稳态层流下的内皮细胞形态特征变化

Changes in the morphometric feature of endothelial cells under steady flow state

目的：探讨稳态层流下的内皮细胞特征变化规律和血流切应力改变对血管内膜结构的影响。方法：采用胶原酶消化法分离培养小牛肺动脉内皮细胞（ＰＡＥＣ），模拟体内流动环境，建立平行平板流动腔模型。利用细胞定量图像分析系统和ＦＩＴＣ－若丹明－次毒蕈环肽标记法，观察内皮细胞在稳态层流下形态与排列变化。结果：内皮细胞在１．０×１０－ ５，２．５×１０－ ５ ｋＰａ 剪切１８ ｈ 后，细胞趋于切应力作用方向排列，与静息态的细胞相比，长短轴比值变化显著［（１．２０±０．１０） ｖｓ（１．１２±０．０８），（１．３８±０．１５） ｖｓ（１．１２±０．０８），Ｐ＜ ０．０１）］，细胞面积无显著变化；在２．５×１０－ ５ ｋＰａ 剪切作用６ ｈ 后，细胞面积和长短轴比值无显著变化，而细胞内骨架沿切应力方向重组；在２．５×１０－ ５ ｋＰａ剪切作用后的ＰＡＥＣ再次处于静息培养２４ ｈ 后，细胞向静息态细胞转变。结论：血流动力学特性中切应力是引起血管内皮细胞形态发生变化的主要原因，细胞形态的改变是细胞胞质内骨架蛋白分子重组的表现；同时，切应力引起内皮细胞的应变是可逆的，当切应力撤除后，细胞亦趋于低切应力下的形态特征。

Objective: To study the regular rule of changes in endothelial cells morphometric feature under steady flow state, and to investigate the effects of shear stress on the structure of vascular intima. Methods: Bovine pulmonary arterial endothelial cells(PAEC) were isolated and cultured with collagenase digestion. The parallel plated flow chamber device was established to simulate blood flow environment in vitro . Changes in the cell morphometric parameters were analyzed quantitatively before and after shear flow exposure. The patterns of microfilament distribution in cells were observed by stained with rhodamine phalloidin. Results: After 1.0×10 -5 and 2.5×10 -5 kPa shear flow exposure, PAEC were elongated and oriented to the direction of the flow, although there were no changes of cell area, the axis ratio(ratio of major axis and minor axis) changed significantly [(1.20±0.10) vs (1.12±0.08), (1.38±0.15) vs (1.12±0.08), P <0.01]. After 2.5×10 -5 kPa shear flow exposure for 6 h the microfilaments were oriented to the direction of flow with no changes of the cell shapes. PAEC which exposed 2.5×10 -5 kPa shear flow for 18 h were cultured under no flow condition for 24 h again, the axis ratio decreased significantly[(1.12±0.11) vs (1.38±0.15), P <0.05].Conclusion:Shear stress,one of the flow mechanical characters,is major factor which affects vascular endothelial cell morphometric.The changes of cells morphometric is a manifestation of cytoskeleton protein remodeling.It is reversion after withdrawn shear force.