3.7. Cannabis affects cell proliferation and viability but not
cell differentiation
Cell proliferation was evaluated by immunostaining cells for the
specific Ki67 marker (Fig. 6D-E) and there was an increase in the number
of proliferative ON cells from cannabis users relative to the controls
(t = 4.212, p = 0.0029, Fig. 6B). This increase was consistent with the
proteomic analysis in which there was an up-regulation of cell growth
and proliferation proteins in cells from cannabis users (Fig. 4C) and
proteins involved in cell cycle regulation (Supplementary Fig. 1).
Despite the increase in proliferation, the total number of cells in
culture was no different between the two groups (t = 0.393, p = 0.704,
Fig. 6C).
To evaluate whether the changes in cell proliferation may affect
differentiation, the expression of the NeuN neuronal marker and the GFAP
astrocyte marker was assessed (Fig. 6G-J). After 7 days in culture,
40.22% of the cells were NeuN+ in both groups,
whereas no GFAP+ cells were detected, indicating a
high degree of mature cells expressing a neuronal, but not a glial
marker. However, no differences were observed between groups in
NeuN+ labelling (t = 0.0889 p = 0.9313, Fig. 6F).
Finally, proteomic results indicated a potential down-regulation of cell
death, based on the differential expression of the proteins identified
(Fig. 4B). We observed a significant decrease in the proportion of
Annexin V+/PI- and Annexin V+/PI+ cells derived from cannabis users
relative to the controls, indicating a decrease in both early and late
apoptosis in ON cells from cannabis users (t = 2.975, p = 0.0177 and t =
2.827, p = 0.0222 respectively: Fig. 6K-O), consistent with the
proteomic data.