (1) cell transplantation group in which diabetic experimental rats were subcutaneously injected with bone marrow mesenchymal stem cells, (2) non-diabetic control group and (3) diabetic control group. Diabetic foot ulcer model was established by removing a 3 mm × 7 mm rectangular full-thickness skin from two feet of diabetic rats. The wound healing and gene and protein expression levels were evaluated at 2, 5, 8 and 11 days after bone marrow mesenchymal stem cell transplantation.
RESULTS AND CONCLUSION: The wound healing rates were as follows: non-diabetic controls > cell transplantation group > diabetic controls, while there were no significant differences between cell transplantation group and diabetic control group. The intensity and distribution of the fluorescence was highest on day 5 after transplantation to the frozen sections in the cell transplantation group. Results of hematoxylin-eosin staining revealed that the granulation tissues in the non-diabetic control group were thicker than that in other groups on day 5, and granulation tissue in cell transplantation group was thicker than that in the diabetic control group. Immunohistochemistry of CD31 demonstrated that the mean number of small blood vessels in the cell transplantation group was greater than that in the diabetic control group on day 8 and day 11 (P < 0.05). However, the mean number of Ki-67-positive cells in the cell transplantation group was greater than that in the diabetic control group at each time point (P < 0.01). The results of enzyme-linked immunosorbent assay and reverse transcription-PCR showed that the expression levels of vascular endothelial growth factor in the non-diabetic control group were higher than those in the cell transplantation group and diabetic control group, while the expression levels of vascular endothelial growth factor in the cell transplantation group were significantly higher than those in diabetic control group on day 5 and 8 (P < 0.01). The subcutaneous transplantation of bone marrow mesenchymal stem cells around the wounds can significantly promote wound healing of diabetic foot ulcers in rats. This effect may be followed by the increased expression of vascular endothelial growth factor in wound tissues.

Key words: stem cells, stem cell transplantation, bone marrow mesenchymal stem cells, diabetic foot, wound healing, ulcers, granulation tissue, vascular endothelial growth factor, cellular proliferation, angiogenesis, other grants-supported paper, stem cell photographs-containing paper

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