同型半胱氨酸通过上调Rap1A表达促进小鼠巨噬细胞增殖及M1极化  

Homocysteine promotes proliferation and M1 polarization of mouse macrophages through up-regulating Rap1A expression

作　　者：杨亚丽 田荣 袁茵 王艳佳 杨晓玲[1,2,3] YANG Ya-li;TIAN Rong;YUAN Yin;WANG Yan-jia;YANG Xiao-ling(School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China;National Health Commission Key Laboratory of Metabolic Cardiovascular Diseases Research,Yinchuan 750004,China;Ningxia Key Laboratory of Vascular Injury and Repair Research,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学基础医学院,宁夏银川750004 [2]国家卫生健康委员会代谢性心血管疾病研究重点实验室,宁夏银川750004 [3]宁夏血管损伤与修复研究重点实验室,宁夏银川750004

出　　处：《中国病理生理杂志》2021年第12期2113-2121,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81760095,No.81960063);宁夏科技创新领军人才项目(No.KJT2016009)。

摘　　要：目的:研究Ras相关蛋白1A(Rap1A)对同型半胱氨酸(Hcy)诱导的ANA-1小鼠腹腔巨噬细胞增殖和M1极化的影响,并探讨其作用机制。方法:体外培养ANA-1细胞,建立高Hcy血症细胞模型,并将细胞分成对照(control)组、Hcy组、Hcy+Rap1A干扰腺病毒(Ad-shRNA/Rap1A)组和Hcy+阴性对照腺病毒(Ad-shRNA/GFP)组。采用CCK-8法和EdU染色检测各组细胞的增殖能力;ELISA检测细胞培养液中炎症因子白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的浓度;RT-qPCR检测各组细胞Rap1A、增殖细胞核抗原(PCNA)、细胞周期抑制蛋白p27、M1极化标志物CD80和CD86及M2极化标志物CD163和CD200R的mRNA表达;Western blot检测Rap1A、PCNA和p27蛋白表达。结果:与control组相比,Hcy组细胞活力、EdU阳性细胞数目、细胞培养液中IL-6和TNF-α浓度、CD80和CD86的mRNA表达水平及Rap1A和PCNA的mRNA和蛋白表达水平均显著升高(P<0.05),而CD163和CD200R的mRNA表达水平及p27的mRNA和蛋白表达水平均显著降低(P<0.05)。与Hcy+Ad-shRNA/GFP组相比,Hcy+AdshRNA/Rap1A组细胞活力、EdU阳性细胞数目、CD80和CD86的mRNA表达水平及Rap1A和PCNA的mRNA和蛋白表达水平均显著降低(P<0.05),CD163和CD200R的mRNA表达水平及p27的mRNA水平和蛋白表达水平均显著升高(P<0.05)。结论:Hcy能够促进小鼠巨噬细胞增殖及M1极化,其机制与Hcy上调Rap1A表达有关。AIM:To investigate the effect of Ras-related protein 1A(Rap1A)on the proliferation and M1 polarization of ANA-1 mouse macrophages induced by homocysteine(Hcy),and to explore the related mechanism.METHODS:An in vitro ANA-1 cell culture model was established for studying hyperhomocysteinemia,and the cultured cells were randomly divided into control group,Hcy group,Hcy+Rap1A interfering adenovirus(Ad-shRNA/Rap1A)group and Hcy+negative control(Ad-shRNA/GFP)group.CCK-8 assay and EdU staining were employed to detect cell proliferation.The concentrations of inflammatory factors interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in cell culture supernatant were measured by ELISA.RT-qPCR was used to detect the mRNA expression of Rap1A,proliferating cell nuclear antigen(PCNA),cell cycle inhibitory protein p27,CD80/CD86(M1 polarization markers),and CD163/CD200R(M2 polarization markers).Western blot was used to detect the protein expression of Rap1A,PCNA and p27.RESULTS:Compared with control group,the cell viability,the numbers of EdU-positive cells,the concentrations of IL-6 and TNF-α,the mRNA expression of CD80 and CD86,and the expression of Rap1A and PCNA at both mRNA and protein levels in Hcy group were significantly increased(P<0.05),while the mRNA expression of CD163 and CD200R,and the expression of p27 at both mRNA and protein levels were significantly decreased(P<0.05).Compared with Hcy+Ad-shRNA/GFP group,the cell viability,the numbers of EdU positive cells,the mRNA expression of CD80 and CD86,and the mRNA and protein expression of Rap1A and PCNA in Hcy+Ad-shRNA/Rap1A group were significantly reduced(P<0.05),while the CD163 and CD200R mRNA expression,and the p27 expression at both mRNA and protein levels were significantly increased(P<0.05).CONCLUSION:Homocysteine promotes the proliferation and M1 polarization of mouse macrophages,and the corresponding mechanism is involved in the up-regulation of Rap1A expression induced by Hcy.

关 键 词：同型半胱氨酸 Ras相关蛋白1A 巨噬细胞 细胞增殖 M1极化 

分 类 号：R343[医药卫生—基础医学] R363