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目的探讨体外联合培养条件下雪旺细胞对成肌细胞生物学特性的影响,为神经化人工肌肉的构建提供理论依据。方法碘酒、酒精消毒后,剥离并获取新生SD乳鼠的臂丛、坐骨神经及小腿三头肌,手术显微镜下彻底剥除其外膜、血管和脂肪,充分剪碎神经和肌肉组织,胰酶、胶原酶混合消化分离,DMEM培养基联合培养雪旺细胞与成肌细胞。倒置相差显微镜观察联合培养状态下两种细胞的形态及生长情况;3HTdR同位素标记掺入及液闪计数,每分钟射线绝对衰变数(DPM)判断雪旺细胞分泌物对成肌细胞增殖的影响;统计不同培养时间的肌管形成率,并采用横纹肌肌动蛋白免疫组织化学显色(SABC)及图像分析检测雪旺细胞对成肌细胞功能分化程度的影响。结果联合培养的成肌细胞生长增殖旺盛,肌管出现早、外形粗长,DPM峰值远高于对照组;横纹肌肌动蛋白反应阳性细胞呈棕褐色,联合培养的成肌细胞胞浆中肌动蛋白灰度值明显高于单独培养的成肌细胞。结论与雪旺细胞联合培养有利于原代培养大鼠成肌细胞的生长、增殖及其功能分化。
Objective To investigate the effect of Schwann cells on the biological characteristics of myoblasts in vitro and to provide a theoretical basis for the construction of neuromuscular muscle. Methods Iodine and alcohol were disinfected. The brachial plexus, sciatic nerve and triceps brachii of neonatal SD rats were dissected and dissected. The adventitia, blood vessels and fat were completely removed under the operation microscope. The nerves and muscle tissue were completely excised and trypsin , Collagenase mixed digestion, DMEM culture medium and cultured Schwann cells and myoblasts. Inverted phase contrast microscope was used to observe the morphology and growth of the two kinds of cells under the condition of co-culture. 3HTdR isotope incorporation, liquid scintillation count and absolute decay per minute (DPM) were used to determine the effect of Schwann cell secretions on myoblast proliferation. The rate of myotubes formation at different culture time was counted. The effect of Schwann cells on the function differentiation of myoblasts was detected by SABC and image analysis. Results The cultured myoblasts proliferated vigorously with the appearance of the myotubes in the early and long shape of the myotubes. The DPM peak value was much higher than that of the control group. The striated actin positive cells were tan, and the co-cultured myoblasts in the cytoplasm Protein gray value was significantly higher than cultured myoblasts alone. Conclusion Co-culture with Schwann cells is beneficial to the growth, proliferation and differentiation of primary cultured rat myoblasts.