Martin J, Dinsdale D, White I N
MRC Toxicology Unit, Carshalton, Surrey, U.K.
Biochem J. 1993 Oct 1;295 ( Pt 1)(Pt 1):73-80. doi: 10.1042/bj2950073.
A novel procedure for the isolation of Clara cells from rat lung is described. Single-cell suspensions from male F344/TOX rat lungs, prepared by subtilisin digestion, were treated with monochlorobimane (10 microM) and anlaysed by fluorescence-activated flow cytometry. A sub-population of about 2.8% of the total, showing the highest blue fluorescence and by inference the highest GSH concentration, was isolated as Clara cells of > 95% purity. Type II cells of similar purity were sorted after treatment with Phosphine 3R. Both sub-populations were > 90% viable, as judged by Trypan Blue exclusion. Comparison of CYP (cytochrome P-450) isoenzymes between these subpopulations, using Western blotting, showed CYP1A1 to be barely detectable. In Clara cells, CYP2B1 was 10-fold higher than in Type II cells. Mono-oxygenase activity towards the O-deethylation of 3-cyano-7-ethoxycoumarin was 2-fold higher in Clara cells. No activity was detected in macrophages. Pretreating rats with the mono-oxygenase inducers phenobarbitone, 3-methylcholanthrene or Aroclor 1254 showed the last-named to be the most potent inducer of CYP1A1. In Clara cells, CYP1A1 concentration and mono-oxygenase activities were induced > 2000- and 50-fold respectively, whereas in Type II cells increases of 300- and 3.6-fold were seen. Clara cells isolated from the lungs of control rats had a concentration of GSH (2.7 nmol/10(6) cells) that was 9- and 2-fold higher than that in Type II cells or macrophages respectively. GSH depleted by monochlorobimane treatment was restored after 2-3 h incubation with 0.2 M N-acetylcysteine. gamma-Glutamyltranspeptidase activity in Clara cells was 6- and 50-fold higher than in Type II cells or macrophages respectively.
本文描述了一种从大鼠肺中分离克拉拉细胞的新方法。通过枯草杆菌蛋白酶消化制备雄性F344/TOX大鼠肺的单细胞悬液，用单氯联苯胺（10微摩尔）处理后，通过荧光激活流式细胞术进行分析。分离出占总数约2.8%的亚群，其显示出最高的蓝色荧光，据此推断谷胱甘肽浓度最高，纯度>95%，即为克拉拉细胞。用三苯基膦处理后，分选得到纯度相似的II型细胞。通过台盼蓝排斥法判断，两个亚群的活力均>90%。使用蛋白质印迹法比较这些亚群之间的细胞色素P-450（CYP）同工酶，结果显示CYP1A1几乎检测不到。在克拉拉细胞中，CYP2B1比II型细胞高10倍。克拉拉细胞对3-氰基-7-乙氧基香豆素O-脱乙基的单加氧酶活性比II型细胞高2倍。巨噬细胞中未检测到活性。用单加氧酶诱导剂苯巴比妥、3-甲基胆蒽或多氯联苯混合物1254预处理大鼠，结果显示最后一种是CYP1A1最有效的诱导剂。在克拉拉细胞中，CYP1A1浓度和单加氧酶活性分别诱导增加>2000倍和50倍，而在II型细胞中分别增加300倍和3.6倍。从对照大鼠肺中分离的克拉拉细胞的谷胱甘肽浓度（2.7纳摩尔/10^6个细胞）分别比II型细胞或巨噬细胞高9倍和2倍。用单氯联苯胺处理耗尽谷胱甘肽后，与0.2M N-乙酰半胱氨酸孵育2-3小时后可恢复。克拉拉细胞中的γ-谷氨酰转肽酶活性分别比II型细胞或巨噬细胞高6倍和50倍。
