摘要
【目的】观察经补肾益精法处理后人脐静脉内皮细胞(HUVEC)来源的外泌体对系统性硬皮病血管损伤的治疗作用。【方法】(1)体外研究:制备大鼠补肾益精中药复方含药血清,通过血管内皮细胞成管实验观察含药血清与对照血清对HUVEC成管的影响,细胞免疫荧光染色法检测血小板内皮细胞黏附分子1(CD31)表达。采用超速离心法提取HUVEC细胞上清液中外泌体,透射电镜观察外泌体形态,Nano Analyzer法检测外泌体径粒与浓度,Western Blot法检测外泌体标志物的表达。设置对照血清干预HUVEC来源的外泌体组(对照血清-外泌体组)和含药血清干预HUVEC来源的外泌体组(含药血清-外泌体组),用外泌体处理HUVEC后,通过成管实验观察HUVEC成管能力,细胞免疫荧光染色法检测CD31表达。(2)体内研究:设置正常组、模型组及外泌体组。采用皮下注射博来霉素(100μL·mg^(-1)·d^(-1))28 d构建硬皮病小鼠模型。此外,模型组及外泌体组小鼠在造模第1、7天分别额外给予外泌体悬液及含药血清-外泌体局部皮下注射。4周后,观察小鼠皮肤组织病理变化,免疫组织化学法检测小鼠皮肤组织血管生成素1(ANG-1)、血管内皮细胞生长因子(VEGF)、血管性假血友病因子(vWF)、CD31表达水平。【结果】与对照组比较,含药血清组与含药血清-外泌体组的HUVEC成管能力及CD31表达均显著增强。电镜下可见类圆形高亮膜性微囊泡,外泌体的标志物表达增强。HE染色结果显示,与模型组比较,外泌体组小鼠皮肤胶原纤维含量有所减少,胶原间隙增宽,血管数量增多;免疫组织化学结果显示外泌体组小鼠皮肤ANG-1、VEGF、v WF、CD31表达水平显著高于模型组,较正常组有所降低。【结论】补肾益精法可通过HUVEC来源的外泌体途径发挥对系统性硬皮病的血管保护作用。
Objective To observe the therapeutic effect of human umbilical vein endothelial cell(HUVEC)-derived exosomes after treatment with Bushen Yijing(tonifying kidney and replenishing essense qi)therapy on vascular injury in systemic scleroderma(SSc).Methods(1)In vitro study:the drug-containing serum was prepared in rats,and the effect of drug-containing serum and control serum on HUVEC tube formation was observed by vascular endothelial cell tube formation assay,and the expression of platelet-endothelial cell adhesion molecule 1(CD31)was detected by cell immunofluorescence staining.Exosomes were extracted from the supernatant of HUVEC cells by ultracentrifugation,and the morphology of exosomes was observed by transmission electron microscopy,the exosome size and concentration were detected by Nano Analyzer,and the expression of exosome markers was detected by Western Blot method.A control serum-interfered HUVEC-derived exosome group(control serum-exosome group)and a drug-containing serum-interfered HUVEC-derived exosome group(drug-containing serum-exosome group)were set up to observe the tube formation ability of HUVEC after treatment with exosomes,and CD31 expression was detected by cell immunofluorescence staining.(2)In vivo study:normal group,model group and exosome group were set up.A mouse model of scleroderma was constructed by subcutaneous injection of Bleomycin(100μL·mg^(-1)·d^(-1))for 28 days.In addition,the mice in the model group and exosome group were additionally given local subcutaneous injection of exosome suspension and drug serum-exosome on the 1st and 7th day of modeling,respectively.After 4 weeks,the histopathological changes in the mouse skin tissues were observed,and immunohistochemistry was performed to detect the expression levels of angiopoietin 1(ANG-1),vascular endothelial growth factor(VEGF),vascular pseudohemophilic factor(vWF)and CD31.Results HUVEC tube-forming ability and CD31 expression were significantly increased in the drug-containing serum group and drug-containing serum-exosome group compared to the control group.The circular and bright membranous microvesicles were observed,and the expression of exosome markers was increased under the electron microscope.HE staining results showed that compared to the model group,the skin collagen fibre content was reduced,the collagen gap was widened and the number of blood vessels was increased in the exosome group;the immunohistochemistry results showed that the expression levels of ANG-1,VEGF,vWF and CD31 in the exosome group were significantly higher than those in the model group,and lower than those in the normal group.Conclusion Bushen Yijing therapy exerts vasoprotective effects in SSc through the exosome pathway derived from HUVEC.
作者
朱珂
谢颂苗
杨海诗
朱佳玲
李东海
齐庆
ZHU Ke;XIE Song-Miao;YANG Hai-Shi;ZHU Jia-Ling;LI Dong-Hai;QI Qing(The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405 Guangdong,China;Shenzhen Longgang Central Hospital,Shenzhen 518116 Guangdong,China;Dongguan Hospital of Traditional Chinese Medicine,Dongguan 523000 Guangdong,China;The Second Hospital Affiliated to Guangzhou Medical University,Guangzhou 510260 Guangdong,China)
出处
《广州中医药大学学报》
CAS
2022年第5期1143-1150,共8页
Journal of Guangzhou University of Traditional Chinese Medicine
基金
国家自然科学基金资助项目(编号:8170150856)
关键词
补肾益精法
系统性硬皮病
外泌体
人脐静脉内皮细胞
小鼠
Bushen Yijing therapy
systemic scleroderma(SSc)
exosome
human umbilical vein endothelialcel(lHUVEC)
mice
