近年,神経の分化や生存維持などにかかわる種々の因子が同定され,そのなかで特に神経栄養因子と呼ばれる一群の物質が様々な効果を神経に及ぼしていることがわかってきた.私達は視神経の再生を目標としてその一助となる薬剤の評価をするための実験系を組み立て,神経栄養因子の評価を行った.成熟ラットの網膜を用い,コラーゲンゲル中で網膜神経節細胞の組織培養を行った.培養液は無血清培養液を用い,その中に各種神経栄養因子nerve growth factor(NGF),brain-derived neurotrophic factor(BDNF),neurotrophin-3(NT-3),neurotrophin-4(NT-4)およびciliary neurotrophic factor(CNTF)を添加した.その後,再生してくる網膜神経節細胞の神経突起数を経時的に計測,比較することにより,各神経栄養因子が網膜神経節細胞の神経突起再生に与える効果を評価した.その結果,培養2日目から単離した網膜神経節細胞より再生神経突起が伸長するのが観察された.免疫組織染色によりこれらは中枢神経である網膜神経節細胞の再生神経突起であることがわかった.この再生神経突起の本数を比較したところ,BDNFおよびNT-4添加培養液では培養期間を通して神経突起の再生促進効果がみられた.NGF添加培養液では,培養早期には促進効果がみられたが,徐々にその効果は減弱した.NT-3およびCNTF添加培養液では,神経突起再生促進効果はみられなかった.BDNFを培養初期に投与したものと培養中期以降に投与したものを比較検討したところBDNFは培養初期に投与された時に神経突起再生促進効果が比較的大きいことが示唆された.Recent studies identified different factors involved in differentiation or survival of nerve cells. A particular group of substances, I.e., neurotrophic factors, has been found to affect nerve cells in various ways. Targeting the regeneration of optic nerve cells, we developed an assay system to evaluate drugs that support optic nerve regeneration and evaluated the neurotrophic factors. Retinal ganglion cells were removed from mature rats and incubated in collagen gels. A serum-free culture medium was spiked with various nerve growth factors (NGFs) including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and ciliary neurotrophic factor (CNTF). Regenerated neurites were counted within retinal ganglion cells and the number was compared with baseline values to evaluate the effect of each nerve growth factor on neurite regeneration in retinal ganglion cells. Regenerated neurites extended from isolated retinal ganglion cells from Day 2 of incubation. Immunohistological staining revealed that these regenerated neurites originated from retinal ganglion cells that are a part of the central nerves. Comparing the number of regenerated neurites, neurite regeneration was stimulated throughout the incubation period in culture media spiked with BDNF or NT-4. In the NGF-added medium, regeneration was promoted soon after the start of incubation, but the effect gradually diminished. In media spiked with NT-3 or CNTF, neurite regeneration was not stimulated. Comparing the effect of BDNF addition between early and intermediate/late stages of incubation, neurite regeneration was more stimulated extent when the medium was spiked with BDNF soon after the start of incubation
