摘要
目的:探究微小核糖核酸(mi R)-152-3p调控果蝇Notch同源物1(Notch1)/Delta样配体4(DLL4)通路对家兔深II度烧伤创面血管生成的影响。方法:将50只新西兰家兔随机分为对照组、模型组、mi R-152-3p拮抗剂(antagomir)组、mi R-152-3p antagomir阴性对照+空载组、mi R-152-3p antagomir+Notch1敲低组,每组10只,除对照组外其余各组家兔构建深II度烧伤模型,分组给药处理后,实时荧光定量聚合酶链式反应(qRT-PCR)检测各组家兔创面组织mi R-152-3p与Notch1、DLL4 m RNA表达;检测各组家兔创面愈合率及微循环血流灌注值(MPD);免疫组织化学染色检测各组家兔创面微血管密度(MVD);酶联免疫吸附反应(ELISA)检测各组家兔血清血管内皮细胞生长因子(VEGF)及促血管生成素1(Ang1)水平;免疫印迹检测各组家兔创面组织VEGF、Ang1与Notch1/DLL4通路蛋白表达;双荧光素酶报告基因实验检测兔脐静脉内皮细胞中mi R-152-3p对Notch1及DLL4的靶向调节。结果:与对照组相比,模型组家兔创面组织mi R-152-3p与Notch1、DLL4 m RNA表达升高(P<0.05),创面MPD及MVD、血清VEGF及Ang1水平、创面组织VEGF与Ang1蛋白表达降低(P<0.05)。与模型组相比,mi R-152-3p antagomir组家兔创面组织mi R-152-3p m RNA表达降低(P<0.05),创面愈合率、创面MPD及MVD、血清VEGF及Ang1水平、创面组织Notch1、DLL4 m RNA及蛋白表达、创面组织VEGF与Ang1蛋白表达升高(P<0.05);mi R-152-3p antagomir阴性对照+空载组家兔各指标无明显差异(P>0.05);与mi R-152-3p antagomir组相比,mi R-152-3p antagomir+Notch1敲低组家兔创面组织mi R-152-3p m RNA表达无明显差异(P>0.05),创面愈合率、创面MPD及MVD、血清VEGF及Ang1水平、创面组织Notch1、DLL4 m RNA及蛋白表达、创面组织VEGF与Ang1蛋白表达降低(P<0.05)。mi R-152-3p可靶向下调兔脐静脉内皮细胞中Notch1及DLL4的表达。结论:敲低mi R-152-3p可通过上调Notch1/DLL4通路而增强家兔深II度烧伤创面血管生成,进而促进其创面愈合。
Objective: To investigate the influences of microRNA(mi R)-152-3p on angiogenesis of rabbits with deep II-degree burn wounds by regulating Drosophila Notch homolog 1(Notch1)/Delta-like ligand 4(DLL4) pathway. Methods: 50 new Zealand rabbits were randomly divided into control group, model group, mi R-152-3p antagomir(antagomir) group, mi R-152-3p antagomir negative control combined empty load group, and mi R-152-3p antagomir combined Notch1 knockdown group, with 10 rabbits in each group, except the control group, the rabbits in the other groups were constructed with deep II-degree burn models. After group administration, real-time fluorescence quantitative PCR(qRT-PCR) was performed to detect the expressions of mi R-152-3p and Notch1, DLL4 m RNAs in wound tissue of rabbits in each group. The wound healing rate and microcirculation perfusion value(MPD) of rabbits in each group were detected.The immunohistochemical staining was performed to detect the wound microvessel density(MVD) of rabbits in each group. The levels of serum vascular endothelial growth factor(VEGF) and angiopoietin 1(Ang1) of rabbits in each group were detected by enzyme-linked immunosorbent assay(ELISA). Western blot was performed to detect the expressions of VEGF, Ang1 and Notch1/DLL4 pathway proteins in the wound tissues of rabbits in each group. The targeted regulation of Notch1 and DLL4 by mi R-152-3p in rabbit umbilical vein endothelial cells was detected by dual-luciferase reporter gene assay. Results: Compared with the control group, the expressions of mi R-152-3p, Notch1 and DLL4 m RNA in the wound tissues of the rabbits in the model group were increased(P<0.05), the MPD and MVD in the wound, the levels of serum VEGF and Ang1, and the expressions of VEGF and Ang1 proteins in the wound tissues were decreased(P<0.05). Compared with the model group, the expression of mi R-152-3p in the wound tissues of rabbits in the mi R-152-3p antagomir group was decreased(P<0.05), the wound healing rate, MPD and MVD in the wound, the levels of serum VEGF and Ang1,the protein expressions of Notch1 and DLL4 m RNA in wound tissues, and the protein expressions of VEGF and Ang1 in wound tissues were increased(P<0.05). There was no obvious difference in each index of rabbits in mi R-152-3p antagomir negative control combined empty load group(P>0.05). Compared with the mi R-152-3p antagomir group, there was no obvious difference in the expression of mi R-152-3p m RNA in the wound tissues of the rabbits in the mi R-152-3p antagomir combined Notch1 knockdown group(P>0.05), the wound healing rate, MPD and MVD in the wound, the levels of serum VEGF and Ang1, the protein expressions of Notch1 and DLL4m RNA in wound tissues, and the protein expressions of VEGF and Ang1 in wound tissues were decreased(P<0.05). The mi R-152-3p could target down-regulate the expressions of Notch1 and DLL4 in rabbit umbilical vein endothelial cells. Conclusion: Knockdown of mi R-152-3p can enhance the angiogenesis of rabbits with deep II-degree burn wounds by up-regulating the Notch1/DLL4 pathway, thereby promoting wound healing.
作者
倪少俊
王成
方鸿
杨军
吴柏磊
NI Shao-jun;WANG Cheng;FANG Hong;YANG Jun;WU Bai-lei(Plastic Burn Surgery,The Fifth Affiliated(Zhuhai)Hospital of Zunyi Medical University,Zhuhai,Guangdong,519100,China)
出处
《现代生物医学进展》
CAS
2023年第3期440-447,共8页
Progress in Modern Biomedicine
基金
珠海市科技计划医疗卫生项目(ZH22036201210084PWC)
广东省医学科学技术研究基金项目(A201811302)。
