摘要
采用活体给药、非连续梯度离心分离和体外培养引入细胞动力阻断剂的技术，建立了裸鼠皮肤表皮基底细胞微核检测方法。实验用６周龄雄性裸鼠，在一次性皮肤涂抹受试化合物４８ｈ后，剥离全层皮肤。经１％胰蛋白酶溶液处理，分离出表皮层，剪碎，置ＤＮＡｓｅ溶液中室温下搅拌，过滤，离心收集细胞。细胞悬液移于Ｐｅｒｃｏｌ溶液，离心留下含基底细胞的底层溶液。细胞置３７℃二氧化碳培养箱培养，４８ｈ后更换新鲜培养基（含细胞松弛素Ｂ３μｇ／ｍｌ），在同样条件下，继续培养４８ｈ后，按常规方法收获细胞。使用氨基甲酸乙酯作为阳性试验物，双核细胞微核率达９７．３‰，丙酮阴性对照物仅为９‰。实验在选定的浓度范围内可获得线性剂量反应曲线；镜下可见单个分离的细胞，细胞边界清楚，胞浆染色均匀，细胞核与微核的染色结构清晰，微核易于判断。
The detection method for micronucleus of cuticular layer basal cell of nude mouse was established by viviadministration of drugs and nonconsecutive gradient centrifugating separation,and the technique of cytodynamic blocking agent led in by culture in vitro. The experimental animals were nude mice aged 6 weeks,after 48 hours of giving them a single application of tested chemical compound, their full layers of skin were stripped and dealt with 1% trypsin solution. The cuticular layer were separated and sheared to pieces and put them into DNAse solution under room temperature and stirred, filtered, and the cells were collected with centrifugation. The cell suspension was put into Percoll solution and centrifugalized to leave basal layer solution contained basal cells. Then put these cells into CO2incubator(37℃) to culture. After 48 hours, these cells were cultured in fresh medium(contained cytochalasin B 3μg/ml) under the same condition for 48 hours and were collected according to routine method. Using urethan as positive test agent,the micronucleus rate of binuclear cell attained to 97.3‰, while the acetone negative control was only 9‰. In the scope of selected concentration, the experiment might obtain linear doseresponse curve; under microscope,the single separated cell might be recognized: the cell border was clear, the cytoplasma staining was homogeneous, the staining structures of nucleus and micronucleus were distinct and the micronucleus was liable to judge.
出处
《华夏医学》
1998年第2期4-6,共3页
Acta Medicinae Sinica
关键词
基底细胞
表皮层
微核检测
鼠
basal cell
cuticular layer
micronucleus detection
nude mouse
