移植研究中X-gal染色的影响因素及优化条件

THE INFLUENCING ELEMENTS AND OPTIMIZATION OF X-gal STAINING METHOD IN THE STUDY OF TRANSPLANTATION

目的 研究在细胞移植过程中 ,利用X gal染色定位lacZ转基因细胞时 ,消除宿主内源性 β 半乳糖苷酶 (β galactosidase ,β Gal)非特异背景的条件。 方法 分别取成年及新生大小鼠 ,侧脑室注入LacZ转基因神经干细胞C17 2后 ,于两种不同灌注条件下取脑 ,在 5个pH点 ,6个孵育时间点行x Gal染色 ,计数不同条件下海马区非特异性着色细胞数 ,得出X gal染色的最佳条件。 结果 X gal染色中非特异染色随pH增高及孵育时间缩短而减少 ,在pH为 9 5 ,孵育时间为 1h时较为理想。并与宿主的种属 ,年龄有密切关系。 结论 X gal染色必须从动物灌注、pH和孵育时间等诸方面加以优化并设立同种属同年龄的对照才可获得客观可信的结果。

Objective To study the influencing elements in x-Gal staining method and optimize the reactive conditions so that nonspecific background can be eliminated and grafted exogenous cells carrying LacZ gene can be discerned correctly. Methods C17 2 cells (carrying lzcZ gene) were injected into the right lateral ventricle both in the adult and newborn animals. After one week they were perfused using two methods, then the slices were stained at different pH and incubating time respectively. The X-gal positive cells in hippocampus were counted under light microscope. Results Background staining in this method has close correlation with the species and age of the host animals and it decreases when pH is higher or incubating time is shorter. The results are the best when pH 9 5 and the incubating time is 1*!h.Conclusion The reliability of X-gal staining method depends on optimization of several parameters, including pH, incubating time, perfusion etc. It is necessary to establish the correspondent controls.;