摘要
目的 探讨负载小鼠胎盘来源间充质干细胞(placental derived mesenchymal stem cells, PDMSCs)甲基丙烯酰化明胶(methacrylated gelatin, GelMA)水凝胶对糖尿病小鼠创面愈合的作用及其机制。方法 采用光固化法制备PDMSCs-GelMA水凝胶,通过扫描电子显微镜观察PDMSCs-GelMA水凝胶的微观结构,采用5-乙炔基-2′-脱氧尿嘧啶核苷(5-ethynyl-2-deoxyuridine5, EdU)检测水凝胶中PDMSCs的增殖情况;采用活细胞/死细胞双染色法检测水凝胶中PDMSCs的存活情况;采用酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)检测含有PDMSCs-GelMA水凝胶的浸提液中表皮生长因子(epidermal growth factor, EGF)和血管内皮生长因子(vascular endothelial growth factor, VEGF)表达情况,依据是否含有水凝胶将培养的PDMSCs分为水凝胶组和无水凝胶组,水凝胶浸提液组和无水凝胶浸提液组。利用打孔器建立小鼠背部全层皮肤损伤模型,并将小鼠分为磷酸盐缓冲溶液(phosphate buffered saline, PBS)组、PDMSCs组和PDMSCs-GelMA组,每组9只小鼠,各组小鼠分别予以等体积PBS、PDMSCs及PDMSCs-GelMA处理,在第1、7、14天分别观察小鼠背部创面愈合情况,同时采用苏木精-伊红(hematoxylin-eosin, HE)染色和Masson染色分别检测第7天创面皮肤组织重塑和真皮胶原组织情况,免疫组织化学染色检测第7天皮肤创面表皮干细胞、新生血管以及炎症情况。结果 成功制备PDMSCs-GelMA水凝胶,扫描电子显微镜观察到PDMSCs-GelMA呈多孔且相互连接的内部结构,细胞生长良好且充满孔隙。EdU法检测证实含有水凝胶组的PDMSCs增殖率明显高于无水凝胶组(P<0.05)。活细胞/死细胞双染色法结果显示有水凝胶组的PDMSCs活性略高于无水凝胶组,但两组比较差异无统计学意义(P>0.05)。ELISA结果显示,培养第3、7天,水凝胶浸提液组中的EGF、VFGF浓度均低于无水凝胶浸提液组(P均<0.05)。与PBS组比较,PDMSCs组和PDMSCs-GelMA组小鼠的伤口愈合率显著提高(P均<0.05);与PDMSCs组比较,PDMSCs-GelMA组小鼠的伤口愈合率显著提高(P<0.05)。HE染色和Masson染色结果提示,PDMSCs-GelMA组创面新生肉芽组织和胶原沉积率明显高于PBS组和PDMSCs组。术后第7天,与PBS组比较,PDMSCs组和PDMSCs-GelMA组的Brdu和CD11b的阳性数均升高,且PDMSCs-GelMA组高于PDMSCs组(P均<0.05);CD45阳性数均降低,且PDMSCs-GelMA组低于PDMSCs组(P均<0.05)。结论 PDMSCs-GelMA水凝胶具备良好的营养渗透性,PDMSCs-GelMA水凝胶通过促进糖尿病创面肉芽组织增生、新生微血管形成和上皮化,同时减轻炎症反应,最终促进创面愈合。
Objective To explore the effect and mechanism of methacrylated gelatin(GelMA)hydrogel loaded with mice placental derived mesenchymal stem cells(PDMSCs)in promoting wound healing in diabetic mellitus mice.Methods PDMSCs-GelMA hydrogel was prepared by light curing method,the microstructure of PDMSCs-GelMA hydrogel was observed through scanning electron microscope,5-e-thynyl-2-deoxyuridine5(EdU)was used to detect the cell proliferation of PDMSCs in hydrogel;live-dead cell stai-ning was used to detect the cell survival rate of PDMSCs in hydrogel;the expressions of epidermal growth factor(EGF)and vascular endothelial growth factor(VEGF)of leach liquor in PDMSCs-GelMA hydrogel was detected by en-zyme-linked immunosorbent assay(ELISA),the cultured PDMSCs were divided into hydrogel group and non-hydrogel group,hydrogel extract group and non-hydrogel extract group depending on whether it contained hydrogel or not.The full-thickness skin injury model was established on the back of mice by using hole punch,and the mice were divided into phosphate buffered saline(PBS)group,PDMSCs group and PDMSCs-GelMA group,with 9 mice in each,the mice in each group was treated with equal volumes of PBS,PDMSCs and PDMSCs-GelMA respectively,the healing of the back wound of mice was observed on the 1st,7th and 14th days of the treatment,hematoxylin-eosin(HE)staining and Mas-son staining were used to detect the collagen tissue in the epidermis and dermis of the wound skin tissue at the 7th day,immunohistochemistry staining was used to detect epidermal stem cells,neovascularization and inflammation in the skin wound at the 7th day.Results PDMSCs-GelMA hydrogel was successfully prepared,scanning electron microscopy ob-served that PDMSCs-GelMA exhibited highly porous and interconnected internal structure and the cells grew well and full of pores.EdU assay confirmed that the proliferation rate of PDMSCs with hydrogel was significantly higher than that in non-hydrogel group(P<0.05).Live-dead cell staining showed that cytoactive of PDMSCs in hydrogel group was slight-ly higher than that in the non-hydrogel group,but there was no statistically significant difference between the two groups(P>0.05).ELISA results showed that on the 3rd and 7th days of the culture,the concentrations of EGF and VFGF in the hydrogel extract group were lower than those in the non-hydrogel extract group(all P<0.05).Compared with the PBS group,the wound healing rate of mice in PDMSCs group and PDMSCs-GelMA group increased(all P<0.05);com-pared with PDMSCs group,the wound healing rate of PDMSCs-GelMA group significantly increased(P<0.05).The re-sults of HE staining and Masson staining showed that the new granulation tissue and collagen deposition rate in PDMSCs-GelMA group were significantly higher than those in PBS group and PDMSCs group.On the 7th day after operation,compared with PBS group,the positive numbers of Brdu and CD11 b in PDMSCs group and PDMSCs-GelMA group in-creased,and the expression of PDMSCs-GelMA group was higher than that of PDMSCs group(all P<0.05);the CD45 positive number decreased,and the number of PDMSCs-GelMA group was lower than those of PDMSCs group(all P<0.05).Conclusion PDMSCs-GelMA hydrogel has good permeability of nutrients,PDMSCs-GelMA hydrogel promotes diabetic mellitus wound healing by promoting granulation tissue proliferation,neovascularization and epithelialization,while reducing inflammatory response.
作者
吴海斌
秦晶
张翌
WU Haibin;QIN Jing;ZHANG Yi(Department of General Surgery,General Hospital of Central Theater Command,Wuhan Hubei 430070,China)
出处
《联勤军事医学》
2025年第9期748-755,共8页
Military Medicine of Joint Logistics
基金
湖北省自然基金面上项目(2023AFB1044)
武汉市知识创新项目(2023020201020524)。
关键词
胎盘来源间充质干细胞
甲基丙烯酰化明胶
水凝胶
创面愈合
糖尿病
炎症反应
Placental derived mesenchymal stem cells
Methacrylated gelatin
Hydrogel
Wound healing
Diabe tes mellitus
Inflammatory response
