HGF/c-Met axis regulates human adipose tissue-derived stem cells in burn wound healing

doi:10.3969/j.issn.2095-4344.0929

Abstract

Abstract:

BACKGROUND: Studies have found that hepatocyte growth factor (HGF) can promote proliferation and migration of adipose-derived stem cells (ADSCs) and accelerate wound healing.
OBJECTIVE: To investigate the mechanism of HGF/c-Met on the proliferation and migration of ADSCs and its effects on burn wound healing.
METHODS: ADSCs were separated from subcutaneous adipose tissues and primary cultured. ADSCs untreated were considered as control group. Cells in experimental groups were treated with HGF (10 μg/L, 24 hours) or c-Met siRNA combined with HGF (10 μg/L, 24 hours). Then, western blot assay was used to detect expressions of c-Met, p-AKT, and AKT in the cells. The proliferation and migration abilities of ADSCs were observed by cell counting kit-8 and Transwell assay. Twelve nude mice were burned by copper bar at 99 ^oC for 5 seconds in order to establish animal models of skin III degree burns with the diameter of 1 cm, and then these model mice were randomly divided into experimental and control group (n=6 per group). In control group, only 1×10⁶ ADSCs were injected around the wound, while in experimental group, 1×10⁶ ADSCs transfected with Ad-HGF were injected around the wound. All the mice were killed at 7 days of the experiment, and fresh full-thickness skin wound tissues were taken. Wound healing rate was calculated and the thickness between dermis and epidermis was measured by hematoxylin-eosin staining.
RESULTS AND CONCLUSION: Significant expression of c-Met was induced by HGF (P < 0.05). The proliferation and migration of ADSCs were promoted by HGF but suppressed by c-Met siRNA (P < 0.05). p-AKT expression could be promoted by HGF but inhibited by c-Met siRNA. Compared with the control group, the experimental group showed higher wound healing rate and thickened re-epithelium (P < 0.05). To conclude, the HGF/c-Met axis can promote the proliferation and migration of ADSCs by activating PI3K/AKT pathway, and HGF can accelerate wound healing of burn nude mice.

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: Adipose Tissue, Mesenchymal Stem Cells, Hepatocyte Growth Factor, Wound Healing, Tissue Engineering

CLC Number:

R394.2

Li Xue-yang, Zheng Wan-ling, Yang Chao, Xia Si-zhan, Xing Xin. HGF/c-Met axis regulates human adipose tissue-derived stem cells in burn wound healing[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 3975-3980.

Figures/Tables 2

2.1 HGF诱导c-Met蛋白的表达采用0，4，8，10，12 μg/L HGF处理ADSCs 12 h后通过Western blot检测c-Met蛋白的表达，差异有显著性意义(P < 0.05)，与0 μg/L组相比较，4 μg/L组c-Met表达无明显变化(P=0.235)，8，10 μg/L组c-Met的表达逐渐上调，12 μg/L组c-Met表达较10 μg/L组下降(P < 0.05，图1A)。因此，选择10 μg/L HGF处理ADSCs 0，4，12，24，38 h后通过Western blot检测c-Met蛋白的表达，差异有显著性意义(P < 0.05)，与0 h组相比较，4 h组c-Met表达无明显变化(P=0.523)，12，24 h组c-Met的表达明显逐渐上调，38 h组较24 h组c-Met表达下调(P < 0.05，图1B)，综上实验结果作者认为以10 μg/L HGF处理ADSCs 24 h为最适浓度及时间。 2.2 c-Met siRNA抑制c-Met蛋白的表达 3组ADSCs中c-Met蛋白的表达差异有显著性意义(P < 0.05)，HGF(10 μg/L)处理ADSCs 24 h后，HGF组与空白对照组比较，c-Met蛋白表达明显上调(P < 0.05)；与HGF组比较，HGF+c-Met siRNA组c-Met蛋白表达明显下调(P < 0.05，图2)。 2.3 HGF促进ADSCs增殖，c-Met siRNA抑制ADSCs增殖通过重复测量数据的方差分析，不同因素处理ADSCs后，比较不同时间点的细胞增殖情况，差异有显著性意义(P < 0.05)；与空白对照组比较， HGF组细胞增殖能力升高，差异有显著性意义(P < 0.05)；与HGF组比较，HGF+c-Met siRNA组细胞增殖能力降低，差异有显著性意义(P < 0.05)，见图3。 2.4 HGF促进ADSCs迁移，c-Met siRNA抑制ADSCs迁移采用重复数据方差分析，不同因素处理ADSCs后，细胞迁移能力差异有显著性意义(P < 0.05)。与空白对照组比较，HGF组细胞迁移能力增强，差异有显著性意义(P < 0.05)；与HGF组比较，HGF+c-Met siRNA组细胞迁移能力降低，差异有显著性意义(P < 0.05)，见图4。 2.5 HGF/c-Met激活PI3K/AKT信号通路 HGF(10 μg/L)处理c-Met siRNA ADSCs及ADSCs 24 h，AKT的表达均未见明显改变；与空白对照组比较，HGF组p-AKT表达明显增多；与HGF组比较，HGF+c-Met siRNA组p-AKT表达明显减少(图5)。 2.6 HGF预处理ADSCs移植对裸鼠烫伤创伤愈合的影响在移植后第7天进行裸鼠模型的创面比较(图6)。实验组在创伤后第7天的创面愈合率和上皮化厚度显著高于对照组，差异有显著性意义(P < 0.05)。"

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