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目的获得胚胎大鼠脊髓运动神经元与C2C12肌管共培养的条件,在体外建立稳定的神经-肌肉共培养体系,并形成功能性的神经肌肉接头。方法 C2C12成肌细胞株体外扩增培养至60%~70%融合时,用分化培养液诱导分化;取孕15~16d的SD大鼠,提取胚胎大鼠脊髓前角运动神经元细胞,种植到分化5d的C2C12肌管细胞中,在神经元基础无血清培养液Neurobasal+2%B27中共培养。倒置显微镜下观察各个阶段神经元形态及突起长度的变化、肌管形态变化及收缩特性、神经肌肉接头的形成,应用免疫荧光染色技术检测突触后膜乙酰胆碱受体(acetylcholine receptor,AChR)特异性结合物α-银环蛇毒素(α-bungarotoxin,α-BTX),并采用屏幕录像技术记录共培养体系中肌肉收缩现象。结果在共培养体系中,原代脊髓运动神经元与C2C12肌管细胞均能存活并进一步分化成熟。3d时,可见运动神经元伸出的轴突延伸至肌管膜表面或包绕肌管;1周时,肌管按同一方向排列,出现广泛的节律性收缩,同时免疫荧光染色结果显示α-BTX特异性结合突触后膜AChR;共培养10d后,运动神经元开始凋亡,肌管细胞逐渐出现萎缩现象。结论在体外培养条件下,无需特殊培养基和各种营养因子,运动神经元和骨骼肌细胞即可共同生存、生长并进一步发育,建立突触连接,触发一系列神经肌肉接头信号转导,引发肌管节律性收缩。
Objective To obtain the conditions of co-culture of spinal cord motor neuron and C2C12 myotubes in embryonic rat, establish a stable neuromuscular co-culture system in vitro and form a functional neuromuscular junction. Methods C2C12 myoblasts were expanded and cultured in vitro to 60% -70% confluency, and then differentiated in differentiation medium. Sprague-Dawley (SD) rats of 15-16 days old were used to extract the spinal cord anterior horn motor neurons. C2C12 myotube cells differentiated for 5 days were co-cultured in Neurobasal + 2% B27 neuron-based serum-free medium. The morphological changes of neurons and their length, the changes of myotubes and the contractile properties and the formation of neuromuscular junctions were observed under an inverted microscope. The specificity of acetylcholine receptor (AChR) in the postsynaptic membrane was detected by immunofluorescence staining Conjugate α-bungarotoxin (α-BTX) was used and the phenomenon of muscle contraction in the co-culture system was recorded by screen recording technique. Results In the co-culture system, primary spinal motor neurons and C2C12 myotubes could survive and further differentiate. 3d, we can see the axons of motor neurons extend to the surface of myotubes or wrap around the myotubes. At 1 week, the myotubes are arranged in the same direction with extensive rhythmic contractions. At the same time, the results of immunofluorescence staining showed that α- BTX specific binding to postsynaptic membrane AChR; co-cultured 10d, motor neurons began to apoptosis, myotubation gradually atrophy phenomenon. Conclusion In vitro culture conditions, without special media and various nutritional factors, motor neurons and skeletal muscle cells can coexist, grow and further develop, establish synaptic connections, trigger a series of neuromuscular junction signal transduction, triggering Myotube rhythmic contraction.