人脐带间充质干细胞条件培养基促进放创复合伤创面愈合及其机制

Effect and mechanism of action of human umbilical cord mesenchymal stem cell conditioned medium in promoting the healing of combined radiation and wound injury

摘要

目的 探索人脐带间充质干细胞条件培养基（human umbilical cord mesenchymal stem cell conditional medium，uMSC-CM）对放创复合伤（combined radiation and wound injury，CRWI）的促愈作用及相关机制。 方法 42只雄性C57BL小鼠被随机分成单纯创伤组、放创复合伤对照组和放创复合伤治疗组（n=14）；以4 Gy γ射线全身辐照联合直径1 cm全层皮肤缺损创面构建小鼠皮肤放创复合伤模型，放创复合伤治疗组隔天1次腹腔注射5 mg/kg uMSC-CM，其余2组注射等体积无血清DMEM培养基，采用拍照及HE染色评估创面愈合情况；CD31免疫荧光染色检测血管形成；α-SMA免疫组化染色检测细胞迁移；Masson染色检测胶原沉积；Ki67和TUNEL染色检测增殖和凋亡；Western blot检测凋亡相关蛋白和 PI3K/AKT信号通路蛋白表达水平；小鼠皮肤原代成纤维细胞分为对照组、辐照组和辐照治疗组；采用Edu染色和集落形成实验检测增殖；细胞划痕和Transwell实验检测迁移；流式细胞术检测凋亡；Western blot检测PI3K/AKT信号通路蛋白表达水平。 结果 uMSC-CM对CRWI创面具有明显促愈效果（P<0.05）；组织学结果提示，uMSC-CM可促进CRWI创面血管生成、细胞迁移和胶原纤维沉积，进一步分析显示uMSC-CM处理后创面细胞增殖增加且凋亡减少（P<0.05）；组织Western blot结果显示，uMSC-CM促进CRWI创面PI3K、AKT蛋白磷酸化表达水平（P<0.05）；体外细胞实验结果显示，uMSC-CM促进辐照后小鼠真皮成纤维细胞增殖和迁移并减少凋亡（P<0.05）；Western blot结果显示，uMSC-CM促进辐照后成纤维细胞PI3K、AKT蛋白磷酸化（P<0.05）。 结论 uMSC-CM加速CRWI创面愈合，这可能与激活PI3K/AKT信号通路促进成纤维细胞的增殖、迁移并抑制凋亡有关。

Abstract

Objective To investigate the effect and mechanism of action of human umbilical cord mesenchymal stem cell conditional medium（uMSC-CM） in promoting the healing of combined radiation and wound injury（CRWI）. Methods A total of 42 male C57BL mice were randomly divided into trauma group，CRWI control group，and CRWI treatment group，with 14 mice in each group. A mouse model of skin CRWI was established by whole body irradiation with 4 Gy γ-ray combined with full-thickness skin defect wound with a diameter of 1 cm. The mice in the CRWI treatment group were given intraperitoneal injection of 5 mg/kg uMSC-CM once every other day，while those in the other two groups were given injection of an equal volume of serum-free DMEM medium. Photographs and HE staining were used to assess wound healing；CD31 immunofluorescent staining was used to observe neovascularization；α-SMA immunohistochemical staining was used to observe cell migration；Masson staining was used to evaluate collagen deposition；Ki67 and TUNEL staining were used to measure proliferation and apoptosis；Western blot was used to measure the expression levels of apoptosis-related proteins and PI3K/AKT signaling pathway proteins. Primary skin fibroblasts of mice were divided into control group，irradiation group，and irradiation treatment group. Edu staining and colony formation assay were used to detect cell proliferation；cell scratch assay and Transwell assay were used to detect cell migration；flow cytometry was used to measure cell apoptosis；Western blot was used to measure the expression levels of PI3K/AKT signaling pathway proteins. Results In this study，uMSC-CM significantly promoted the healing of CRWI（P<0.05），and the histological results showed that uMSC-CM could promote angiogenesis，cell migration，and collagen fiber deposition in CRWI. Further analysis showed that uMSC-CM increased cell proliferation and reduced cell apoptosis in CRWI（P<0.05）. Western blot showed that uMSC-CM promoted the protein expression levels of phosphorylated PI3K and phosphorylated AKT（P<0.05）. In vitro cell experiments showed that uMSC-CM promoted the proliferation and migration of mouse dermal fibroblasts and reduced apoptosis after irradiation（P<0.05），and Western blot showed that uMSC-CM promoted the protein expression levels of phosphorylated PI3K and phosphorylated AKT after irradiation（P<0.05）. Conclusion This study shows that uMSC-CM accelerates CRWI wound healing，possibly by activating the PI3K/AKT signaling pathway to promote the proliferation and migration of fibroblasts and inhibit their apoptosis.

关键词

人脐带间充质干细胞 / 条件培养基 / 放创复合伤 / 创面愈合 / PI3K/AKT信号通路

Key words

human umbilical cord mesenchymal stem cell / conditioned medium / combined radiation and wound injury / wound healing / PI3K/AKT signaling pathway

基金资助

国家自然科学基金重点资助项目(82030056)