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

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 endothelialization-promoting TEBVs displayed better biocompatibility for favorable of cell adhesion and growth.In 30 and 90 d after transplantation,radiological studies displayed that the NPs-treated vessels showed better vascular patency than the other groups.HE and Masson staining indicated that in 90 d after transplantation,the collagen and blank CS groups had blood vessels deformed,without normal structures and blocked due to the thrombosis and collagen fibrosis,while the NPs group had intact blood vessels and better patency.Immunofluorescence assay for CD31 and vWF showed that the lumen in the vessles from the NPs group had ECs well-covered,suggesting better endothelialization when compared with other groups.Conclusion Endothelialization-promoting TEBVs shows better promoting effect on endothelialization,and is helpful in maintenance of long-term patency of blood vessels after transplantation.