SCF/c-Kit反应轴调控ADSCs在糖尿病创面修复中的作用研究

Role of SCF/c-Kit axis on regulation of human adipose tissue-derived stem cells in diabetic wound healing

摘要:

探讨干细胞因子（SCF）/ 干细胞因子受体（c-Kit）信号调节脂肪间充质干细胞（ADSCs）增殖、迁移的作用机制，及其在糖尿病创面愈合中的作用。方法分离培养ADSCs。将其分为ADSCs 组、ADSCs+SCF（4 ng/ml，24 h）组和ADSCs+SCF+c-Kit siRNA（4 ng/ml，24 h）组；利用Western blot检测c-Kit、p-AKT、AKT蛋白的表达；采用CCK-8、细胞克隆形成检测增殖情况；采用Transwell法检测迁移情况。采用链脲佐菌素腹腔注射复制糖尿病小鼠模型，并复制背部直径为2cm 的全层皮肤缺损模型。将6 只糖尿病裸鼠随机分为实验组和对照组，对照组小鼠于创面周围以皮内注射方式注射1×106个未处理的ADSCs，实验组小鼠注射等量的经SCF（4 ng/ml）预处理24 h后的ADSCs。10 d后处死各组裸鼠，计算创面愈合率，苏木精- 伊红染色法检测真皮层与表皮层间的厚度，比较两组的差异。结果SCF 诱导c-Kit的表达呈时间、浓度依赖性（p <0.05）。SCF促进ADSCs的增殖、迁移，c-Kit siRNA抑制ADSCs的增殖、迁移（p <0.05）。SCF促进p-AKT的表达，c-Kit siRNA抑制c-Kit、p-AKT 的表达。实验组糖尿病裸鼠创面愈合率更高，真皮层与表皮层厚度大（p <0.05）。结论SCF/c-Kit 反应轴激活PI3K/AKT 信号通路，促进ADSCs 增殖、迁移。SCF 预处理能够促进糖尿病裸鼠创面愈合。

Abstract:

To investigate the mechanism of stem cell factor (SCF) / stem cell factor receptor (c-Kit) in regulation of proliferation and migration of adipose-derived stem cells (ADSCs) and its effect on diabetic wound healing. Methods ADSCs were separated from subcutaneous adipose tissues and primarily cultured. ADSCs without any procession were considered as control group. For experimental groups, the cells were treated with SCF (4 ng/ml, 24 h) or c-Kit siRNA combined with SCF (4 ng/ml, 24 h). Then, Western blot was used to detect the expressions of c-Kit, p-AKT, and AKT. The proliferation and migration abilities of ADSCs were observed by CCK-8, colony formation and Transwell assay. The models of diabetic nude mice were established by intraperitoneal injection of STZ and the full-thickness skin of the back (2 cm in diameter) was cut to create injury. All six diabetic mice were randomly divided into experimental group and control group. For the control group, only 1×106 untreated ADSCs were injected around the wound bed. And for the experimental groups, 1×106 ADSCs pretreated with SCF (4 ng/ml) for 24 h were also injected in the woundbed via the same way. The wound healing rate was calculated and the thickness between dermis and epidermis was measured by hematoxylin eosin staining 10 d after model establishment. The differences were compared between the two groups. Results The expression of c-Kit was induced by SCF in a does-and timedependent manner (p < 0.05). Meanwhile, the proliferation and migration of the ADSCs were promoted by SCF but suppressed by c-Kit siRNA (p < 0.05). p-AKT expression was induced by SCF and inhibited by c-Kit siRNA. Compared to the control group, the mice of the experimental groups showed higher wound healing rate and thicker dermis -epidermis distance (p < 0.05). Conclusions The SCF/c -Kit axis can promote the proliferation and migration of ADSCs by activating PI3K/AKT signaling pathway. ADSCs pre-treated with SCF can accelerate wound healing of diabetic nude mice.