The Experimental Study Of Cytotoxicity By Free Fatty Acids In Primary Cultured Rat Pancreatic Islet Cells

Objective: To study the effects of insulin secretion stimulated by free fatty acids(FFAs) in primary cultured SD rat pancreatic cells and the apoptosis evoked by palmitic acids to SD rat pancreatic islet cells in vitro, explore the cytotoxicity of free fatty acids to rat pancreatic cells and its probable mechanisms.Methods: 1. Pancreatic tissues were short-term digested by collagenase in different times, the cells were inoculated for 18 h and then diverted to a new culture plate. The concentration of the insulin and glucose-stimulated insulin secretion(GSIS) were detected with radio immunological methods in the cell culture media. Pancreatic islet cells intravital staining and immunohistochemistry staining were adopted to evaluate organism viability of islet cells. Pancreatic islet cells growth condition were observed under invert microscope. 2. Pancreatic islet cells were treated with FFAs (PA, OA and AA) at different concentrations for 48 h, and at 0.25mmol/L PA for different time respectively, the insulin secretion levels and their changes were analyzed by radio immunological methods described above. 3. Apoptosis were analyzed by Hoechst33342/PI double-staining, electron-microscope, DNA ladder, and the changes of protein concentration were detected by West-blot.Results: 1. Pancreatic tissues could be well digested by collagenase with a short-term time for multi-time methods, diverted culture at 18 h decrease fibroblast interfere for islet cells. More than 90% cells were alive tested by typan blue staining. The secretion function kept normal whencultured for 7-11 days. 2. FFAs at any concentrations have no effect on insulin basic secretion of islet cells or secretion stimulated by low glucose (2.8mmol/L). 0.25mmol/L and 0.5mmol/L of PA and OA could inhibit insulin secretion, but 0.125mmol/L did not show effect on insulin secretion by high glucose(27.8mmol/L). PA at 0.25mmol/L concentration showed inhibition impact on islet cell secretion at 48 h. 3. Typical apoptotic changes, such as nuclear shrek, membran blebbing, apoptotic bodies could be found either by light microscope or electron-microscope. DNA ladders were showed at 0.25 mmol/L and 0.5 mmol/L concentrations of PA, Bcl-2 protein expression decrease and Bax increased in these process.Conclusion: 1. Islet cells can be obtained by repeatedly short-term digesting and diverting culture. 2. FFAs shows influence on insulin secretion, the degree of impact is related not only to the kinds but also to the concentration of FFAs. PA and OA have no effect on basic secretion of insulin, but inhibition on stimulations of high dose glucouse can be observed in a dosage dependent manner. 3. PA can induce SD rat islet cells apoptosis in vitro and the apoptosis rate is associated to its concentrations.