Park-Matsumoto Y C, Askanas V, Engel W K
Ron Stever Tissue Culture Laboratory, USC Neuromuscular Center, University of Southern California School of Medicine, Los Angeles 90017.
J Neurocytol. 1992 May;21(5):329-40. doi: 10.1007/BF01191701.
In contrast to aneurally cultured human muscle, which is immature in regard to its morphologic phenotype and only rarely and weakly contracts spontaneously, innervated cultured human muscle fibres have: (1) nearly continuous, d-tubocurarine-inhibitable contractions; (2) well-developed cross-striations, basal lamina, t-tubules, and postsynaptic folds of the neuromuscular junctions; (3) the majority of their nuclei peripheralized; and (4) acetylcholinesterase-positive sites present only at the neuromuscular junctions. To see whether the expression of the muscle morphologic phenotype is induced only by neural factors generated from the spinal cord explants or also by their frequent contractile activity, we paralyzed innervated cultured human muscle fibres with 2 microM tetrodotoxin for four weeks, either from the first day of muscle contractions or following four weeks of muscle contractions. In both experimental designs, by light microscopy tetrodotoxin paralysis abolished cross-striations and caused prominent internalization of muscle nuclei; however, it did not influence the intensity of acetylcholinesterase staining at the neuromuscular junctions. By electron microscopy, there was no difference between paralyzed and contracting muscle fibres in development of t-tubules, basal lamina and postsynaptic folds. Our study demonstrates that in human muscle contractile activity: (1) regulates peripheral migration of nuclei and development of cross-striations; and (2) does not influence development of the neuromuscular junction, basal lamina, and t-tubules, which are mainly regulated by neural influences. This culture model may be useful for studying detailed mechanisms of human muscle fibre development and structural abnormalities in human neuromuscular diseases.
与无神经培养的人类肌肉相比，无神经培养的人类肌肉在形态表型上不成熟，且很少自发收缩且收缩微弱，而有神经支配的培养人类肌纤维具有以下特点：(1) 近乎持续的、可被筒箭毒碱抑制的收缩；(2) 发育良好的横纹、基膜、横小管以及神经肌肉接头的突触后皱襞；(3) 大多数细胞核位于周边；(4) 乙酰胆碱酯酶阳性位点仅存在于神经肌肉接头处。为了探究肌肉形态表型的表达是仅由脊髓外植体产生的神经因子诱导，还是也由其频繁的收缩活动诱导，我们用2微摩尔的河豚毒素使有神经支配的培养人类肌纤维麻痹四周，要么从肌肉收缩的第一天开始，要么在肌肉收缩四周后进行。在这两种实验设计中，通过光学显微镜观察，河豚毒素麻痹消除了横纹，并导致肌肉细胞核显著内陷；然而，它并未影响神经肌肉接头处乙酰胆碱酯酶染色的强度。通过电子显微镜观察，麻痹的肌纤维和收缩的肌纤维在横小管、基膜和突触后皱襞的发育方面没有差异。我们的研究表明，在人类肌肉中，收缩活动：(1) 调节细胞核的周边迁移和横纹的发育；(2) 不影响神经肌肉接头、基膜和横小管的发育，这些主要受神经影响调节。这种培养模型可能有助于研究人类肌纤维发育的详细机制以及人类神经肌肉疾病中的结构异常。
