人参皂苷Rg1促进移植大鼠的组织工程血管内皮化  

Ginsenoside Rg1 promotes endothelialization of tissue-engineered blood vessels in transplanted rats

作　　者：王恬然 聂志强 邹灏 霍达 许有前 朱楚洪[1] WANG Tianran;NIE Zhiqiang;ZOU Hao;HUO Da;XU Youqian;ZHU Chuhong(State Key Laboratory of Trauma,Burns and Combined injury,Department of Human Anatomy,National and Regional Engineering Laboratory for Tissue Engineering,National and Regional Engineering Laboratory for Vascular Implants,Key Laboratory of Biomechanics and Tissue Engineering of Ministry of Education,Chongqing Key Laboratory for Biomechanics and Tissue Engineering,College of Basic Medical Sciences,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]陆军军医大学(第三军医大学)基础医学院人体解剖学教研室,组织工程国家地方联合工程实验室,血管植入物国家地方联合工程实验室,烧伤、创伤与复合伤国家重点实验室,教育部生物力学与组织工程重点实验室,重庆市生物力学与组织工程重点实验室,重庆400038

出　　处：《第三军医大学学报》2020年第5期444-452,共9页Journal of Third Military Medical University

基　　金：国家重点研发计划(2016YFC1101100)。

摘　　要：目的包载人参皂苷Rg1(Ginsenoside Rg1,Rg1)构建促内皮化的组织工程血管(tissue-engineered blood vessels,TEBVs),并移植大鼠颈总动脉以观察其通畅情况和内皮化效果。方法CCK-8和流式细胞凋亡检测培养48 h后Rg1对内皮细胞(endothelial cells,ECs)增殖和凋亡的影响,同时确定药物适宜工作浓度;将ECs分为对照组(普通培养基)、Rg1处理组(15.63μmol/L Rg1培养基)和联合处理组[15.63μmol/L Rg1+0.5μg/mL血管内皮生长因子受体-2(VEGFR2)抑制剂XL184培养基],检测各组细胞培养48 h后增殖和划痕愈合情况;构建壳聚糖包被的Rg1-(2-羟丙基)-γ-环糊精颗粒(Rg1-HGC/CS NPs,以下简称NPs)并修饰至胶原血管内腔面,进行FTIR、DLS和SEM检测;体外条件下,将ECs与脱细胞血管和构建的TEBVs共培养48 h,观察其生物相容性;动物实验分为胶原组(胶原处理的血管)、空白CS组(空白CS处理的胶原组血管)和实验组(NPs处理的胶原组血管),将其移植至SD大鼠的左侧颈总动脉;分别于30、90 d后使用超声和Micro-CT检查血管通畅情况;使用HE、Masson和(CD31+vWF)免疫荧光观察90 d后血管的内皮化效果。结果Rg1通过VEGFR2对ECs产生促进增殖和迁移的作用(P<0.05),未见明显的凋亡影响;所构建的NPs粒径约为130 nm,大小均一(PDI<0.3),并已成功修饰至血管内腔面;共培养实验表明,所构建的TEBVs生物相容性较好,利于细胞贴附生长;移植30、90 d后的影像学检测显示,实验组较其他组通畅度良好;移植90 d后的HE、Masson切片染色显示,胶原组和空白CS组的血管发生形变,失去正常结构,且因血栓和胶原纤维增生而发生阻塞,而实验组血管外形完整且无明显阻塞;CD31和vWF免疫荧光显示,实验组血管内腔面均匀覆有ECs,内皮化效果较其他组良好。结论所构建的促内皮化TEBVs可促进血管的内皮化进程,维持血管移植后的远期通畅。Objective To prepare the endothelialization-promoting tissue-engineered blood vessels(TEBVs)with encapsulated Ginsenoside Rg1(Rg1)to promote endothelialization,and to evaluate its patency and endothelialization after implantation into the rat common carotid artery.Methods CCK-8 assay and flow cytometry were used to detect the effects of Rg1 on the proliferation and apoptosis of endothelial cells(ECs)after 48 hours’treatment,and the optimal dose of the agent was also determined.ECs was divided into control group,Rg1 treatment group(15.63μmol/L Rg1),and Rg1+XL184(15.63μmol/L Rg1+0.5μg/mL XL184,VEGFR2 inhibitor)treatment group.Cell proliferation and wound healing of ECs were examined after the treatment for 48 h.Chitosan coated Rg1-(2-hydroxypropyl)-cyclodextrin particles(Rg1-HGC/CS NPs,abbreviated as NPs)were prepared and modified to the lumen of blood vessels(after decellularization and collagen treatment),and then Fourier transform infrared(FTIR)spectroscopy,dynamic light scattering(DLS)and scanning electron microscopy(SEM)were used to detect these obtained samples.ECs were co-cultured with acellular vessels and TEBVs for 48 h to observe the biocompatibility.For in vivo study,the acellular vessels were divided into collagen group(collagen treatment),blank CS group(collagen and chitosan treatment),and NPs group(modified with NPs).Then these vessel samples were transplanted into the left common carotid artery of SD rats respectively.Ultrasound and micro-CT scanning were performed to observe the vascular patency in 30 and 90 d after transplantation.HE and Masson staining and immunofluorescence assay for CD31 and vWF were used to observe the endothelialization in each group in 90 d after transplantation.Results Rg1 promoted the proliferation and migration of ECs via VEGFR2(P<0.05),and showed no significant effect on apoptosis.The prepared NPs were about 130 nm in diameter and uniform in shape(PDI<0.3),and could be successfully modified to the lumen of blood vessels.The co-cultured study showed that endothelializ

关 键 词：组织工程血管 内皮细胞 人参皂苷RG1 内皮化 

分 类 号：R285.5[医药卫生—中药学] R329.28[医药卫生—中医学]