細胞培養された鶏胚網膜神経層に対するメチルB12の効果

EFFECT OF METHYLCOBALAMINE (CH3-B12) ON THE GROWTH AND DIFFERENTIATION OF CULTURED NEURAL RETINAL CELLS OF CHICKEN EMBRYOS

はじめに

　ラット小脳を培養すると髄鞘の形成が観察されるが，米沢はメチルB₁₂が低濃度では髄鞘形成をやや促進し，高濃度では阻害すると報告している^1）。中沢は，メチルB₁₂が，分離したグリア細胞におけるLecithine合成を神経細胞のそれに比べて十倍以上促進したと報告している^2）。これらの報告はメチルB₁₂が，神経組織のなかでも殊にグリア細胞に強い影響を及ぼすことを示唆していろ。

　この報告では，中枢神経系と由来を同じくする網膜神経層をin vitroに移して細胞培養し，その増殖と分化にメチルB₁₂がどのような効果を及ぼすかを検討した。発生中の鶏胚網膜神経経層をいつたん個々の細胞にまで解離して培養すると，扁平な大型の上皮様細胞はさかんに増殖して細胞シートを形成し，神経細胞はそのシート上にまるい細胞集合塊をつくる。この大型の細胞はグリア性の非神経細胞と考えられ，小型の神経細胞と容易に区別して追跡，観察できるので，メチルB₁₂がこれらの二つのタイプの網膜細胞に及ぼす作用について検討した。今回，メチルB₁₂存在下におけるグリア性細胞，神経細胞の，培養の経過にともなう細胞数の変動を測定するとともに，それぞれの細胞の微細構造に及ぼす効果についても検討を加えた。

Neural retinal cells of chick embryos were cultivated as monolayer culture and the effect of methylcobalamine on the growth of cultured cells was examined. The effect on the morphological appearances was also investigated by an electron microscope.

Isolated neural retinas uncontaminated by cells of other tissues were dissociated by treatment with EDTA and 0.2% trypsin. About 7×10⁶ or 1×10⁷ cells were inoculated into each Falcon culture dish (3cm in diameter) using Eagle's minimum essential medium supplemented with 6% fetal calf serum.

Neural retinal cells grown in vitro can be clas-sified into two groups. The larger cells of more than 15μ in diameter attached to the culture dish to form an epithelial cell sheet and increased in number during the culture period. The smaller round cells of less than 10μ in diameter constituted an aggregate on the cell sheet of large cells, and decreased in number owing to detachment from the cell sheet.

When methylcobalamine was administered to the neural retinal culture of 8.5-day-old embryos, small cells were well maintained on the cell sheet at a low concentration of the drug ; that is, when the drug was added into the medium at 10^-5mg/ml, the number of small cells in the 10-day culture was 1.6 times as large as that of the control culture, and the number of large cells was almost the same. At a higher concentration of 10^-2mg/ml, the num-ber of small cells was about half of the control culture, and large cell number was 0.86 times less than the control. On the other hand, when the drug was added to the culture of 6-day-old embryos at the concentration of 0, 10^-5, and 5×10^-2mg/ml no significant differences in the number of small cells as well as large cell were observed.

Electron microscopic observations revealed that large epithelial cells had numerous microvilli and desmosome-like junctions, and were rich in micro-filaments as well as 10nm filaments. Small cells in aggregates elongated their cytoplasmic processes to form a"plexiform area", where many nerve ter-minals were found. These ultrastructural features were fundamentally the same, regardless of the concentration of the drug.

As a result, in the culture of neural retinal cells of 8.5-day-old chick embryos, methylcobalamine had a promoting effect on the retention of small neuronal cells on the epitheilal cell sheet at a low concentration, but had little effect on the growth of large glial cells.