Bray D, Bunge M B, Chapman K
Exp Cell Res. 1987 Jan;168(1):127-37. doi: 10.1016/0014-4827(87)90422-8.
Sensory neurons were dissociated from lumbar dorsal root ganglia of embryonic chick and put into culture, either directly or after removing non-neuronal cells by density gradient centrifugation. The cells were grown on culture substrata of various kinds in medium containing nerve growth factor (NGF). After 24 h the cultures were fixed, mounted and analysed. Lengths of neurites were measured, and the numbers of primary processes formed at the cell body and of growth cones were counted. From these values, the rates of growth cone advance and frequency of growth cone branching were calculated. Neuronal outgrowths increased strikingly in length and complexity with embryonic age; there was a 3.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones when neurons from 15-day embryos (E15) were compared with those from 8-day embryos (E8) grown on the same substratum (glass). Growth was markedly greater on surfaces prepared with laminin or conditioned medium compared with plain glass or air-dried collagen. When E15 neurons grown on glass were compared with those grown on laminin, for example, a 2.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones was observed. Calculations showed that a major factor in these changes was an increase in the frequency of growth cone branching. The number of initial processes emanating from the cell body changed with age, but not with the different substrata tested. Non-neuronal cells when present in low numbers and in contact with neurons did not appear to influence neuronal geometry in a systematic way. Our results document the fact that both external factors (in this case, the nature of the culture substratum) and intrinsic factors (stage of development of the neuron) can influence the geometry of neurite outgrowth.
感觉神经元从胚胎鸡的腰背部背根神经节中分离出来，直接进行培养，或者通过密度梯度离心去除非神经元细胞后再进行培养。细胞在含有神经生长因子（NGF）的培养基中，生长在各种类型的培养基质上。24小时后，将培养物固定、封片并进行分析。测量神经突的长度，计算细胞体形成的初级突起数量和生长锥数量。根据这些数值，计算生长锥前进的速率和生长锥分支的频率。随着胚胎年龄的增长，神经元的生长显著增加，长度和复杂性都有所提高；当将15天胚胎（E15）的神经元与在相同基质（玻璃）上培养的8天胚胎（E8）的神经元进行比较时，总神经突长度增加了3.5倍，生长锥数量增加了3倍。与普通玻璃或风干胶原蛋白相比，用层粘连蛋白或条件培养基处理的表面上的生长明显更大。例如，当将在玻璃上生长的E15神经元与在层粘连蛋白上生长的神经元进行比较时，观察到总神经突长度增加了2.5倍，生长锥数量增加了3倍。计算表明，这些变化的一个主要因素是生长锥分支频率的增加。从细胞体发出的初始突起数量随年龄变化，但不受所测试的不同基质的影响。当非神经元细胞数量较少且与神经元接触时，似乎不会以系统的方式影响神经元的形态。我们的结果证明了这样一个事实，即外部因素（在这种情况下，是培养基质的性质）和内在因素（神经元的发育阶段）都可以影响神经突生长的形态。
