中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (26): 4202-4208.doi: 10.12307/2024.392

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

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

李斯锦，冯骁腾，王怡茹，刘  萍   

  1. 上海中医药大学附属龙华医院，上海市  200032
  • 收稿日期:2023-05-26 接受日期:2023-07-04 出版日期:2024-09-18 发布日期:2023-10-07
  • 通讯作者: 刘萍，教授，主任医师，上海中医药大学附属龙华医院，上海市 200032
  • 作者简介:李斯锦，女，河南省郑州市人，上海中医药大学在读博士，主要从事中西医结合防治动脉粥样硬化等心脑血管疾病研究。
  • 基金资助:
    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

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

Li Sijin, Feng Xiaoteng, Wang Yiru, Liu Ping   

  1. Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
  • Received:2023-05-26 Accepted:2023-07-04 Online:2024-09-18 Published:2023-10-07
  • Contact: Liu Ping, Professor, Chief physician, Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
  • About author:Li Sijin, MD candidate, Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
  • Supported by:
    National Natural Science Foundation of China, Nos. 82074200 and 81873117 (both to LP); National Natural Science Foundation of China for the Youth, No. 82204849 (to WYR)

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摘要：


文题释义：

特异性启动子：是一段位于结构基因5’-端上游区的DNA序列，能活化RNA聚合酶，使之与模板DNA准确地结合，并具有转录起始的特异性。在该启动子调控下，外源基因的表达一般只发生在某些特定来源的细胞或组织部位。
血管内皮生长因子C：是一种高度特异性的促淋巴管内皮细胞生长因子，也是第一个被发现的促淋巴管生成因子，具有促进淋巴管通透性增加、细胞外基质变性、淋巴管内皮细胞增殖迁移和淋巴管形成等作用。


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5.0×1011 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体1荧光强度随着时间延长逐渐增加，其中主动脉淋巴管内皮透明质酸受体1的mRNA水平、主动脉窦周围及心肌的淋巴管内皮透明质酸受体1荧光强度在28 d时表达量最高(P < 0.05)，其余均在21 d时表达最高，28 d后逐渐减少(P < 0.05)。结果表明，rAAV9-SP146-C1-VEGFC可有效转染动脉粥样硬化模型小鼠骨髓巨噬细胞，并促进淋巴管增生。

https://orcid.org/0000-0002-3230-5170(李斯锦)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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

Abstract: 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 5.0×1011 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 VEGFC in aortic sinus plaques were significantly increased in the 7-day transfection group compared with the control group (P < 0.05, P < 0.01). Serum VEGFC level of mice transfected with rAAV9-SP146-C1-VEGFC gradually increased with time and began to decrease at 28 days. mRNA levels of VEGFC, VEGFR3, Podoplanin and LYVE-1 in mouse aorta and bone marrow-derived macrophages, VEGFC protein level in bone marrow-derived macrophages, VEGFC fluorescence intensity in aortic sinus plaques, LYVE-1 fluorescence intensity around the aortic sinus and in the myocardium gradually increased with time (P < 0.05). In addition, the mRNA level of LYVE-1 in the aorta and the fluorescence intensity of LYVE-1 around the aortic sinus and in the myocardium were the highest at 28 days (P < 0.05), and gradually decreased (P < 0.05). The expression of the other indicators reached the peak at 21 days. To conclude, rAAV9-SP146-C1-VEGFC could effectively transfect bone marrow-derived macrophages and promote lymphatic hyperplasia in atherosclerotic mice.

Key words: atherosclerosis, macrophage, vascular endothelial growth factor C, macrophage specific promoter, aorta

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