Etherington D J, Wardale R J
J Cell Sci. 1982 Dec;58:139-48. doi: 10.1242/jcs.58.1.139.
The mononuclear cell fraction of rat hind-limb muscle was obtained by digestion with clostridial collagenase in the presence of calcium ions, filtration through nylon screens and washing to remove the enzyme. Final traces of contaminant myofibrillar debris were separated by isopycnic centrifugation in a Percoll density gradient. Whole muscle, washed cells and Percoll-fractionated cells were extracted in the presence of non-ionic detergent and the supernatants assayed for the lysosomal enzymes cathepsins B + L, N-acetyl-beta-glucosaminidase, beta-glucuronidase and protein. The enzyme levels were highest in the muscle from young rats, but the percentage of recovered activity in the mononuclear cell fraction was little altered by the age of the animal. The values obtained were: cathepsin B + L, 2.4-4.0%; N-acetyl-beta-glucuronidase, 4.3-7.6%; and beta-glucuronidase, 6.3-10.3%. Because of unavoidable losses in preparation these are minimal values and the actual levels of activity from the mononuclear cell fraction in muscle would be higher. The specific activity values of the cell lysates were raised after isopycnic centrifugation and were nearly constant over the age range 65-180 days. Substantially higher specific activity values were obtained for the cells from rats of 38 days. When grown in culture the mononuclear cell fractions were seen to contain mainly fibroblasts and myoblasts with only few leucocytes. The cultures reached confluence by the second week, at which time numerous myotubes had formed. In addition there were groups of large, circular cells with a prominent centrosphere. The origin of these latter cells is uncertain. It was concluded that although total lysosomal enzyme activity was higher in young rats there was little effect of age on the distribution of activity between muscle fibres and mononuclear cells in the muscle.
在钙离子存在的情况下，用梭菌胶原酶消化大鼠后肢肌肉，通过尼龙筛过滤并洗涤以去除酶，从而获得大鼠后肢肌肉的单核细胞部分。通过在Percoll密度梯度中进行等密度离心分离最终残留的污染性肌原纤维碎片。在非离子去污剂存在的情况下提取全肌肉、洗涤后的细胞和经Percoll分级分离的细胞，并测定上清液中的溶酶体酶组织蛋白酶B + L、N - 乙酰 - β - 葡糖胺酶、β - 葡糖醛酸酶和蛋白质。酶水平在幼鼠的肌肉中最高，但单核细胞部分中回收活性的百分比受动物年龄的影响很小。获得的值为：组织蛋白酶B + L，2.4 - 4.0%；N - 乙酰 - β - 葡糖胺酶，4.3 - 7.6%；以及β - 葡糖醛酸酶，6.3 - 10.3%。由于制备过程中不可避免的损失，这些是最小值，肌肉中单核细胞部分的实际活性水平会更高。等密度离心后细胞裂解物的比活性值升高，并且在65 - 180天的年龄范围内几乎恒定。38天大的大鼠的细胞获得了明显更高的比活性值。当在培养中生长时，单核细胞部分主要含有成纤维细胞和成肌细胞，只有少数白细胞。培养物在第二周达到汇合，此时形成了许多肌管。此外，还有一群大的圆形细胞，有一个突出的中心球。这些后一种细胞的起源尚不确定。得出的结论是，尽管幼鼠的总溶酶体酶活性较高，但年龄对肌肉纤维和肌肉中单核细胞之间的活性分布影响很小。
