流体剪切力对喉鳞癌Hep2细胞形态和紧密连接的影响

Effect of Fluid Shear Stress on the Cellular Morphology and Tight Junction of Laryngeal Squamous Carcinoma Hep2 Cells

为研究流体剪切力对喉鳞癌细胞(Hep2)紧密连接的影响,对Hep2细胞施加1.4dyn/cm2的流体剪切力,倒置显微镜观察不同时间点细胞的形态学变化;透射电镜(TEM)观察剪切力加载和松弛后细胞胞间紧密连接的变化规律;Western blot检测紧密连接蛋白(Occludin、Claudin-5和ZO-1)的表达;共聚焦显微镜下观察Claudin-5分布及表达情况。结果表明,在流体剪切力作用下,细胞形态由多边形呈梭形变化,撤销力学刺激后,细胞呈向初始形态恢复的趋势;透射电镜观察随剪切力加载时间的增加,细胞间连接更为紧密;Western blot结果显示剪切力诱导紧密连接蛋白Occludin、Claudin-5和ZO-1的表达增加,而撤销剪切力后,其表达均呈现出显著下调;免疫荧光结果表明Claudin-5表达趋势与Western blot结果一致,静态时Claudin-5均匀分布在胞浆,加载剪切力后其分布易位于胞间,撤销力学刺激后,在胞间和胞浆区域仍有Claudin-5的表达。因此,剪切力可改变Hep2细胞形态,并且通过上调Occludin、Claudin-5和ZO-1蛋白增加细胞间的紧密连接。

This paper is aimed to investigate the effect of fluid shear stress on the tight junction of laryngeal squamous carcinoma（Hep2）cells and to explore the potential molecular mechanism.Hep2 cells were selected and subjected to the fluid shear stress of 1.4dyn/cm^2 for different time,respectively.The morphological changes of Hep2 cells under shear stress were observed using inverted microscope.The cell-cell junctions were examined by transmission electron microscope（TEM）.The expressions of tight junction proteins（including Occludin,Claudin-5and ZO-1）and the distribution of Claudin-5were examined by Western blot assay and laser scanning confocal microscope,respectively.The results indicated that Hep2 cells turned to spindle-like shapes after exposed to shear stress,and showed the trend of the recovering to original shapes when the shear stress was cancelled.The cell-cell junctions were tight under the shear flow condition,and the permeability was reduced under the condition of 1.4dyn/cm2 shear flow.The expressions of tight junction proteins were enhanced with increased duration of shear flow,but reduced after removing shear flow.The result of Claudin-5expression by immufluorescence assay was consistent with that by Western blot.The Claudin-5 mainly distributed in the cytoplasm under static condition,while it located at the intercellular after shear flow stimulation,and it appeared intercellular and cytoplasm after stopping shear flow stimulation.Therefore,it can be concluded that shear stress changes the morphology of laryngeal squamous carcinoma Hep2 cells,and upregulates the tight junction.