HO1工程化无细胞提取液联合石墨烯远红外光促糖尿病溃疡修复的疗效及机制  

Efficacy and mechanism of HO1 engineered cell-free extract combined with graphene far-infrared radiation in promoting the repair of diabetic ulcer

作　　者：温晴 范睿诚 杨艺 李红丽 熊玮 WEN Qing;FAN Rui-cheng;YANG Yi;LI Hong-li;XIONG Wei(Department of Geriatrics and Special Service Medicine,First Affiliated Hospital of Army Medical University,Chongqing 400038,China;Department of Histology and Embryology,College of Basic Medicine,Army Medical University/Chongqing Key Laboratory of Neuroscience,Chongqing 400038,China;Basic Education and Experimental Center,College of Basic Medicine,Army Medical University,Chongqing 400038,China)

机构地区：[1]陆军军医大学第一附属医院老年与特勤医学科,重庆400038 [2]陆军军医大学基础医学院组织学与胚胎学教研室/重庆市神经科学重点实验室,重庆400038 [3]陆军军医大学基础医学院基础医学教学实验中心,重庆400038

出　　处：《局解手术学杂志》2023年第4期285-292,共8页Journal of Regional Anatomy and Operative Surgery

基　　金：国家重点研发计划(2018YFC2000301)。

摘　　要：目的探讨HO1工程化无细胞提取液(CE)联合石墨烯远红外光(FIR)在糖尿病溃疡创面修复中的疗效及机制。方法将40只6~8周龄雄性C57BL/6小鼠建立正常创面模型(对照组,n=8)及糖尿病溃疡模型(n=32),其中将糖尿病溃疡模型小鼠随机分为糖尿病组、HO1BMSCs-CE组、FIR组、联合组。构建过表达血红素氧合酶1(HO1)的骨髓间充质干细胞(BMSCs),获取工程化富含HO1蛋白的无细胞提取液(HO1BMSCs-CE),运用Western blot、ELISA、RT-PCR、Masson染色、免疫组化等检测各组创面愈合率、组织修复情况、创面氧化应激及炎症水平等。结果Western blot及ELISA检测结果显示,HO1BMSCs-CE中HO1、表皮生长因子(EGF)、转化生长因子-β(TGF-β)表达水平显著高于BMSCs-CE(P<0.05)。治疗后第5、11天,联合组的创面愈合率均显著高于HO1BMSCs-CE组和FIR组(P<0.05)。与HO1BMSCs-CE组及FIR组相比,联合组在治疗后第3、7、11天创缘组织中Ca2+浓度较高,TNF-α、IL-6表达水平较低,而ROS水平仅在治疗后第3、7天较低(P<0.05)。治疗后第14天,联合组创面组织的表皮厚度较厚,真皮Masson染色胶原容积分数和VEGF免疫阳性信号OD值均显著高于FIR组和HO1BMSCs-CE组(P<0.05);治疗后第7天,联合组创面组织中凋亡相关蛋白Caspase-3表达水平显著低于FIR组和HO1BMSCs-CE组(P<0.05)。结论糖尿病溃疡创面中运用工程化富含HO1蛋白的HO1BMSCs-CE联合石墨烯FIR治疗可明显抑制局部组织氧化应激反应,减少炎症因子释放和细胞凋亡发生,加快创面区域皮肤组织上皮化形成、真皮胶原和血管增生,较HO1BMSCs-CE或FIR治疗的促愈疗效更为突出,为探索临床糖尿病难愈性创面的新型联合治疗提供了基础研究证据。Objective To investigate the efficacy and mechanism of HO1 engineered cell-free extract(CE)combined with graphene far-infrared radiation(FIR)in the repair of diabetic ulcer wounds.Methods A total of 40 male C57BL/6 mice(6 to 8 weeks old)were used to establish normal wound model(the control group,n=8)and diabetic ulcer model(n=32).The diabetic ulcer model mice were randomly divided into the diabetes group,the HO1BMSCs-CE group,the FIR group and the combined group.The bone marrow mesenchymal stem cells(BMSCs)overexpressing heme oxygenase-1(HO1)was constructed to obtain engineered cell-free extract rich in HO1 protein(HO1BMSCs-CE).Western blot,ELISA,RT-PCR,Masson staining and immunohistochemistry were used to detect the wound healing rate,tissue healing status,wound oxidative stress and inflammation levels in each group.Results The results of Western blot and ELISA showed that the expression levels of HO1,epidermal growth factor(EGF)and transforming growth factor-β(TGF-β)in HO1BMSCs-CE were significantly higher than those in BMSCs-CE(P<0.05).The wound healing rate 5 and 11 days after treatment in the combined group were higher than those in the HO1BMSCs-CE group and the FIR group(P<0.05).Compared with the HO1BMSCs-CE group and the FIR group,the Ca2+concentration of the wound margin tissue 3,7 and 11 days after treatment in the combined group were higher,and the expression levels of TNF-αand IL-6 were lower,while the expression levels of ROS 3 and 7 days after treatment were lower(P<0.05).The thickness of epidermis of the wound tissue 14 days after treatment in the combined group was thick,and the Masson staining collagen volume fraction in dermis and the OD value of VEGF immunopositive signal were significantly higher than those in the FIR group and the HO1BMSCs-CE group(P<0.05).The expression levels of apoptosis-related protein Caspase-3 of the wound tissue 7 days after treatment in the combined group was significantly lower than those in the FIR group and the HO1BMSCs-CE group(P<0.05).Conclusion The application

关 键 词：糖尿病溃疡 骨髓间充质干细胞 无细胞提取液 血红素氧合酶1 石墨烯远红外光 

分 类 号：R587.2[医药卫生—内分泌]