摘要
目的:探讨神经生长因子(NGF)改善糖尿病大鼠随意皮瓣缺血坏死的分子机制。方法:①建立Wistar大鼠I型糖尿病模型,将这些大鼠随机分为模型组(DM组)和实验组(DM+NGF组)两组,设计9 cm×3 cm的改良McFarlane皮瓣并进行皮瓣移植,观察两组皮瓣的存活情况并统计存活面积。采集大鼠皮瓣组织,利用HE染色和免疫组化染色研究皮瓣存活与血管生成的关系,通过Western blot实验研究两组CD31、VEGF血管指标蛋白表达差异以及AGE-RAGE/MAPK-ERK1/2信号通路的变化。②利用建立的高糖人脐静脉内皮细胞(HUVEC)模型,将细胞实验分为细胞模型组(DM组)和细胞实验组(DM+NGF)组两组,通过CCK-8增殖实验、MCM2+细胞免疫荧光实验、细胞划痕实验、Transwell实验和血管成管实验,分析在高糖环境下NGF对HUVEC细胞的增殖、迁移和血管形成的影响。利用STRING数据库探讨NGF对VEGF、SDF1α、HIF-1α、PDGFA、TGF-β1等常见血管生成相关因子的互作情况以及功能富极化分析;通过RT-qPCR和Western blot验证NGF对其表达的影响。通过RNA-seq测序和Western blot分析NGF对HUVEC的影响机制。结果:动物实验方面:DM+NGF组比DM组皮瓣存活面积多(P<0.01);CD31免疫组化染色发现DM+NGF组CD31阳性微血管密度比DM组高(P<0.01),且DM+NGF组CD31和VEGF表达均较DM组高(P<0.05);而皮瓣组织AGE-RAGE/MAPK-ERK1/2信号通路的RAGE下降(P<0.05),而p-ERK1/2上调(P<0.05)。细胞实验方面:成功建立了25 mmol/L高糖模型,100 ng/mL NGF能促进HUVEC的增殖(P<0.01);与DM组比,DM+NGF组的细胞迁移和细胞血管生成能力明显加强(P<0.01),且DM+NGF组的VEGF、PDGFA、HIF-1α、Arg-1和bFGF相对表达量更高(P<0.05),而KEGG和GO功能富集化富集结果主要与血管形成和AGE-RAGE/MAPK-ERK1/2信号通路途径等相关。结论:NGF可能通过影响AGE-RAGE/MAPK-ERK1/2信号通路促进糖尿病大鼠内皮细胞血管生成,从而改善随意皮瓣的缺血坏死。
Objective:To explore the molecular mechanism of nerve growth factor(NGF)in improving the ischemic necrosis of random skin flap in diabetic rats.Methods:A Wistar rat model of type 1 diabetes was established,who were randomly divided as the DM group(model group)and the DM+NGF group(experimental group).A modified McFarlane flap measuring 9 cm×3 cm was designed and manufactured for skin flap transplantation.The survival of the flaps in both groups was observed,and the survival area was measured.Rat flap tissues were collected to investigate the relationship between flap survival and angiogenesis using HE staining and immunohistochemical staining.Western blot experiments were carried out to study the differences in CD31 and VEGF protein expression between the two groups and the changes in the AGE-RAGE/MAPK-ERK1/2 signaling pathway.Using the established high-glucose human umbilical vein endothelial cell(HUVEC)model,the cell experiment was divided into two groups:cell model group(DM group)and experimented cell group(DM+NGF group).The effects of NGF on cell proliferation,migration,and angiogenesis in HUVECs under high-glucose conditions were analyzed.This was done through CCK-8 proliferation assays,MCM2+cell immunofluorescence assays,scratch assays,Transwell assays,and angiogenesis assays.The interaction and functional enrichment analysis of NGF with common angiogenesis-related factors such as VEGF,SDF1α,HIF-1α,PDGFA,and TGF-β1 was made using the STRING database.The effect of NGF on their expression was validated through RTqPCR and Western blot.The impact of NGF on HUVECs was further investigated through RNA-seq and Western blot experiments to explore the underlying mechanisms.Results:After successful animal modeling,it was observed that the DM+NGF group had a 10%-20%larger flap survival area compared with the DM group(P<0.01).Immunohistochemical staining of CD31 revealed a higher microvessel density in the DM+NGF group(284.2±44.76 mm2)compared with the DM group(P<0.01),along with increased expression of CD31 and VE
作者
王晓武
朱仙懂
王永强
吴志炫
温知楷
吴大洲
陈吉彩
WANG Xiaowu;ZHU Xiandong;WANG Yongqiang;WU Zhixuan;WEN Zhikai;WU Dazhou;CHEN Jicai(Department of Hepatobiliary and Pancreatic Surgery,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China;Department of Thyroid Surgery,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China;Department of Breast Surgery,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China;Department of Hernia and Abdominal Wall Surgery,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China)
出处
《温州医科大学学报》
CAS
2024年第2期87-98,共12页
Journal of Wenzhou Medical University
基金
浙江省自然科学基金项目(84120045G)
温州市基础性科研项目(Y2020938)。
