作者简介：周法庭 男,硕士在读, 主要研究方向:肿瘤细胞信号系统与作用靶标
通信联系人：刘肖珩 男 1968 年出生， 教授, 博士生导师。主要研究方向：⑴ 组织、细胞生物力学与工程及临床应用; ⑵ 组织工程与人工器官;⑶ 心血管疾病的机理研究;⑷ 生物医学电子学.
(四川大学 华西基础医学与法医学院 生物医学工程实验室(成都 610041))
摘要：研究剪切力对喉鳞癌细胞通透性变化的影响，初步探讨力学刺激影响细胞生理功能的分子机制。方法对喉鳞癌Hep 2 细胞株施加1.4dyn/cm2 的剪切力刺激，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)观察加载力学刺激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 剪切力刺激后，细胞形态呈梭形，撤销力学刺激后，细胞恢复至初始形态;TEM 观察随剪切力加载时间的增加细胞间连接更为密，通透性降低;剪切力诱导Occludin、Claudin-5 和ZO-1 的表达增加，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。
The Effect of Fluid Shear Stress on the Permeability of Laryngeal Squamous Carcinoma Hep2 Cells
Zhou Fating, Liu Xiaoheng, Shen Yang, LIU Shuangfeng, HONG Jinyong, XIA Qing
(Institute of Biomedical Engineering，West China School of Preclinical and Forensic Medicine，Sichuan University,Chengdu 610041)
Abstract: This paper is aimed to investigate the effect of fluid shear stress on the permeability of laryngeal squamous carcinoma (Hep2) cells and to explore the potential molecular mechanism. Hep2 cells were selected and subjected to the 1.4 dyn/cm2 fluid shear stress for different durations, respectively. The morphologic changes of Hep2 cells under shear stress were observed using inverted microscope. The cell-cell junctions were examined by transmission microscope (TEM). The
expressions of tight junction proteins (including Occludin, Claudin-5 and ZO-1) and the distribution of Claudin-5 were examined by Western blot assay and laser scanning confocal microscope, respectively. The results showed that Hep2 cells showed spindle shapes after shear stress stimulation, and recovered to original polygon when shear stress was cancelled. The cell-cell junctions was tight under shear flow condition, the permeability was reduced under the condition of 1.4 dyn/cm2 shear flow. The expressions of tight junction proteins (Occludin, Claudin-5 and ZO-1) were enhanced with increased duration of shear flow, but reduced after removing shear flow. The result of Claudin-5 expression 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 cell-cell junctions after shear flow stimulation. Therefore, we can conclude that shear stress mediates the change of the permeability of laryngeal squamous carcinoma Hep2 cells by by upregulation of the tight junction proteins such as Occludin,
Claudin-5 and ZO-1.
Key words: Fluid shear stress; Laryngeal squamous cell carcinoma; Permeability; Tight junction