胚胎大鼠脊髓运动神经元与C2C12肌管体外共培养体系的建立  

Establishment of in vitro co-culture system for embryonic rat spinal motoneurons and C2C12 myotubes

作　　者：高颖娜[1] 李晓雨[1] 宋先敏 陈世彩[1] 陈东辉[1] 汤维芳[1] 郑宏良[1] 

机构地区：[1]第二军医大学长海医院耳鼻咽喉头颈外科,上海200433

出　　处：《第二军医大学学报》2013年第12期1277-1280,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(81070775)~~

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一肌肉共培养体系，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（acetyleholinereceptor，AChR）特异性结合物”银环蛇毒素（ccbungarotoxin，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 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 vftro. 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 ]unction signal transduction, and causing rhythmic contraction of the myotubes.

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 

分 类 号：R329.29[医药卫生—人体解剖和组织胚胎学]