具有免疫调节功能的多能干细胞复合凝胶的设计及其修复糖尿病创面的机制研究

Design of Pluripotent Stem Cell Composite Gel with Immunomodulatory Function and Its Mechanism in Repairing Diabetes Wounds

目的探究具有免疫调节功能的多能干细胞复合凝胶的设计及其修复糖尿病创面的机制。方法中国医学科学院医学实验动物研究所于2023年10月采购60只SPF级9~10周龄、250 g雄性大鼠纳入研究。体外实验制备具有免疫调节功能的多能干细胞复合凝胶,提取大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),体外验证BMSCs在丝蛋白微颗粒(silk protein microparticles,LPs)上增殖及形态变化,并检测LPs对BMSCs干性维持及旁分泌功能的影响。随后进行体内实验,构建糖尿病大鼠背部皮肤缺损模型,荧光素酶+/绿色荧光蛋白+慢病毒转染BMSCs。分析丝蛋白纳米纤维(silk fibroin nanofiber,SFN)-LP-BMSCs促进创面血管化、炎症调节、创面的快速上皮化。结果采用Western Blotting检测培养1、7、14 d后的空白对照组(TCP组)、丝蛋白组(LP组)中的BMSCs,LP培养的BMSCs水平的干性指标(Sox2、Oct4、Klf4)较TCP组增加,差异有统计学意义(P均<0.05)。与TCP组相比,LP共培养下BMSCs表达的血管生成素-1、血管内皮生长因子-α、基质细胞衍生因子-1、肝细胞生长因子、碱性成纤维细胞生长因子水平均上升,差异有统计学意义(P均<0.05)。与空白对照组(Blank)、高晶丝蛋白纳米纤维组(SFN)、丝蛋白微颗粒组(SFN-LPs)、骨髓间充质干细胞组(BMSCs)、SFN-BMSCs组相比,SFN-LPs-BMSCs组愈合所用时间最短,差异有统计学意义(P<0.05)。组间两两对比及多组间进行CD31、CD68、M2型巨噬细胞水平比较,差异有统计学意义(P均<0.05)。创面上皮程度随着时间的增加而增加。结论多能干细胞复合凝胶应用于糖尿病创面可以加速创面的愈合及上皮化程度,实现皮肤损伤修复。

Objective To explore the design of pluripotent stem cell composite gel with immunomodulatory function and its mechanism of repairing diabetic wounds.Methods In October 2023,the Institute of Experimental Animal Medicine,Chinese Academy of Medical Sciences purchased 60 SPF 9-10 weeks of age,250 g male rats for inclusion in the study.In vitro,the pluripotent stem cell composite gel with immunomodulatory function was prepared,and rat bone marrow mesenchymal stem cells(BMSCs)were extracted.The proliferation and morphological changes of BMSCs on silk protein microparticles(LPs)were verified in vitro,and the effects of LPs on the stemness maintenance and paracrine function of BMSCs were detected.Subsequently,in vivo experiments were performed to construct a back skin defect model of diabetic rats,and luciferase+/green fluorescent protein+lentivirus was transfected into BMSCs.Silk fibroin nanofiber(SFN)-LP-BMSCs promotes wound vascularization,inflammation regulation and rapid epithelialization of wounds was analyzed.Results Western Blotting was used to detect the BMSCs in the blank control group(TCP group)and fibroin group(LP group)group after 1,7 and 14 days of culture.The stemness indexes(Sox2,Oct4,Klf4)of BMSCs cultured in LP were higher than those in TCP group,and the differences were statistically significant(all P<0.05).Compared with TCP group,the levels of angiopoietin-1,vascular endothelial growth factor-α,stromal cell-derived factor-1,hepatocyte growth factor and basic fibroblast growth factor expressed by BMSCs under LP co culture increased,and the differences were statistically significant(all P<0.05).Compared with the blank control group(Blank),high-crystal fibroin nanofiber group(SFN),silk fibroin microparticles group(SFN-LPs),bone marrow mesenchymal stem cell group(BMSCs)and SFN-BMSCs group,the healing time of SFN-LPs-BMSCs group was the shortest,and the difference was statistically significant(P<0.05).There were significant differences in the levels of CD31,CD68 and M2 macrophages between the two groups and between the groups(all P<0.05).The degree of wound epithelium increased with time.Conclusion The application of pluripotent stem cell composite gel to diabetic wounds can accelerate wound healing and epithelialization,and achieve skin damage repair.