Hefley T J, Stern P H, Brand J S
Exp Cell Res. 1983 Nov;149(1):227-36. doi: 10.1016/0014-4827(83)90394-4.
The enzymatic isolation of cells with bacterial collagenase has proved to be a powerful technique for the study of a wide variety of tissues. Unfortunately, for some applications such as the isolation of cells from membranous bone, the cellular damage that results from the exposure of the cells to cytotoxic contaminants of bacterial collagenase has limited the usefulness of this approach. The use of chromatographically purified collagenase alone is often ineffective or very slow to release cells from tissue. We have found that two enzymes are necessary and sufficient to isolate cells from neonatal mouse calvaria: purified collagenase and neutral protease. These two enzymes can be chromatographically purified on a preparative scale to yield 100 mg amounts of each enzyme. The purified enzymes can be recombined in amounts which will digest calvaria at the same rate as the crude bacterial collagenase from which they were derived. The cells that are isolated using the purified enzymes are undamaged, as indicated by the measurement of their equilibrium density on gradients of Ficoll and sodium metrizoate. Cells isolated with crude collagenase never reach an equilibrium density upon isopyknic centrifugation, whereas cells isolated with the purified enzymes reach an equilibrium density of 1.074 g/ml in 90 min.
用细菌胶原酶进行细胞的酶解分离已被证明是研究多种组织的一项强大技术。不幸的是，对于某些应用，如从膜性骨中分离细胞，细胞因暴露于细菌胶原酶的细胞毒性污染物而导致的损伤限制了这种方法的实用性。单独使用经色谱纯化的胶原酶通常在从组织中释放细胞方面效果不佳或非常缓慢。我们发现，从新生小鼠颅骨中分离细胞需要两种酶，且这两种酶就足够了：纯化的胶原酶和中性蛋白酶。这两种酶可以在制备规模上进行色谱纯化，每种酶可产生100毫克的量。纯化后的酶可以按一定量重新组合，其消化颅骨的速度与它们所源自的粗制细菌胶原酶相同。使用纯化酶分离得到的细胞未受损伤，这通过在Ficoll和甲泛葡胺梯度上测量它们的平衡密度得以表明。用粗制胶原酶分离的细胞在等密度离心时从未达到平衡密度，而用纯化酶分离的细胞在90分钟内达到1.074克/毫升的平衡密度。
