摘要
目的:探讨流体切应力(SS)对体外大鼠脑血管平滑肌细胞(VSMCs)表型转化的影响及相关自噬调控机制。方法:运用倒置相差显微镜和免疫细胞化学方法对大鼠VSMCs进行鉴定后,并给予15.28 dyne/cm 2流体切应力冲击VSMCs 6、 12、24 h。为探讨自噬在VSMCs表型转化中作用,于冲击前6 h给予自噬激动剂(Rapa)和抑制剂(3-MA)干预。实验分为control组、SS组、SS+3-MA组和SS+Rapa组。于冲击后6、12、24 h采用Real-time PCR法检测VSMCs收缩表型及合成表型的标志基因的表达水平;免疫细胞化学和免疫印迹法检测自噬标志蛋白LC-3和Beclin-1的定性定量表达。结果:与control组相比,SS组中VSMCs收缩表型标志基因α-SMA及SM-22a的表达显著下调( P <0.05);合成表型标志基因 MMP-2和 TNF-α的水平显著上调( P <0.05);自噬蛋白LC-3和Beclin-1的表达显著增强( P <0.05);与SS组相比,自噬抑制剂3-MA可显著逆转流体切应力诱导的VSMCs表型转化( P <0.05),与之相反,自噬激动剂Rapa可促进VSMCs的表型转化( P <0.05)。结论:流体切应力可能通过激活细胞自噬诱导体外VSMCs的表型转化,进而参与颅内动脉瘤的发生发展。
Objective: To explore the effects of shear stress(SS)on phenotypic modulation of vascular smooth muscle cells(VSMCs)and related autophgy mechanism through establishing an cell model of intracranial aneurysms in vitro. Methods: VSMCs were identified by inverted phase contrast microscope and immunocytochemical staining,and shear stress of 15.28 dyne/cm 2 was applied on the surfaces in parallel and vertical directions.To investigate the role of autophagy in the phenotypic transition of cultured VSMCs,we pre-incubated cells with an autophagy inhibitor(3-MA)or an autophagy inducer(rapamycin) 6 h before SS treatment.Then,VSMCs were divided into control group,SS group,SS+3-MA group,SS+Rapa group.The expressions of VSMCs contractile and synthetic marker genes were determined by Real-time PCR analysis.Autophagy marker protein LC-3 and Beclin-1 levels were tested by immunocytochemical staining and Western blot assay. Results: Shear stress significantly reduced the expression of contractile marker genes such as α-SMA and SM-22a,whereas increased the levels of matrix metalloproteinase-2(MMP-2)and tumour necrosis factor(TNF-α)in cultured VSMCs.The dramatically elevated LC-3 and Beclin-1 levels indicated autophagy activation in VSMCs exposed to shear stress.Inhibition of autophagy (3-MA)reversed shear-induced VSMCs phenotypic modulation,in contrast,the activation of autophagy(Rapa)promoted this process. Conclusion: Shear stress induced phenotypic modulation of VSMCs via activating autophagy process,which could involved in the development of intracranial aneurysms.
作者
袁广胜
吴海东
陈胜利
李小梅
邵楠
王纪斌
张婷
YUAN Guang-Sheng;WU Hai-Dong;CHEN Sheng-Li;LI Xiao-Mei;SHAO Nan;WANG Ji-Bin;ZHANG Ting(Dongying Shengli Hospital,Dongying 257055,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2019年第11期1337-1340,1353,共5页
Chinese Journal of Immunology
基金
山东省东营市科技计划项目
关键词
颅内动脉瘤
流体切应力
血管平滑肌细胞
表型转化
自噬
Intracranial aneurysms
Shear stress
Vascular smooth muscle cells
Phenotypic modulation
Autophagy
