Uterine smooth muscle cells in primary culture
Uterine smooth muscle cells in primary culture
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原代培养的子宫平滑肌细胞
作者：
L. Palmberg;J. Thyberg
摘要从成年大鼠和人子宫肌层中酶解分离出平滑肌细胞（SMC），并进行原代培养。透射电镜观察细胞精细结构和细胞骨架组织，细胞化学显示肌动蛋白丝、微管和中间丝，胸腺嘧啶放射自显影研究DNA合成起始。在培养的最初几天里，细胞在基质上扩散，并经历了形态转变，包括肌丝的丢失，随后形成了广泛的粗糙内质网和一个大的高尔基复合体。肌动蛋白丝聚集在应力纤维中，跨越整个细胞的长度，微管和中间丝形成起源于核旁区域的辐射系统。在体内，SMC含有对蛋白有反应的中间丝，但早在培养的第一天就表达了蛋白。至少在5天内，所有细胞都保持两种蛋白的阳性，但desmin的染色减少，而vimentin的染色增加。这种结构修饰伴随着DNA合成的开始，在第3天达到峰值（45-55%标记的细胞核）。未融合、生长受阻的原代培养对纯化的血小板来源的生长因子和血清反应较弱，在继代培养中对有丝分裂刺激没有反应。观察结果表明，体外培养的子宫SMC经历了从收缩型到合成型的转变，类似于先前在相同条件下描述的动脉SMC的转变。然而，子宫细胞的增殖潜能明显降低。这些发现支持了这样的观点，即过渡到合成表型是SMC DNA合成起始的必要条件，但不是充分条件，并且内脏和血管SMC代表了不同的分化途径。
SummarySmooth muscle cells (SMC) were enzymatically isolated from the myometrium of adult rat and human uteri and grown in primary culture. Cell fine structure and cytoskeletal organization were followed by transmission electron microscopy and cytochemical demonstration of actin filaments, microtubules and intermediate filaments, and initiation of DNA synthesis was investigated by thymidine autoradiography. During the first few days in culture the cells spread out on the substrate and went through a morphological transformation including loss of myofilaments followed by formation of an extensive rough endoplasmic reticulum and a large Golgi complex. Actin filaments aggregated in stress fibers spanning the entire length of the cells and microtubules and intermediate filaments formed a radiating system originating in the juxtanuclear region. In vivo, the SMC contained intermediate filaments reactive for desmin, but as early as the first day of culture expressed vimentin as well. For five days at least, all cells remained positive for both proteins, but the staining for desmin decreased while that for vimentin increased. This structural modification was accompanied by initiation of DNA synthesis, with a peak on day 3 (45–55% labeled nuclei). Subconfluent, growth-arrested primary cultures responded weakly to purified platelet-derived growth factor and serum, and in secondary cultures no response to the mitogenic stimulation was obtained. The observations indicate that uterine SMC cultivated in vitro undergo a transformation from contractile to synthetic phenotype, similar to the transformation described previously for arterial SMC under the same conditions. The proliferative potential of the uterine cells is, however, markedly lower. The findings support the notions that the transition into synthetic phenotype is a necessary but not sufficient requirement for initiation of DNA synthesis in SMC and that visceral and vascular SMC represent separate differentiation pathways.