Enhancement of neural regeneration and neural survival in peripheral nervous system by Novel Liver Derived Neuronal Activator

新的肝源性神经元激活剂（LDNA）已被证明可以促进成年大鼠背根神经节（DRG）神经终末的再生，并支持其存活。本研究证明了在无血清培养条件下，LDNA能促进成年大鼠背根节神经细胞的存活。培养6天后，将外植体在液氮中快速冷冻，然后用10微米厚的低温恒温器切片。切片中的尼氏体用尼氏法或姬姆萨法染色。LDNA使Nissle阳性神经元占总神经元的相对数量从33%提高到40%。这些结果表明，LDNA改善神经突再生和它们的存活，从而抑制神经元细胞死亡。我们证明了LDNA的另一个重要功能，即LDNA促进与肝脏接触的模糊神经或交感神经的神经元再生。当将LNA施加到迷走神经时，与无LNA的培养物相比，该因子增强了从横断的迷走神经末梢的神经突再生。在上级颈神经节切断的神经末梢的轴突再生中也观察到类似的增强作用。这些自主神经被认为在肝脏的代谢或再生中起重要作用。肝细胞分泌LDNA可能通过促进自主神经再生与肝细胞接触而激活肝细胞的代谢功能或再生能力，并提出了从1000 L肝细胞条件培养液中测定LDNA氨基酸序列的方案。该因子的纯化进展顺利，预计氨基酸序列将在早期确定。然后我们将开始克隆因子。

Novel liver derived neuronal activator (LDNA) has been proved to enhance neurite regeneration from transected-nerve terminals of adult rat DRG with associated nerve bundles and support their survival. In this project we proved that LDNA promotes neuronal cell survival in adult rat DRG explant after in serum-free culture. After 6 days in culture the explants were rapidly frozen in liquid nitrogen and then sectioned by a cryostat with 10 micron thickness. Nissle bodies in the sections were stained with either Nissle method or Giemsa method. LDNA improved the relative number of Nissle-positive neurons to total neurons from 33% to 40%. These results suggest that LDNA improves neurite regeneration and their survival resulting in inhibition of neuronal cell death. We demonstrated another important function of LDNA that LDNA promotes neuronal regeneration from either vague nerve or > sympathetic nerve both of which contacts with liver. When LDNA was applied to the vagus nerve, this factor enhanced neurite regeneration from the transected-vagus nerve terminals in comparison with that in LDNA-free culture. Similar enhancement by LDNA was seen in neurite regeneration from transected-nerve terminals of superior cervical ganglion. These autonomic nerves are thought to play important roles in either metabolism or regeneration in liver. It is plausible that liver may secrete LDNA and activate either its metabolic function or regeneration capability by promoting autonomic nerve regeneration to contact liver itself.On the other hand we advances the project to determine amino acid sequence of LDNA from 1000 L of hepatocyte conditioned medium. Purification of the factor is going well and it is expected that the amino acid sequence will be determined in an early opportunity. Then we will start to cloning the factor.