Isolation and characterization of terminally differentiated chicken and rat skeletal muscle myoblasts.
Isolation and characterization of terminally differentiated chicken and rat skeletal muscle myoblasts.
复制标题
终末分化的鸡和大鼠骨骼肌成肌细胞的分离和表征。
DOI：
10.1016/0012-1606(82)90003-3
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发表时间：
1982
影响因子：
2.7
通讯作者：
Coleman,JR
中科院分区：
文献类型：
--
作者：
Konieczny,SF;McKay,J;Coleman,JR
在鸡胚腿肌、大鼠成肌细胞系L 6和L 8以及小鼠成肌细胞系G8的培养物中研究了骨骼肌成肌细胞在没有自发融合的情况下分化的能力。在低Ca 2+（25 μm）、Mg 2+耗尽的培养基中培养48-96小时后，鸡成肌细胞仅表现出3-5%的融合，而在对照培养物中高达64%的细胞融合。Mg 2+的消耗导致成纤维细胞的优先消除，结果是在120小时时剩余的97%的单核细胞表现出双极形态，并用针对M-肌酸激酶、骨骼肌肌球蛋白和结蛋白的抗体染色。单核成肌细胞很少表现出可见的横纹或M线染色与抗肌裂素，除非培养基中补充有0.81 mMMg 2+，这表明Mg 2+在肌节组装中起作用。条件下的Ca 2+和Mg 2+耗尽抑制成肌细胞融合的啮齿动物细胞系，以及，但单核成肌细胞未能在这些条件下分化。从大鼠细胞系和鸡细胞培养物中获得分化的单个成肌细胞，当融合被细胞松弛素B（CB）中的生长抑制时。CB处理的大鼠成肌细胞培养物中积累的MM-CK比活性几乎是年龄相当的广泛融合对照培养物中发现的两倍。CB处理过程中积累的球形细胞被分离出来，并显示出含有近8倍的CK比活性存在于来自相同培养物的非球形细胞中。大约90%的这些细胞表现出免疫荧光染色与骨骼肌肌球蛋白的抗体，未能纳入[3 H]胸苷或形成克隆传代培养的集落，因此代表终末分化的大鼠成肌细胞。对单个细胞核的定量显微荧光DNA测量表明，在这些实验中从鸡和大鼠获得的终末分化成肌细胞含有2cDNA水平，表明在细胞周期的G 0期阻滞。
The ability of skeletal muscle myoblasts to differentiate in the absence of spontaneous fusion was studied in cultures derived from chicken embryo leg muscle, rat myoblast lines L6 and L8, and the mouse myoblast line G8. Following 48–96 hr of culture in a low-Ca2+(25 μm), Mg2+-depleted medium, chicken myoblasts exhibited only 3–5% fusion whereas up to 64% of the cells fused in control cultures. Depletion of Mg2+led to preferential elimination of fibroblasts, with the result that 97% of the mononucleated cells remaining at 120 hr exhibited a bipolar morphology and stained with antibodies directed against M-creatine kinase, skeletal muscle myosin, and desmin. Mononucleated myoblasts rarely showed visible cross-striations or M-line staining with anti-myomesin unless the medium was supplemented with 0.81 mMMg2+, suggesting that Mg2+plays a role in sarcomere assembly. Conditions of Ca2+and Mg2+depletion inhibited myoblast fusion in the rodent cell lines as well, but mononucleated myoblasts failed to differentiate under these conditions. Differentiated individual myoblasts from rat cell lines and from chicken cell cultures were obtained when fusion was inhibited by growth in cytochalasin B (CB). CB-treated rat myoblast cultures accumulated MM-CK to nearly twice the specific activity found in extensively fused control cultures of comparable age. Spherical cells which accumulated during CB treatment were isolated and shown to contain nearly eight times the CK specific activity present in nonspherical cells from the same cultures. Approximately 90% of these cells exhibited immunofluorescent staining with antibodies to skeletal muscle myosin, failed to incorporate [3H]thymidine or to form colonies in clonal subculture, and thus represent terminally differentiated rat myoblasts. Quantitative microfluorometric DNA measurements on individual nuclei demonstrated that the terminally differentiated myoblasts obtained in these experiments from both chicken and rat contain 2cDNA levels, suggesting arrest in the G0stage of the cell cycle.