Department of Cell Biology, UT Southwestern Medical Center at Dallas, Dallas, TX, USA.
Skelet Muscle. 2011 Mar 8;1(1):12. doi: 10.1186/2044-5040-1-12.
A hallmark of muscular dystrophies is the replacement of muscle by connective tissue. Muscle biopsies from patients severely affected with facioscapulohumeral muscular dystrophy (FSHD) may contain few myogenic cells. Because the chromosomal contraction at 4q35 linked to FSHD is thought to cause a defect within myogenic cells, it is important to study this particular cell type, rather than the fibroblasts and adipocytes of the endomysial fibrosis, to understand the mechanism leading to myopathy.
We present a protocol to establish clonal myogenic cell lines from even severely dystrophic muscle that has been replaced mostly by fat, using overexpression of CDK4 and the catalytic component of telomerase (human telomerase reverse transcriptase; hTERT), and a subsequent cloning step. hTERT is necessary to compensate for telomere loss during in vitro cultivation, while CDK4 prevents a telomere-independent growth arrest affecting CD56+ myogenic cells, but not their CD56- counterpart, in vitro.
These immortal cell lines are valuable tools to reproducibly study the effect of the FSHD mutation within myoblasts isolated from muscles that have been severely affected by the disease, without the confounding influence of variable amounts of contaminating connective-tissue cells.
肌肉萎缩症的一个标志是肌肉被结缔组织取代。严重患有面肩肱型肌营养不良症（FSHD）的患者的肌肉活检可能包含很少的肌源性细胞。由于与 FSHD 相关的 4q35 染色体收缩被认为导致肌源性细胞内的缺陷，因此研究这种特定的细胞类型很重要，而不是研究肌内膜纤维化的成纤维细胞和脂肪细胞，以了解导致肌病的机制。
我们提出了一种使用 CDK4 和端粒酶的催化成分（人端粒酶逆转录酶；hTERT）过表达以及随后的克隆步骤，从几乎完全被脂肪取代的严重萎缩肌肉中建立克隆性肌源性细胞系的方案。hTERT 对于在体外培养过程中补偿端粒丢失是必要的，而 CDK4 可防止体外影响 CD56+肌源性细胞但不影响其 CD56-对应物的端粒非依赖性生长停滞。
这些永生化细胞系是从受疾病严重影响的肌肉中分离出的成肌细胞中研究 FSHD 突变影响的有用工具，而不会受到混杂的结缔组织细胞数量的影响。
