Establishment of immotalized cell lines from transgenic mouse nephron segments

根据我们的计划，我们可以得到的结果如下。1)转基因小鼠肾脏永生化培养细胞系的建立。将ts-SV40大t抗原基因注射到C57BL/6小鼠受精卵原核中，获得第一代小鼠。从胶原酶处理的肾脏中显微解剖不同的肾元段并进行培养。这样制造的细胞可以维持100代以上。2)建立细胞系的特性。并与新分离的肾元段细胞系进行了肾元段特异性酶、代谢和激素受体等性状的比较。除了γ - gtp活性、糖异生和甲状旁腺激素受体外，这些细胞系保持了大多数位点特异性特征。3)这些细胞系在药理学和毒理学研究中的应用。来源于末端近端小管的细胞可成功用于顺铂致肾毒性的评价。由于ATP嘌呤受体在外髓集管细胞中高度表达，这些细胞可能是克隆ATP受体蛋白cDNA的合适样本。这项试验目前正在调查中。

The results that we could obtain according to our plan are as follows.1)Establishment of immortalized cultured cell lines from transgenic mouse kidneys.A ts-SV40 large T-antigen gene was injected into pronuclei of fertilized eggs of C57BL/6 mice, and mice of first generation were abtained. Various nephron segments were microdissected from collagenase-treated kidneys and cultivated. Cells thus made can be maintained over 100 passages.2)Characterization of established cell lines.Several characters like nephron segment-specific enzymes, metabolisms, and hormone recepters in the cell lines were compared with those in freshly isolated respective nephron segments. These cell lines maintained most site-specific characters except gamma-GTP activity, gluconeogenesis and porathyroid hormone receptor.3)Application of these cell lines for pharmacological and toxicological studies.Cells originated from the terminal proximal tubule could be successfully applied for the evaluation of cisplatin-induced nephrotoxicity. Since ATP-purinoceptor was highly expressed in cells form outer medullary collecting duct, these cells might be suitable sample for cloning cDNA of ATP receptr protein (s) . This trial is now under investigation.