摘要
目的 探究siRNA沉默微管乙酰化转移酶1(alpha tubulin acetyltransferase 1,α-TAT1)对肝肺综合征(hepatopulmonary syndrome,HPS)中内皮细胞迁移行为的影响。方法 通过在线数据库Tabula Muris分析肺组织各细胞亚群α-TAT1的表达。24只雄性SD大鼠按照随机数字表法分为对照组(Sham组,n=6)、胆管结扎组(HPS组,n=18)。造模后在第2、4周分别取材HPS组大鼠,通过免疫荧光共定位检测2周和4周HPS大鼠肺血管内皮细胞中α-TAT1表达水平变化。收集Sham和HPS大鼠血清处理人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs) 12 h和24 h,之后进行小分子RNA(small interfering RNA,siRNA)干扰实验,分为Sham血清+siRNA NC组、Sham血清+siRNA α-TAT1组、HPS血清+siRNA NC组、HPS血清+siRNA α-TAT1组。Western blot检测各组HUVECs中α-TAT1、Ace-α-tubulin表达水平变化。免疫荧光观察各组诺考达唑(10μmol/L)预处理后HUVECs中α-Tubulin微管聚合水平,评估微管的结构稳定性;细胞划痕实验联合细胞免疫荧光观察HUVECs的细胞迁移能力和高尔基体的定位。体外成血管实验检测HUVECs成血管能力。结果 体内及体外实验表明HPS中肺血管内皮细胞α-TAT1表达显著增加(P<0.01)。siRNA α-TAT1干扰组中α-TAT1和Ace-α-tubulin表达量均显著下调,且微管稳定性减弱(P<0.01)。另外,siRNA α-TAT1干扰组中GM130标记的高尔基体在核周细胞突出段方向的分布比例降低,且迁移能力下降(P<0.01)。同时,成血管长度及网络水平显著减弱(P<0.01)。结论 沉默α-TAT1可减弱HPS中内皮细胞迁移和成血管能力,其机制与减弱微管稳定性有关。
Objective To investigate the effect of silencing alpha tubulin acetyltransferase 1(α-TAT1) on migration behavior of endothelial cells induced by hepatopulmonary syndrome(HPS).Methods Online database Tabula Muris was used to analyze the expression of α-TAT1 in various cell subsets in the lungs.Twenty-four male SD rats were randomly divided into control group(Sham group,n=6) and common bile duct ligation group(HPS group,n=18).The rats in HPS group were euthanasized at 2 and 4 weeks after modelling,and then the expression of α-TAT1 in pulmonary vascular endothelial cells was detected by immunofluorescence colocalization.The sera from the Sham and HPS rats were used to stimulate human umbilical vein endothelial cells(HUVECs) for 12 and 24 h,respectively.Then the obtained HUVECs were divided into 4 groups:Sham serum + siRNA NC group,Sham serum + siRNA α-TAT1 group,HPS serum+ siRNA NC group,HPS serum + siRNA α-TAT1 group.The expression levels of α-TAT1 and Ace-α-tubulin in HUVECs were detected by Western blotting.Immunofluorescence assay was applied to observe the levels of polymerized microtubules of α-Tubulin in HUVECs after nocodazole(10 μmol/L) pretreatment to evaluate the stability of microtubule structure.Cell scratch assay combined with cell immunofluorescence assay was employed to observe the nuclear localization of Golgi apparatus and cell migration ability of HUVECs.The angiogenesis ability of HUVECs was tested by in vitro angiogenesis test.Results In vivo and in vitro experiments showed that the expression of α-TAT1 in endothelial cells was significantly increased after HPS inducement.The expression levels of α-TAT1 and Ace-α-tubulin were significantly down-regulated,and the stability of microtubules was weakened in the siRNA α-TAT1 interference group(P<0.01).In addition,the distribution of GM130 labeled Golgi apparatus in the protrusion of HUVECs was down-regulated in the siRNA α-TAT1 interference group,as well as the migration ability(P<0.01).And the length of angiogenesis and network level w
作者
刘畅
朱佳熙
刘亚楠
徐木
陈璟莉
LIU Chang;ZHU Jiaxi;LIU Yanan;XU Mu;CHEN Jingli(Department of Anesthesiology,Wuhan Central Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan,Hubei Province,431600,China;Hubei Provincial Key Laboratory of Molecular Diagnosis,Wuhan,Hubei Province,431600,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2024年第3期215-224,共10页
Journal of Army Medical University
基金
国家自然科学基金青年科学基金(82000584)。
关键词
肝肺综合征
内皮细胞
病理性血管新生
α-TAT1
迁移极性
hepatopulmonary syndrome
endothelial cells
pathological angiogenesis
α-TAT1
polarity of migration
