THE MADIN DARBY CANINE KIDNEY (MDCK) EPITHELIAL-CELL MONOLAYER AS A MODEL CELLULAR-TRANSPORT BARRIER
THE MADIN DARBY CANINE KIDNEY (MDCK) EPITHELIAL-CELL MONOLAYER AS A MODEL CELLULAR-TRANSPORT BARRIER
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DOI：
10.1023/a:1015807904558
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发表时间：
1989-01-01
影响因子：
3.7
通讯作者：
SCIESZKA, JF
中科院分区：
文献类型：
--
作者：
CHO, MJ;THOMPSON, DP;SCIESZKA, JF
两种 Madin Darby 犬肾 (MDCK) 细胞株在具有 3-μm 孔的聚碳酸酯膜上生长，无需任何细胞外基质处理。该膜直径为 2.45 厘米，是市售预灭菌培养插入物的一部分，当放置在常规六孔培养板中时，可提供两个室。人们发现该装置可方便地研究一些选定的液相标记物在 MDCK 细胞单层上的运输。来自美国典型培养物保藏中心 (ATCC) 的菌株和所谓的高抗性菌株 I，在 65 至 70 之间的连续传代中，均显示出接种浓度依赖性滞后期，随后是 21 小时倍增时间的生长期。当播种5次时。 104 个细胞/cm2，在含有 10% 胎牛血清的改良 Eagle's 最低必需培养基 (MEM) 中，在 5% CO2 气氛下，5 天后即可达到细胞汇合。同样，跨上皮电阻（TEER）也在 5 天内达到平台值。光学和电子显微镜检查都显示出明确的连接结构。主要在 37°C 下研究了液相标记物、蔗糖、荧光黄 CH (LY)、胰岛素和葡聚糖穿过 MDCK 细胞单层的转运。 C 遵循顶端到基底外侧以及基底外侧到顶端的方向。对于细胞层制备物之间的给定标记物，观察到稳态传输速率的巨大变化。因此，本研究提出了一种“内部标准”程序，用于对运输速率进行有意义的比较。当标准化为蔗糖速率时，蔗糖:LY:菊粉:葡聚糖的速率比为1.00:0.80:0.67:0.15。该比率实际上与温度、细胞应变、标记物迁移方向和 TEER 值无关，表明存在共同的转运机制。观察到的速率比似乎反映了分子大小和电荷。理论上，本研究中观察到的运输包括细胞旁路分流和跨细胞囊泡运输。对整个运输中的每个运输机制进行定量评估一直很困难。本研究中观察到的最慢运输的[3H]葡聚糖的初始吸收仍比文献值快 300 倍。这似乎表明本研究中观察到的转运主要是通过细胞旁路分流途径。
Two strains of Madin Darby canine kidney (MDCK) cells were grown on a polycarbonate membrane with 3-.mu.m pores without any extracellular matrix treatment. The membrane, 2.45 cm in diameter, which is part of a commercially obtained presterilized culture insert, provides two chambers when placed in a regular six-well culture plate. This device was found to be convenient for investigating transport of a few selected fluid-phase markers across the MDCK cell monolayer. Both the strain from the American Type Culture Collection (ATCC) and the so-called highly resistant strain I, at a serial passage between 65 and 70, showed a seeding concentration-dependent lag phase followed by a growth phase with a 21-hr doubling time. When seeded at 5 .times. 104 cells/cm2, cell confluence was achieved in 5 days in a modified Eagle''s minimum essential medium (MEM) containing 10% fetal bovine serum under a 5% CO2 atmosphere. Similarly, transepithelial electrical resistance (TEER) also reached a plateau value in 5 days. Both light and electron microscopic examinations revealed well-defined junctional structures. Transport of the fluid-phase markers, sucrose, lucifer yellow CH (LY), insulin, and dextran across the MDCK cell monolayers was studied primarily at 37.degree. C following the apical to-basolateral as well as the basolateral-to-apical direction. Large variations in the steady-state transport rate were observed for a given marker between the cell layer preparations. Thus, the present study proposes an "internal standard" procedure for meaningful comparisons of the transport rate. When normalized to the rate of sucrose, the rate ratio was 1.00:0.80:0.67:0.15 for sucrose:LY:inulin:dextran. This ratio was virtually independent of temperature, cell strain, direction of the marker migration, and TEER value, suggesting a common transport mechanism. The observed rate ratio appears to reflect molecular size and charge. The transport observed in the present study would consist, in theory, of both paracellular shunt and transcellular vesicular transport. Quantitative assessment of each transport mechanism in the overall transport has been difficult. The initial uptake of [3H]dextran estimated for the slowest transport observed in the present study was still 300-fold faster than a literature value. This appears to indicate that the transport observed in the present study is largely through the paracellular shunt pathway.