视网膜神经节细胞的纯化和体外存活

Purification and survival of retinal ganglion cell]

We had used a specific anti-Thy 1.1 antibody binding method and a nylonmembrane sieve method to isolate and purify retinal ganglion cells from neonatal rats in order to compare the effect of tectal extract on these purified cells retinal ganglion cells. Isolated retinal cell suspension with retinal ganglion cells retrograde-prelabelled with Fast Blue were seeded on culture dishes coated with the specific anti-Thy 1.1 antibody for 30 minutes before nonadherent cells were removed. The percentage purity of the adherent retinal ganglion cells determined microscopically to be 95%. However, the percentage purity of the Fast Blue-labelled retinal ganglion cells recovered using the nylon membrane of pore size 15 microns was only 60 +/- 5%. Retinal ganglion cells purified by both methods could survive and grow into large, active neurons with neurite outgrowths in the presence of tectal extract. A MTT colorimetric microassay was used to quantify the survival growth activity of these purified retinal ganglion cells after culture for 24 hours. The result showed that the optical density ratio (+Te/-Te) of the retinal ganglion cells purified by anti-Thy 1.1 antibody binding method was 12.3 (0.111/0.009) and by the nylon membrane method was 6.4 (0.102/0.016), and the optical density ratio of the non-purified retinal cells was 3.8 (0.095/0.025), p less than 0.01 for all 3 sets of results. It was concluded that in the absence of other cells, the purified retinal ganglion cells responded specifically to the trophic activity in tectal extract, the purer the retinal ganglion cells and the clearer the effect.