| 摘要: |
| 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15～16 d 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5 d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。3 d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10 d 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。 |
| 关键词: 运动神经元 成肌细胞 肌收缩 共同培养技术 神经肌肉接头 |
| DOI： |
| 投稿时间：2013-06-19修订日期：2013-08-28 |
| 基金项目:国家自然科学基金(81070775). |
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| Establishment of in vitro co-culture system for embryonic rat spinal motoneurons and C2C12 myotubes |
| GAO Ying-na,LI Xiao-yu,SONG Xian-min,CHEN Shi-cai,CHEN Dong-hui,TANG Wei-fang,ZHENG Hong-liang* |
(Department of Otolaryngology and Head-Neck Surgery, Changhai Hospital,Second Military Medical University, Shanghai 200433, China
*Corresponding author.) |
| Abstract: |
| Objective To identify the conditions for co-culturing embryonic rat spinal motoneurons and C2C12 myotubes, establish a stable co-culture system, and to form functional neuromuscular junction in vitro. Methods The C2C12 myoblasts were cultured to 60%-70% confluence and then were induced by differentiation medium. The embryonic spinal cord anterior horn motor neurons were obtained from 15-16 d pregnant SD rats, and were implanted in the myotubes after differentiating for 5 days; the products were co-cultured in the basic serum-free culture medium Neurobasal+2% B27. The neuronal morphology and projection length at each stage, myotube morphological and contraction characteristics, and formation of the neuromuscular junction were observed under an inverted microscope. The α-bungarotoxin (α-BTX), which can specifically bind to acetylcholine receptor (AChR) of the postsynaptic membrane, was examined by immunofluorescence technique and the muscle contraction in the co-culture system was recorded by screen recording technology. Results Both the primal spinal motoneurons and the C2C12 myotubes survived in the co-culture system, with further differentiation and maturation. On day 3 the axons extended to the myotube membrane surface or surrounded the myotubes. On day 7 the myotubes were arranged in the same direction, with wide rhythmic contraction, and immunofluorescence showed that α-BTX specifically bound to AChR of the postsynaptic membrane. On day 10 of co-culture, the motor neurons began to have apoptosis and the myotube cells gradually shrank. Conclusion Under in vitro culture condition, motor neurons and skeletal muscle cells can co-exist and grow, establishing synaptic connections, triggering a series of neuromuscular junction signal transduction, and causing rhythmic contraction of the myotubes. |
| Key words: motor neurons myoblasts muscle contraction co-culture techniques neuromuscular junction |
