改良的小鼠原代肝细胞分离纯化方法

An improved method for isolation hepatocytes of mice

目的:建立一种简便、经济、高效的小鼠肝细胞分离和纯化方法。方法:对传统的两步原位胶原酶灌流法进行改进,缩短消化时间和改变灌注方式;分离的小鼠肝细胞,采用低速离心(300 r/min,1min,3～5次)纯化,台盼兰染色检测活率,糖原染色鉴定肝细胞,并与单密度梯度离心纯化法进行比较。结果:原代肝细胞经低速离心纯化后,活率达到90%左右,纯度达95%以上,与密度梯度离心法效果相近。结论:建立了一种新的小鼠原代肝细胞的分离纯化方法。

Objective：To develop a convenient,inexpensive and efficiency method for separate high-purity primary hepatocytes isolation of murine, Methods：The improved method was used with low concentration type Ⅳ collagenase. Purified hepatocytes were separated from the dissociative cells by low-speed eentrifugation （300 r/min, l min,3-5 times）. The survival rate was measured by typan blue exclusion and the purity was measured by PAS staining. The time,viability and purity of two methods were compared. Results： The cell viability was higher than that of cells obtained by traditional method,and the quantity of collagenase and the time used was decreased markedly. The purity was nearly the same as the method of percoll purity. Conclusion：A new method for separate highpurity primary hepatoeytes isolation was sullessfully established.