重症急性胰腺炎腹水对正常肾细胞影响

The effects of ascites in severe acute pancreatitis on the normal kidney cells

目的通过观察重症急性胰腺炎(SAP)腹水对正常肾细胞影响,探讨SAP时急性肾功能损伤的机制。方法SD大鼠54只分成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6h、12h、24h采集血清、腹水。ELISA法检测血清、腹水TNFα水平,RT PCR检测肾细胞TNFαmRNA表达。将24h采集的血清和腹水处理体外培养NRK细胞,MTT法检测细胞活性,流式细胞术测定NRK细胞凋亡率。结果SAP组6h、12h、24h血清和腹水TNFα水平渐进性升高,各时间段差异显著(P<0.05)且较假手术后明显升高(P<0.01)。SAP组大鼠血清和腹水处理后NRK细胞活性均低于假手术组。SAP组大鼠腹水和血清处理24h后,NRK细胞培养液中TNFα水平较假手术组明显升高(P<0.01);NRK细胞TNFαmRNA表达上调,显著高于假手术组;凋亡率分别为29.67%±4.12%和59.95%±2.11%,假手术组大鼠血清和腹水处理24h凋亡率分别为3.72±2.90%和4.11±3.30%,差异显著(P<0.01)。结论SAP时肾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TNFα升高及肾细胞过多分泌TNFα有关,SAP腹水对肾的直接损伤可能比血清明显。

Objective： To explore the mechanism of renal injury in severe acute pancreatitis （SAP）. Methods ：54 SD rats were randomly divided into sham operation group（n=24） and SAP group（n=30）. SAP model was induced by injection of 5% sodium taurocholate solution into bilopancreatic duct.Rats from each group were killed and the serum and ascite were collected at 6h,12h, 24h after operation. Tumor necrosis factor-α（TNF-α）level of serum and ascite were measured by enzyme linked immunno-sorbent assay （ELISA）. The expressions of TNF-α mRNA were assessed by RT-POR analysis. The serum and ascJtes that had been collected at 24h after development were inJected into medium of NRK cell, then the levels of TNF-α secreted by NRK cell were measured after injection 6h,12h,24h,and the cell apoptosis were also evaluated after injection 24h. NRK cell apoptosis were evaluated by flow cytometry （FCM）. Results ：①The serum and ascites levels of TNF-α were signficantly elevated in SAP groups compared with sham operation groups at any time point（P〈0. 01）, and considerably differed In SAP groups between any two time point（P〈0. 05）.②The levels of TNF-α produced by the NRK cells increased signlficautly at 12h than 6h,also were higher at 24h than 12h after injection of serum and ascites of 24h＇s SAP（P〈0. 05）. Moreover, the ascltes-induced groups were obviously evalated than serum-induced groups. Morphology of NRK cells became thin and long at 6h, intracelluar granules Increased at 12h and cells deformed at 24h when being disposed by the serum and ascites of SAP. Ratio of apoptotic ascites-induced NRK cells was 56. 95%±2.11% ,at 24h of that with serum-induced groups（27.27%±4. 12%） and pre-induoed groups （3.72%±2.90 and 4.11%±3.30%）（P〈0. 01）. Conclusion ：Over apoptosis of NRK cell is possibly involved in the mechanism of renal inJury during SAP, TNF-α is one of and the major factors inducing the NRK cell apoptosis. The effect of ascites plays an important role in the renal injury than serum during SAP.