压应力超负荷诱导BV2小胶质细胞活化的模型建立  

Model establishment of BV2 microglia activation induced by compressive stress overload

作　　者：徐海光 茶健美 张扬[1] 赵子文 郭秋哲 闫芳冰 马嘉[1] XU Hai-guang;CHA Jian-mei;ZHANG Yang;ZHAO Zi-wen;GUO Qiu-zhe;YAN Fang-bing;MA Jia(Department of Ophthalmology,First Affiliated Hospital of Kunming Medical University,Kunming 650000,Yunnan,China;School of Pharmaceutical Science and Yunnan Key Laboratory of Pharmacology for Natural Products,Kunming Medical University,Kunming 650500,Yunnan,China;Department of Cardiac Surgery,Fuwai Yunnan Hospital,Chinese Academy of Medical Sciences,Kunming 650102,Yunnan,China)

机构地区：[1]昆明医科大学第一附属医院眼科,云南昆明650000 [2]昆明医科大学药学院暨云南省天然药物药理重点实验室,云南昆明650500 [3]云南省阜外心血管病医院心脏外科,云南昆明650102

出　　处：《生物医学工程与临床》2024年第6期749-755,共7页Biomedical Engineering and Clinical Medicine

基　　金：云南省科技厅基础研究计划青年项目(202101AU070116);昆明医科大学第一附属医院博士科研基金资助项目(2020BS005)。

摘　　要：目的青光眼是全球第一大不可逆致盲眼病,视网膜小胶质细胞活化所致异常免疫调节和神经节细胞凋亡是其主要机制之一,压应力升高是青光眼特征性应力改变。建立离体BV2小胶质细胞压应力超负荷模型,为进一步研究青光眼力学机制奠定基础。方法选择BV2小胶质细胞系,分为低压应力组、高压应力组和对照组3组,每组样本6个。通过前期构建并改良的离心培养装置,对低压应力组、高压应力组BV2小胶质细胞施加30 mmHg、50 mmHg 48 h持续压力刺激。通过细胞骨架染色计算细胞面积,采用3-(4,5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺基苯基)-2H-四唑鎓(MTS)法检测细胞活性,评估压应力干预对细胞形态和活性的影响。通过Western blot和聚合酶链式反应(PCR)方法检测小胶质细胞活化标志物肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6)mRNA和蛋白表达。结果使用改良的离心装置可以对BV2细胞施加持续的压力刺激,孵箱环境下离心培养48 h后,3组细胞活性差异无统计学意义(OD值1.23±0.36、1.20±0.35、1.28±0.38。P>0.05)。细胞骨架染色以对照组细胞平均面积为基准,对照组、低压应力组、高压应力组细胞相对面积依次增大(1.00±0.22 vs 1.22±0.26 vs 1.36±0.36。P<0.05)。通过PCR观察到高压应力组TNF-α、IL-1β、IL-6 mRNA水平显著高于对照组(P<0.05);通过Western blot也检测到高压应力组细胞TNF-α、IL-6蛋白水平显著升高(P<0.05)。结论通过离心培养方式,可以有效对离体培养的BV2小胶质细胞施加持续压应力刺激,压应力刺激可以诱导BV2细胞活化,该模型为压应力相关的青光眼力学机制研究奠定了方法学基础。Objective To establish compressive stress overload model of BV2 microglia in vitro,and lay the foundation for further study of glaucoma mechanical mechanism,base on the fact that glaucoma is the first irreversible blindness disease in the world,abnormal immune regulation and ganglion cell apoptosis caused by activation of retinal microglia is one of the main glaucoma mechanisms,and elevated compressive stress is the characteristic stress change in glaucoma.Methods BV2 microglia cell line was selected and divided into 3 groups:low-pressure stress stimulation group,high-pressure stress stimulation group and control group,with 6 samples in each group.The pre-constructed and modified centrifugal culture device was used to applied 30 mmHg and 50 mmHg continuous pressure stimulation for 48-hour to BV2 microglia in low-pressure stress stimulation group and high-pressure stress stimulation group.The cell area was calculated by cytoskeleton staining,and cell activity was detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)method to evaluate the effect of compressive stress intervention on cell morphology and activity.The mRNA and protein expressions of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)were detected by Western blot and polymerase chain reaction(PCR).Results The continuous pressure stimulation was applied to BV2 cells with modified centrifugal device.After 48-hour centrifugal culture in incubator environment,there was no significant difference in cell viability in 3 groups[optical density(OD)values 1.23±0.36,1.20±0.35,1.28±0.38.P>0.05].The cytoskeleton staining was based on mean area of cells in control group,and relative cell area of control group,low-pressure stress stimulation group and high-pressure stress stimulation group increased successively(1.00±0.22 vs 1.22±0.26 vs 1.36±0.36.P<0.05);the mRNA levels of TNF-α,IL-1βand IL-6 in high-pressure stress stimulation group were significantly higher than those in contro

关 键 词：青光眼 压应力 视网膜小胶质细胞 体外模型 

分 类 号：R775[医药卫生—眼科]