目的:观察脂多糖(LPS)、雨蛙肽(cerulein)刺激对大鼠离体胰腺组织热休克蛋白60(HSP60)表达的影响.方法: 分离培养大鼠离体胰腺组织,用低、高浓度cerulein (Cer 10-11mol/L,10-5mol/L)或LPS(10 mg/L,20 mg/L)刺激,以生理盐水(NS)作为对照,在刺激前和刺激后1、4h测定胰腺组织的活力(MTT法)、胰蛋白酶原活性肽(TAP)水平(ELISA测定),培养上清液中白介素6(IL-6)水平(ELISA测定) 并用 real-time PCR和Western blotting分别测定胰腺组织HSP60 mRNA和蛋白的表达.结果: 低、高浓度的cerulein和LPS刺激胰腺组织后1 h,组织的活力与NS对照组比较稍有下降(P〉0.05),刺激4h后,组织活力明显降低(P〈0.05) 组织中TAP水平在高浓度cerulein和低、高浓度的LPS刺激下明显升高 其培养上清液中IL-6水平在刺激后1 h变化不明显,4 h时升高,尤其以高浓度的cerulein或LPS作用显著(P〈0.05).胰腺组织HSP60 mRNA和蛋白的表达量随LPS刺激时间的延长和浓度的升高而降低 而低、高浓度的cerulein刺激HSP60 mRNA的表达增加,但蛋白的表达降低,以高浓度、长时间刺激的作用为明显(P〈0.05).结论: LPS及cerulein对离体胰腺组织具有损伤作用,并呈一定时间及浓度依赖性 同时,胰腺组织HSP60蛋白表达降低,提示HSP60细胞保护作用的减弱可能参与该损伤过程.
AIM : To investigate the effects of lipopolysaccharides (LPS) or cerulein on the expression of HSP60 in isolated rat pancreatic tissues. METHODS: The tissue of rat pancreas was isolated by surgical operation and prepared into tissue snips. The isolated pancreatic tissue was cultured, and stimulated with low - and high - concentrations of cerulein (Cer, 10-11 mol/L, 10-5 tool/L) or lipopolysaccharides (LPS, 10 mg/L, 20 rag/L). Normal saline (NS) was used as control reagent. Before stimulation and 1 h or g h after stimulation, the following parameters for evaluating injury were detected: the viability and the level of trypsinogen activation peptide (TAP) in the pancreatic tissues, and the level of interleukin 6 ( IL - 6) in the culture supernatants. Meanwhile, real - time PCR and Western blotting were used to determine the HSP60 expression at mRNA and protein levels respectively. RESULTS: Under the stimulation with LPS or cernlein, the viability of the pancreatic tissues decreased slightly at 1 h and became much lower with prolonged treatment for g h ( P 〈 0. 05 ). The TAP level in the pancreatic tissues increased obviously at both 1 h and 4 h ( P 〈 0. 05 ) after treatment, except LPS at lower dose. The IL -6 level in the culture supernatant showed no significant change at 1 h stimulation, then increased remarkably at 4 h after stimulation, especially under the conditions of stimulating with LPS or cerulein at higher doses (P 〈 0. 05). The expression of HSP60 at mRNA and protein levels decreased sharply with the increase in the concentration of LPS and the prolonged stimulation time; In cerulein stimulation group, the expression of HSP60 mRNA showed an obvious increase (P 〈 0. 01 ), whereas HSP60 protein was reduced remarkably, especially stimulated with cerulein at higher dose and with longer stimulation time ( P 〈 0.05 ). CONCLUSION: LPS or cernlein induce injuries in isolated pancreatic tissues in dose and time dependent manners. Meanwhile, the prot