Protection Effect Of Caspase Inhibitor On Islet Cells

ObjectiveOur aim is to explore the protective effect of caspase inhibitor in islet isolation, purification, and culture.MethodExperimental group and control group were included in our research, in which NICC were collected from 5 neonatal pigs each. In experimental group, caspase inhibitor Z-VAD-FMK was added during NICC purification at the concentration of 20uM/dl, and all the NICC were collected in culture dish where the culturing medium was changed on day 2,4, and 6. Z-VAD-FMK was added when medium was changed at the same concentration. However, in the control group, NICC isolation and culturing condition remained the same except that Z-VAD-FMK was not added. Under the same culture condition, the two groups were examined on day 7 as following, AO/PI fluorescence staining was performed to evaluate the survival rate of NICC and glucose static culture to evaluate insulin secreting efficiency. Flow cytometry was performed to test apoptotic rate of NICC. Bcl-2 and p53 gene expression was evaluated by RT-PCR. Caspase-3 activity was examined by Caspase-3 activity kit.ResultCaspase-3 activity of experiment group remained stably lower than the control group according to Caspase-3 activity test (P<0.001). Meanwhile, RT-PCR test demonstrated Bcl-2 gene expression was significantly upregulated and p53 gene expression was significantly downregulated in experiment group. Flow cytometry results showed that apoptotic cell ratio was significantly lower in the experiment group (P<0.001). According to glucose stimulating test, there was no significant difference on in vitro insulin secreting function of NICC between the experiment and control group.ConclusionCaspase inhibitor (Z-VAD-FMK, at the concentration 20Um/dl) was anti-apoptotic among in vitro NICCs and could also upregulate Bcl-2 gene expression and downregulate p53 gene expression, while it showed no significant influence on the secreting function of NICCs, therefore it played a role in NICC protection, and further insure the therapeutic effect of islet transplantation as the number of NICC with normal function increased greatly.