Objective: To study caspase-3 activities and the neuroprotective effect of caspase-3 inhibitor Ac-DEVD-CMK in primary cultures of rat hippocampal neurons during hypoxia/reoxygenation (H/R). Methods: After pretreated with Ac-DEVD-CMK or the vector DMSO fo 1 h, primary cultures of rat hippocampal neurons were induced to hypoxia for 3 h, followed by 12 to 48 h of reoxygenation. The neuronal viability was estimated by MTT assay, and caspase-3 cellular activities were measured by a colorimetric method using Ac-DEVD-pNA as a substrate. Results: During H/R, the cultured rat hippocampal neuronal viability gradually decreased, and caspase-3 activities in the primary cultures of rat hippocampal neurons were significantly increased, and peaked at 24 h of reoxygenation. Caspase-3 activities were decreased in the neurons pretreated with Ac-DEVD-CMK in a dose dependent manner. Conclusion: Delayed neuronal death is detected in cultured hippocampal neurons during H/R, and apoptosis mediated by caspase-3 may play an important role in it, and caspase-3 inhibitor has a neuroprotective effect on them.

Objective To explore the method of isolation, identification and in vitro differentiation of islet stem cells in neonatal rat. Methods The whole pancreata of neonatal rats were digested with collagenase, then,under the (condition) of pH 7.4～7.6,the digested tissue fragments were cultured with serum and serum-free RPMI 1640 (( bFGF), EGF, N2).The whole formation process of new islet cell-like cell masses was examined.The insulin (release) test was used to detect islet function. The expression of nestin was tested by immunocytochemistry. Results The nestin positive cells can be found within 36 h of culture of the digested pancreatic fragments.After addition of bFGF,ECF,N2,nestin positive cells proliferated fast and formed new islets-like cell masses after 18～24 d, and (insulin)ssion could be confirmed. Conclusions The nestin positive cells of pancreatic cells possess the character of islet stem cells, and can form islet like cell masses through culture in vitro.

OBJECTIVETo regain bile excretory function of hepatocytes cultured in vitro.

METHODSSandwich configuration was used to culture hepatocytes and the structure of bile canaliculi as well as the function of bile excretion was observed by immunocytochemistry and the test of FDA metabolism with a single collagen configuration as a control.

RESULTSFirst, the formation of bile canaliculi was observed by immunocytochemistry. In sandwiched hepatocytes, the gradual development of bile canaliculi-like structures into an anastomotic network was observed. At 24 h after cell culture, the formation of bile canaliculi was observed. With time progressed, the bile canaliculi became more clear and the network was established at 120 h. In contrast, hepatocytes in single collagen configuration showed almost no network of bile canaliculi. Second, the ability for hepatocytes to internalize, metabolize and excrete compounds into bile was indicated by FDA metabolized in the hepatocytes. In sandwiched hepatocytes, the bile excretory function was shown at 96 h, but in single collagen system, no bile excretion was observed.

CONCLUSIONCultured hepatocytes are able to regain bile excretion in a given certain condition. Sandwich-cultured hepatocytes can reestablish bile canalicular structure and regain bile excretory function.

Animals ; Bile ; secretion ; Bile Canaliculi ; physiology ; Cells, Cultured ; Female ; Hepatocytes ; secretion ; Immunohistochemistry ; Rats ; Rats, Sprague-Dawley