Umbilical cord mesenchymal stem cell conditioned medium combined with hyaluronic acid for mouse skin repair

doi:10.3969/j.issn.2095-4344.0405

Abstract

Abstract:

BACKGROUND: Numerous studies have shown that mesenchymal stem cells (MSCs) can effectively promote tissue repair by secreting various bioactive factors.
OBJECTIVE: To observe the effects of umbilical cord cord mesenchymal stem cells conditioned media (UCMSCs-CM) combined with hyaluronic acid (HA) on the repair of skin wounds in mice.
METHODS: The UCMSCs-CM was collected, and added with HA to prepare the gel solution. Forty mice were selected to prepare animal models of full-thickness skin wound. According to different treatments, the mice were randomly divided into four groups, blank control group (0.5 mL of normal saline), UCMSCs-CM group (0.5 mL of UCMSCs-CM), HA group (0.5 mL of HA), UCMSCs-CM+HA group (0.5 mL of UCMSCs-CM+0.5 mL of HA). Each mouse received three treatments a day (in the morning, at noon and at night), for continuous 7 days. The wound size was measured and the wound healing rate was calculated. The factors related to skin repair were detected by immunohistochemstry and qRT-PCR methods.
RESULTS AND CONCLUSION: After treatment, the wound size was reduced to different extents in the treatment groups as compared with the blank control group. The UCMSCs-CM combined with HA significantly improved the wound healing rate as compared with the other groups (P < 0.01). Strongly positive expression of the factors related in skin injury, such as vascular endothelial growth factor and matrix metalloproteinase 9 protein, were found in the UCMSCs-CM+HA group; moreover, the mRNA levels of basic fibroblast growth factor, transforming growth factor β and matrix metalloproteinase 9 protein were significantly higher in the UCMSCs-CM+HA group than the blank control group (P < 0.01). To conclude, UCMSCs-CM combined with HA can accelerate skin wound repair in mice, and exhibit better effects than the UCMSCs-CM or HA alone, which provides theoretical support for stem cells combined with biological scaffolds in skin wound repair.

Key words: Umbilical Cord, Mesenchymal Stem Cells, Culture Media, Conditioned, Hyaluronic Acid, Skin, Wounds and Injuries, Tissue Engineering

CLC Number:

R394.2

Lou Min-ming, Wang Hong-wei, Ma Jie. Umbilical cord mesenchymal stem cell conditioned medium combined with hyaluronic acid for mouse skin repair[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(1): 20-25.

Figures/Tables 1

2.1 脐带间充质干细胞的分离培养与鉴定脐带沃顿胶组织贴壁培养8 d左右时，可见组织块周围有细胞爬出，且呈放射状生长；培养至第16天，镜下可见较多形态均一，呈纤维状、漩涡状生长的细胞，见图1A。当细胞生长至80%-90%融合时，进行消化传代培养，取生长状态良好的第3代细胞进行流式分析，检测培养细胞表面标志物的表达。结果显示细胞高表达CD73、CD90、CD105(>95%)，极低表达CD14、CD19、CD34、CD45、HLA-DR(<0.5%)，符合间充质干细胞的特性，见图1B。 2.2 实验动物数量分析小鼠造模以及涂抹治疗过程中均无死亡，进入结果分析40只小鼠。 2.3 小鼠创面愈合率涂抹第7天，与生理盐水对照组相比，各治疗组创面面积均有不同程度的缩小，其中干细胞条件培养液+透明质酸凝胶治疗组的创面几乎完全愈合，见图2A。分别计算治疗第3天和第5天的创面愈合率，结果显示，干细胞条件培养液+透明质酸凝胶治疗组的创面愈合率明显高于其他治疗组，见图2B。 2.4 干细胞条件培养液和透明质酸凝胶液对小鼠创面组织损伤修复相关因子表达的影响由于干细胞条件培养液+透明质酸凝胶组创面愈合效果显著优于其他治疗组，因此，取干细胞条件培养液+透明质酸凝胶组和生理盐水对照组第5天的创面组织，通过免疫组化检测损伤修复相关因子的表达，进一步探究其作用机制。免疫组化结果显示，与生理盐水对照组相比，干细胞条件培养液+透明质酸凝胶组血管内皮生长因子、基质金属蛋白酶9蛋白呈强阳性表达(图3A)，且血管内皮生长因子在上皮细胞和表皮细胞均呈强阳性表达，基质金属蛋白酶9在基质中呈强阳性表达。取干细胞条件培养液+透明质酸凝胶组和生理盐水对照组第5天的创面组织，qRT-PCR检测血管内皮生长因子、碱性成纤维细胞生长因子、表皮生长因子、转化生长因子β、基质金属蛋白酶9 mRNA的表达，结果显示，与生理盐水对照组相比，干细胞条件培养液+透明质酸凝胶组除表皮生长因子的表达上调无统计学意义外，血管内皮生长因子、碱性成纤维细胞生长因子、转化生长因子β、基质金属蛋白酶9 mRNA的表达均上调且差异有显著性意义(图3B)。"

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