巨噬细胞特异性启动子SP146-C1增强血管内皮生长因子C在动脉粥样硬化小鼠中的表达  

Macrophage-specific promoter SP146-C1 enhances vascular endothelial growth factor C expression in atherosclerotic mice

作　　者：李斯锦 冯骁腾 王怡茹 刘萍[1] Li Sijin;Feng Xiaoteng;Wang Yiru;Liu Ping(Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院,上海市200032

出　　处：《中国组织工程研究》2024年第26期4202-4208,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(82074200,81873117),项目负责人:刘萍;国家自然科学基金青年项目(82204849),项目负责人:王怡茹。

摘　　要：背景:携带巨噬细胞特异性启动子SP146-C1(Synthetic promoter 146-C1)及外源性基因血管内皮生长因子C(vascular endothelial growth factor C,VEGFC)的重组9型腺相关病毒(recombinant adeno-associated virus serotype 9,rAAV9)在动脉粥样硬化组织中的表达效率还不确定。目的:探究rAAV9-SP146-C1-VEGFC在动脉粥样硬化小鼠中的表达效率及对淋巴管生成的影响。方法:取30只ApoE^(-/-)小鼠给予高脂饮食喂养12周构建动脉粥样硬化模型,随机数字表法分为转染7,14,21,28,35 d组各5只,尾静脉注射5.0×10^(11) vg rAAV9-SP146-C1-VEGFC,对照组5只小鼠尾静脉注射等量对照病毒rAAV9-SP146-C1-Scramble。分别于转染后7,14,21,28,35 d时麻醉后处死,留取血清、股骨、胫骨、心脏及主动脉组织,对照组于7 d时同法取材。各组小鼠的股骨和胫骨用于提取骨髓原代巨噬细胞,RT-qPCR检测骨髓原代巨噬细胞及主动脉中VEGFC、血管内皮细胞生长因子受体3、平足蛋白、淋巴管内皮透明质酸受体1的基因表达;Western blot检测骨髓原代巨噬细胞及主动脉中VEGFC的蛋白表达水平,ELISA检测小鼠血清中VEGFC水平,免疫荧光检测主动脉窦中VEGFC的表达,主动脉周围及心肌中淋巴管内皮透明质酸受体1的表达。结果与结论:①与对照组比较,转染7 d组小鼠血清VEGFC水平增加,主动脉及骨髓巨噬细胞中VEGFC、血管内皮细胞生长因子受体3、平足蛋白、淋巴管内皮透明质酸受体1的mRNA表达增高,骨髓巨噬细胞中VEGFC蛋白表达增高,主动脉窦斑块的VEGFC荧光强度增高(P<0.05,P<0.01);②转染rAAV9-SP146-C1-VEGFC的各组小鼠血清VEGFC水平随时间延长逐渐增加,在28 d时开始减少;主动脉及骨髓巨噬细胞中VEGFC、血管内皮细胞生长因子受体3、平足蛋白、淋巴管内皮透明质酸受体1的mRNA水平、骨髓巨噬细胞中VEGFC蛋白水平、主动脉窦斑块的VEGFC荧光强度、主动脉窦周围及心肌的淋巴管内皮透明质酸受�BACKGROUND:The expression efficiency of recombinant adeno-associated virus serotype 9(rAAV9)carrying the macrophage-specific promoter synthetic promoter 146-C1(SP146-C1)and the exogenous gene vascular endothelial growth factor C(VEGFC)in atherosclerosis is uncertain.OBJECTIVE:To investigate the expression efficiency of rAAV9-SP146-C1-VEGFC in atherosclerotic mice and its effect on lymphangiogenesis.METHODS:Thirty ApoE^(-/-) mice were fed high-fat diet for 12 weeks to establish atherosclerosis models and were randomly divided into six groups,five in each group:7-,14-,21-,28-,and 35-day transfection groups and control group.The mice in the transfection groups were transfected with 5.0×10^(11) vg rAAV9-SP146-C1-VEGFC by caudal vein injection.In the control group,the mice were injected with the same amount of control virus rAAV9-SP146-C1-Scramble.Animals in the first five groups were killed under anesthesia at 7,14,21,28 and 35 days after transfection,respectively,and those in the control group were killed under anesthesia at 7 days.Serum,femur,tibia,heart and aorta tissue samples were collected and retained in each group.The femur and tibia of mice in each group were used to extract bone marrow-derived macrophages.The gene expression of vascular endothelial growth factor C(VEGFC),vascular endothelial growth factor receptor 3(VEGFR3),Podoplanin and lymphatic vessel endothelial hyaluronan receptor-1(LYVE-1)in bone marrow-derived macrophages and the aorta were detected by RT-qPCR.VEGFC protein expression levels in bone marrow-derived macrophages and the aorta were detected by western blot,serum level of VEGFC was detected by ELISA,and VEGFC expression in the aortic sinus and LYVE-1 expression around the aorta and in the myocardium was detected by immunofluorescence.RESULTS AND CONCLUSION:The serum level of VEGFC,the mRNA expression of VEGFC,VEGFR3,Podoplanin,and LYVE-1 in bone marrow-derived macrophages and the aorta,the protein expression of VEGFC in bone marrow-derived macrophages,and the fluorescence intensity of V

关 键 词：动脉粥样硬化 巨噬细胞 血管内皮生长因子C 巨噬细胞特异性启动子 主动脉 

分 类 号：R459.9[医药卫生—治疗学] R332[医药卫生—临床医学] R543.1