Research On The Dependablity Of Chronic Pancreatitis And Pancreatic β-cell K_ATP Channel

Objective: To establish model of chronic pancreatitis which induced of oleic acidand to confirm its success, so as to study on changes of pancreaticβ-cell K_ATPchannel function and the influence of some drugs, to discuss mechanism of overtpancreatogenic diabetes developed by chronic pancreatitis, as well as to providefoundation for the medical research of pancreatogenic diabetes. Methods: Usinghealthy Wistar rats which were divided into model group and control group. Anexperimental model of chronic pancreatitis was induced by a retrograde injection ofoleic acid into the rat pancreatic duct. Normal saline (NS) was injected into pancreaticduct of control rats instead. Appearance and pathological changes of pancreas, OGTT,FBG and insulin level changes were investigated over a period of 6 weeks afterinduction of this model. Results: After 6 weeks, model rats displayed impairedglucose tolerance (IGT), FBG markedly increased (p＜0.05) and insulin in serummarkedly decreased (p＜0.05). Histologically, destruction of pancreatic small ducts andacini appeared, with some clearly necrosis and congestion in vascellum. Inflammatorycells most of which were lymphocytes infiltrated with halo around them. Structure ofpancreatic islets was destroyed with decrease ofαandβcells. Some tissuedemonstrated fiber proliferation. Conclusion: The model of chronic pancreatitisinduced by oleic acid demonstrated extensive acinar degeneration and overt damageof islet, accompanying with IGT, abnormality of FBG and serum insulin level. The CPmodel was succeeded and it is suitable for progressive research on dependablitybetween CP and K_ATP channel on pancreaticβ-cells. Objective: To observe the changes of pancreaticβ-cell KATP channels inchronic pancreatitis rats, so as to study on the influence of Western medicine-nateglinide and Chinese medicine -tanshinol to the K_ATP channels, the therapeuticaleffect and the regulating mechanism of these 2 medicines on OGTT, fasting bloodglucose (FBG) and insulin level of CP rats. Methods: Healthy Wistar rats which wereselected and established CP models and control rats according to the method insection one. Then we divided them into model group, nateglinide group, tanshinolgroup and control group, with 10 rats each group. Oral nateglinide was administratedto everyone in nateglinide group by a dose of 37.5mg/kg/day. Oral tanshinol wasadministrated to everyone in tanshinol group by a dose of 0.224mg/kg/day. Water wasadministrated to the other 2 groups by a dose of 0.5ml/100g/day. 4 weeks later, afterobserving OGTT changes, experimental rats were put to death and taken theirpancreas and blood, so as to observe the pathological changes of pancreas, FBG andinsulin level changes, observing immunofluorescence of Kir6.2 and SUR1 comingfrom pancreaticβ-cell KATP channels under CLSM (Confocal Laser ScanningMicroscope). Using western blot to detect the expression of Kir6.2 and SUR1 proteinsand RT-(nested)-PCR to detect the expression of Kir6.2 and SUR1 mRNA. On theother hand, we investigated the influence of the 2 medicine mentioned above. Results:Model rats displayed impaired glucose tolerance (IGT), FBG markedly increased(p＜0.05) and insulin in serum markedly decreased (p＜0.05). Nateglinide and tanshinol may effectively improve those abnormalities (p＜0.05). Nateglinide displayed a bettertherapeutic efficacy. Histologically, destruction of pancreatic small ducts and aciniappeared, with some clearly necrosis and congestion in vascellum. Inflammatory cellsmost of which were lymphocytes infiltrated with halo around them: Structure ofpancreatic islets was destroyed with decrease ofαandβcells. Some tissuedemonstrated fiber proliferation. Reparation of destructive pancreatic islets wasobserved in Nateglinide group with increasedαandβcells, comparing with modelgroup. Decreased number of inflammatory cells and lessening fiber proliferation wasobserved in tanshinol group. Western blot showed that expression of protein Kir6.2and SUR1 in model rats significantly diminished comparing with what in control rats(p＜0.05). Nateglinide displayed up-regulation to the expression in some degree.Tanshinol almost had no influence to expression ofβ-cell K_ATP channels. Theseresults agreed with those showed from immunofluorescence. RT-(nested)-PCRshowed that Kir6.2 and SUR1 mRNA of model rats significantly diminishedcomparing with which of control rats (p＜0.05). Nateglinide displayed up-regulation tothe expression in some degree, but the expression was still notably lower than control.Tanshinol almost had no influence to expression of Kir6.2 and SUR1 mRNA.Conclusion: The model of chronic pancreatitis induced by oleic acid demonstratedIGT, abnormality of FBG and insulin level in serum. Model pancreas displayedextensive acinar degeneration and overt damage of islet, accompanying with chronicinflammation and fibrosis of tissue. Protein and mRNA of K_ATP channels onpancreaticβ-cells diminished in model, and improvemet happened in various degreeand various point of view after treated with nateglinide and tanshinol. Treatment ofintegrated Western and Chinese medicine will showed better curative effect andprospect. Objective: To study on the optimal methods and condition of the isolation,purification and primary cultication of Wistar rat islet cells, supplying islet cells withcomplete membrane and fine activity, so as to provide the basal precondition andfoundation for researching on the electrophysiology and therapeutic effects ofmedicine to chronic pancreatitis islet cells. Methods: After anesthetizing ratintraperitoneal with chloraldurat, isolating and purifying rat islet adoptingCollagenase V retroperfusion, situ-digestion and gradient centrifugation in Ficoll-400solution. Then isolating and cultivating single islet cells. Results: 1. Isolating andpurifying islet cells adopting Collagenase V retroperfusion and gradient centrifugationin Ficoll-400 solution may gain 321±60 pancreatic islet per rat. 2.The isolation andcultivation of islet cells: adhering within 12-24 hours, optimal cells growing withcomplete membrane and fine refraction at 4-5 days. Conclution: Retroperfusion withCollagenase V, situ-digestion and gradient centrifugation in Ficoll-400 solution is aneffective method to isolate and purify islet cells of rats. The cultured islet cells havethe optimal state of complete membrane and fine refraction at 4-5 days, fiting thefurther electrophysiological study.