Genestie I, Morin J P, Guery C, Bode G, Lorenzon G
Département de Toxicologie, Roussel Uclaf, Romainville, France.
In Vitro Cell Dev Biol Anim. 1997 Oct;33(9):692-702. doi: 10.1007/s11626-997-0127-x.
The aim of this study was to better characterize rabbit proximal kidney tubule cells cultured on collagen IV-coated porous inserts, as compared to the same cells seeded in standard plastic wells. Total protein contents in confluent monolayers on permeable membranes were about twofold higher than those measured in confluent cultures in plastic wells. Microscopy examinations suggested that such a difference was probably due to a higher cell density and to an impressive development of the apical brush-border membrane. Moreover, measurement of unidirectional transport of p-aminohippuric acid and tetraethylammonium bromide confirmed the high polarization level of cultures on porous inserts. Results of methyl(alpha-D-[U-14C]glyco)pyranoside uptake suggested that cell phenotype was probably influenced by culture conditions. Analysis of different markers as a function of time in culture showed decreases of alkaline phosphatase (AP), gamma-glutamyltranspeptidase (GGT), and Na(+)-K(+)-ATPase activities as well as increases in LDH, ATP, and glutathione levels, similar to those formerly reported for cells cultured in standard plastic plates. However, comparative data from 6-d-old monolayers have shown that AP, GGT, Na(+)-K(+)-ATPase, glutathione reductase (GRED), and selenium-dependent glutathione peroxidase (Se-GPX) activities were 2.8-, 2.6-, 1.6-, 1.2-, and 2.1-fold, respectively, better preserved on precoated permeable membranes. On the other hand, this paper reports for the first time in the literature that GRED and SE-GPX, two phase II detoxification enzymes, were well maintained in cultures of rabbit proximal kidney tubule cells. Our results show that culturing rabbit proximal kidney tubule cells on collagen IV-coated porous membranes was accompanied by an improvement of both morphological and biochemical properties of the cells.
本研究的目的是，与接种于标准塑料孔板中的相同细胞相比，更好地描述在包被有IV型胶原蛋白的多孔插入物上培养的兔近端肾小管细胞的特征。可渗透膜上汇合单层细胞中的总蛋白含量比塑料孔板中汇合培养物中测得的总蛋白含量高约两倍。显微镜检查表明，这种差异可能是由于细胞密度较高以及顶端刷状缘膜的显著发育。此外，对对氨基马尿酸和溴化四乙铵单向转运的测量证实了多孔插入物上培养物的高极化水平。甲基（α-D-[U-14C]糖）吡喃糖苷摄取结果表明，细胞表型可能受培养条件影响。分析不同标志物随培养时间的变化，结果显示碱性磷酸酶（AP）、γ-谷氨酰转肽酶（GGT）和Na(+)-K(+)-ATP酶活性降低，而乳酸脱氢酶（LDH）、ATP和谷胱甘肽水平升高，这与先前报道的在标准塑料板中培养的细胞情况相似。然而，来自6日龄单层细胞的比较数据表明，在预包被的可渗透膜上，AP、GGT、Na(+)-K(+)-ATP酶、谷胱甘肽还原酶（GRED）和硒依赖性谷胱甘肽过氧化物酶（Se-GPX）活性分别得到了2.8倍、2.6倍、1.6倍、1.2倍和2.1倍的更好保存。另一方面，本文首次在文献中报道，两种II期解毒酶GRED和SE-GPX在兔近端肾小管细胞培养物中得到了良好维持。我们的结果表明，在包被有IV型胶原蛋白的多孔膜上培养兔近端肾小管细胞，细胞的形态和生化特性均得到了改善。
