Cao X, Shoichet M S
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, Canada M5S 3E5.
Neuroscience. 2001;103(3):831-40. doi: 10.1016/s0306-4522(01)00029-x.
The developing axon is believed to navigate towards its target tissue in response to a concentration gradient of neurotrophic factors, among other diffusible and surface-bound stimuli. However, the minimum concentration gradient required for guidance over the maximum distance is still unknown, largely because well-defined systems have not been utilized to address this question. In this study, a linear concentration gradient of nerve growth factor was achieved across a 5-mm agarose membrane that separated a nerve growth factor source compartment from a sink compartment. The concentrations in both compartments were maintained constant (and different). Both concentration and concentration gradient were well defined across the membrane, allowing us to study the relative importance of concentration gradient vs concentration for neurite guidance. The orientation of PC12 cell neurites was studied in response to a series of nerve growth factor concentration gradients in vitro. For effective guidance of PC12 cell neurite outgrowth, a minimum concentration gradient of 133ng/ml per mm was required, below which guidance was ineffective. Higher gradients were effective for guidance yet were limited by the concentration of nerve growth factor in the source compartment. At a nerve growth factor concentration of 995ng/ml, the PC12 cells' receptors were saturated, thereby limiting the maximum effective distance for guidance to less than 7.5mm in response to a diffusible nerve growth factor cue. This distance exceeds the 0.5-2mm distance observed by others for effective neurite guidance. Using this model system, we propose that the minimum concentration gradient can be defined for other cells and growth factors. Ultimately, it is anticipated that such concentration gradients could be included in a device to promote regeneration.
人们认为，发育中的轴突会响应神经营养因子的浓度梯度以及其他可扩散和表面结合的刺激，向其靶组织导航。然而，在最大距离上引导所需的最小浓度梯度仍然未知，这主要是因为尚未利用明确的系统来解决这个问题。在本研究中，在一个5毫米的琼脂糖膜上实现了神经生长因子的线性浓度梯度，该膜将神经生长因子源区室与吸收区室分隔开。两个区室中的浓度保持恒定（且不同）。整个膜上的浓度和浓度梯度都得到了很好的定义，这使我们能够研究浓度梯度与浓度对神经突引导的相对重要性。在体外，研究了PC12细胞神经突对一系列神经生长因子浓度梯度的取向。为了有效引导PC12细胞神经突生长，每毫米需要133纳克/毫升的最小浓度梯度，低于这个梯度引导就无效。更高的梯度对引导有效，但受到源区室中神经生长因子浓度的限制。在神经生长因子浓度为995纳克/毫升时，PC12细胞的受体饱和，从而将对可扩散神经生长因子信号的最大有效引导距离限制在小于7.5毫米。这个距离超过了其他人观察到的有效神经突引导的0.5 - 2毫米距离。使用这个模型系统，我们提出可以为其他细胞和生长因子定义最小浓度梯度。最终，预计这样的浓度梯度可以包含在促进再生的装置中。
