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首页> 外文期刊>American Journal of Physiology >Characterization of a primary cell culture model of the avian renal proximal tubule.
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Characterization of a primary cell culture model of the avian renal proximal tubule.

机译：禽肾近端小管的原代细胞培养模型的表征。

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Methods have been developed for producing functional, transporting monolayers of avian proximal tubule (PT) cells. A highly homogenous fraction of PT fragments was prepared by enzymatic digestion (collagenase + Dispase) of chick (3- to 5-day-old) kidneys, followed by Percoll gradient centrifugation. The PT fraction was enriched in glucose-6-phosphatase, a proximal enzyme marker, and reduced in specific activity of hexokinase, a distal marker. PT fragments were grown to confluence in serum-free media on collagen-coated permeable filter supports. Electron microscopy of confluent monolayers revealed numerous microvilli and mitochondria, central cilia, and tight junctions, all characteristic of PT cells. gamma-Glutamyltranspeptidase, a proximal brush-border enzyme, showed threefold higher activity on apical than on basolateral sides of the monolayer. The electrophysiological characteristics of monolayers were investigated by voltage-clamp techniques. Monolayers displayed low transepithelial resistances (40-60 Omega . cm2), lumen-negative potentials, and baseline currents of 6-12 microA/cm2 (with or without 5 mM glucose). Both alpha-methyl-D-glucose (2 mM), a nonmetabolizable hexose, and phenylalanine (2 mM) significantly stimulated short-circuit current when added to the mucosal side of glucose-free monolayers. Phloridzin, a specific inhibitor of Na+-coupled glucose transport, significantly inhibited short-circuit current, as did 10(-5) M amiloride. Monolayers also expressed net secretory transport of urate. This cell culture preparation may provide a useful working model for the study of avian PT transport.
机译：已经开发出用于产生功能性，运输性禽近端小管（PT）细胞单层的方法。 PT片段的高度同质级分是通过酶消化（3至5天大的小鸡）肾脏的酶（胶原酶+ Dispase），然后进行Percoll梯度离心来制备的。 PT级分富含葡萄糖-6磷酸酶（一种近端酶标记），而其己糖激酶（一种远端标记物）的比活性降低。 PT片段在胶原蛋白涂层的可渗透滤膜支架上的无血清培养基中生长至汇合。融合单层的电子显微镜检查显示出许多微绒毛和线粒体，中央纤毛和紧密连接，这些都是PT细胞的特征。 γ-谷氨酰转肽酶是一种近端的刷状边界酶，其顶端的活性比单层的基底外侧高三倍。通过电压钳技术研究了单层的电生理特性。单分子膜显示出较低的跨上皮电阻（40-60Ω·cm2），管腔负电位和基线电流为6-12 microA / cm2（有或没有5 mM葡萄糖）。当添加到无葡萄糖单层的粘膜一侧时，不可代谢的己糖α-甲基-D-葡萄糖（2 mM）和苯丙氨酸（2 mM）都会显着刺激短路电流。与10（-5）M阿米洛利一样，Phloridzin是Na +偶联的葡萄糖转运的特异性抑制剂，可显着抑制短路电流。单层也表达尿酸盐的净分泌运输。这种细胞培养物的制备可能为研究禽类PT运输提供有用的工作模型。

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