剪切力对脑微血管内皮细胞形态学的影响

　　我们利用自行研制的细胞流动小室对大鼠脑微血管内皮细胞在剪切力作用下的形态学改变进行了初步研究。结果提示脑微血管内皮细胞对剪切力的耐受性要低于大血管内皮细胞，在同样的剪切力和作用时间下，大血管内皮细胞没有明显变化。微血管内皮细胞已有明显的形态学变化，细胞间隙明显增大、细胞皱缩、脱落，这些改变的直接后果是通透性的增加。我们的工作为进一步开展剪切力对微血管内皮细胞功能、代谢等方面的影响提供了实验数据。

　　分类号： 　R318.01;R322.1+3

FLUID SHEAR STRESS-INDUCED MORPHOLOGY CHANGES OF BRAIN

　　MICROVASCULAR ENDOTHELIAL CELL MONOLAYERS IN RAT

Hu Jinlin, Song Xin, Li Xianghong

　　(Laboratory of Microcirculation, Chinese PLA General Hospital, Beijing 100853)

ABSTRACT

　　To determined the effect of fluid shear stress on microvascular endothelial cell monolayers, we designed a flow chamber to investigated the morphology changes after a period of shear stress effect. A coverslip carrying a confluent rat brain microvascular endothelial cell monolayers was inserted into the device and parallel plate flow was given by the chamber. The experiment proceeded with various perfusion rates. The perfusion medium was D-Hank′s balanced salt solution with the viscosity of 0.75mPa.s at 37℃. The shear stress effected on the monolayers were 0.19, 0.38, 0.77 and 1.43 dyn/cm2 by a roller pump for a perfusion period of 1h, 3h and 6h. The whole device was warmed to 37℃ with an air curtain incubator. After a period of perfusion, the coverslip was taken out and then placed on the stage of an inverted tissue culture microscope with camera. The monolayer photos were analyzed by image processing and analysis system. With all the shear stress level, monolayers sharp had no significant change with a period of 1h. After 3h, the intercellular space increased and monolayers were shrinking but the rearrangements of monolayers were not observed. The results showed the significantly injury of monolayers caused by shear stress. After 6h, the monolayers injuries were more exacerbation. Compare to the results of shear stress effects on the great vessels endothelial cell monolayers, our results indicated that the brain microvascular endothelial cell monolayers might take more sensitive to the fluid shear stress. The farther work is needed to demonstrate the relationship between the shear stress and the monolayers permeability and rearrangement.

　　Key words:　Shear stress; Microvessels; Endothelial cell; Flow chamber

　　0　引 言

　　血管的腔侧面被覆一层内皮细胞，血液流动对其形态学及功能都有一定的影响［1～5］，但已有的报道主要研究剪切力对大血管内皮细胞形态、功能的影响，对微血管内皮细胞在剪切力作用下的形态、功能、代谢等方面改变的研究尚少见报道，这可能与微血管内皮细胞的培养较之大血管更为困难有关。我们利用自行设计的剪切力流动小室对大鼠脑微血管内皮细胞在剪切力作用下的形态学改变进行了初步研究，取得了有益的结果，为进一步开展剪切力对微血管内皮细胞功能、代谢等方面的影响提供了实验数据。

　　1　材料和方法

　　1.1　脑微血管内皮细胞的培养

　　1月龄SD大鼠(本院动物中心提供)，6%水合氯醛腹腔麻醉。断头后无菌条件下剥离软脑膜及肉眼可见的大血管，去除大脑白质及小脑，收集大脑皮质，充分剪碎、匀浆后过170μm及80μm滤网，收集滤网上的血管段，离心后加入0.1%Ⅰ型胶原酶(Sigma公司)消化，再经离心、D-Hank′s液冲洗后成内皮细胞悬浮液，接种于培养皿中，待细胞融合达80%后消化传代于8mm×8mm的盖玻片上，传代后7d左右用于实验。

　　1.2　剪切力流动小室的制作

　　流动小室的制作参考Stephen-G等人［1］的实验装置并加以简化改进，小室的剖面图见图1。装置由上下两层有机玻璃制成，下层中央有一个1cm×1cm×0.02cm的正方形槽，可放置长满内皮细胞的盖玻片。此槽上方有一个6cm×1.35cm×0.04cm的长方形槽，与上层有机玻璃共同形成灌流液流动的通道，通道两端各开一个出口用于注入灌流液。上下层有机玻璃之间用医用乳胶垫密封。对于平板槽流，剪切力τ与流量Q及流室几何尺寸的关系为：

　　(1)

　　式中：η为循环液粘度，Q为循环液流量，w为流室宽度，h为流室高度。