【摘要】 目的:通过体内体外观察大鼠和大鼠正常肾细胞(NRK)在重症急性胰腺炎(severe acute pancre-atitis,SAP)状态下凋亡和分泌肿瘤坏死因子α(TNF-α)的情况,探讨SAP时急性肾功能损伤的机制。方法:SD大鼠54只随机分成SAP组(n=30)和假手术组(n=24),SAP组以5%牛磺酸胆酸钠溶液胆管逆行注射诱导模型,术后6、12、24 h采集血清、腹水。ELISA法检测血清、腹水TNF-α水平,TUNEL法检测肾组织凋亡率,RT-PCR检测肾组织TNF-αmRNA表达。将24 h采集的血清和腹水处理后,体外培养NRK细胞,MTT法检测细胞活性,检测培养液TNF-α水平,流式细胞术测定NRK细胞凋亡率,检测NRK细胞TNF-αmRNA表达。结果:SAP组61、2、24 h血清和腹水TNF-α水平呈进行性升高,各时段比较差异有统计学意义(P<0.05),较假手术组在各时段明显升高(P<0.01)。SAP组肾组织TNF-αmRNA表达较假手术组明显上调。SAP组凋亡指数在6、12、24 h时分别为(18.6±5.6)%(、18.1±4.7)%(、25.3±5.1)%,假手术组分别为(19.4±3.1)%、(13.5±2.8)%(、14.6±5.5)%,24 h时SAP组凋亡指数明显高于假手术组(P<0.05)。SAP组24 h肾组织TNF-αmRNA的表达较假手术组明显上调。经SAP组大鼠血清和腹水处理后,NRK细胞活性低于假手术组。经SAP组大鼠血清和腹水处理24 h后,NRK细胞培养液中TNF-α水平较假手术组明显升高(P<0.01),NRK细胞TNF-αmRNA表达上调,显著高于假手术组,NRK细胞凋亡率均较假手术组明显升高(P<0.05)。结论:SAP时肾损害明显,其损伤可能与肾细胞过度凋亡有关,而肾细胞过度凋亡与血清和腹水TNF-α升高及肾细胞过多分泌TNF-α有关,SAP腹水对肾的直接损伤可能比血清明显。更多还原

【Abstract】 Objective: To explore the mechanism of acute renal injury in severe acute pancreatitis(SAP).Methods: Fifty-four SD rats were randomly divided into a sham operation group(n=24) and a SAP group(n=30).The SAP model was induced by injection of 5% sodium taurocholate solution into the bilo-pancreatic duct.Rats from each group were killed,and the serum and ascites were collected 6,12,24 h after the operation.TNF-αlevel of serum and ascites was measured by enzyme linked immuno-sorbent assay(ELISA).The expression of TNF-αmRNA and apoptosis rate of kidney tissue in both groups were assessed by RT-PCR analysis and TUNEL stain.The serum and ascites that had been collected 24 hours after development were injected into a medium of NRK cell,and then the level of TNF-αsecreted by the NRK cells was measured after injection 6,12,24 h,and the cell apoptosis was also evaluated after injection 24 h.The level of TNF-αwas assessed by an enzyme linked immunosorbent assay(ELISA).The expression of TNF-α mRNA was detected by reverse transcription polymerase chain reaction(RT-PCR).NRK cell apoptosis was evaluated byflow cytometry(FCM).Results: ①The serum and ascites levels of TNF-α were significantly elevated in the SAP groups compared with the sham operation groups at all time points(P<0.01),and considerably differed in SAP groups between any two time points(P<0.05).The expression level of TNF-αmRNA in renal tissue was more dramatically up-regulated than that in the sham operation group.The morphology of renal tissue was normal in the sham operation groups,on the other hand,obvious interstital hyperemia and edema and neutrophil infiltration were observed in the SAP group,in particular,being severe at the 24 h time point;②The level of TNF-αproduced by the NRK cells increased more significantly at 12h than that at 6 h,also was higher at 24 h than at 12 h after injection of serum and ascites at 24 h′s SAP(P<0.05).Moreover,the ascites-induced groups were more obviously elevated than the serum-induced groups.NRK cells became thin and long at 6 h,intracelluar granules increased at 12 h and cells deformed at 24 h when being disposed by the serum and ascites of SAP. In SAP groups,the apoptosis ratio of ascites-induced NRK cells was 56.95% and of serum-induced groups was 27.27% at 24 h,and in the sham operation group,they were 3.72% and 4.11%(P<0.001),respectively.Thereafter,the index of apoptosis NRK cells appeared to increase transiently(ascites-induced groups: 12.3%,seruminduced groups: 8.1%,pre-induced groups: 3.8%,P<0.05).Conclusion: Over apoptosis of NRK cells is possibly involved in the mechanism of renal injury during SAP, and TNF-αis one of the major factors inducing the NRK cell apoptosis.Ascites plays a more important role in renal injury than serum during SAP.更多还原