大鼠急性胰腺炎时胰腺腺泡细胞凋亡及Bax,Caspase-8的表达

Role of acinar cell apoptosis and expression of Bax and Caspase-8 in rats with acute pancreatitis

目的:研究大鼠急性胰腺炎腺泡细胞凋亡情况,凋亡调控基因Bax与凋亡蛋白酶Caspase-8的表达及其同疾病严重程度的关系．方法:胆胰管逆行注入不同浓度去氧胆酸钠方法制备大鼠急性胰腺炎模型,设假手术(SO)组、急性水肿性胰腺炎(AEP)组和急性坏死性胰腺炎(ANP)组．采用TUNEL技术检测胰腺腺泡细胞的凋亡,RT-PCR与免疫组化方法分别分析胰腺组织Bax,Caspasc-8 mRNA及蛋白的表达,对胰腺组织进行病理学评分并测定血清淀粉酶及IL-6,TNF-α含量．结果:与SO组比较,AEP与ANP组胰腺组织显示不同程度的病理损害,血清淀粉酶(63 413±6118,1532 134±17 654 nkat／L vs 15 736±483 nkat／L,P<0．01)及TNF-α(1.86±0．13, 2．97±0．14μg／L vs 0．95±0．08μg／L,P<0．01), IL-6(47．10±7．05,170．10±7．59 ng/L vs 29．20±4．47 ng／L,P<0．01)浓度显著升高,且ANP组显著高于AEP组(P<0．05);AEP与ANP组腺泡细胞凋亡指数显著升高(22．09±3．71,6．50±1．58 vs 0．13±0．05,P<0．01),但ANP组显著低于AEP组(P<0．05)．同时发现．AEP与ANP组胰腺组织Bax(mRNA:1．530±0．501,1．046±0．337;蛋白:453．7±30．5,339．4±26．7)和Caspase-8(mRNA:0．595±0．17,0．505±0．173;蛋白:3606±337,3134±231)mRNA及蛋白表达水平均显著高于SO组(Bax mRNA:0．613±0．244,Bax蛋白:245．2±30．0;Caspase-8 mRNA:0．357±0．130,Caspase-8蛋白:2396±266)(P<0．01),ANP组Bax mRNA及蛋白表达水平显著低于AEP组(1．046±0．337,339．4±26．7 vs 1．530±0．501,453．7±30．5,P<0．05), ANP组Caspase-8 mRNA及蛋白表达水平低于AEP组,但差异不具有显著性意义．结论:急性胰腺炎时胰腺腺泡细胞发生凋亡伴随凋亡调控基因Bax与凋亡蛋白酶Caspase-8表达增加,并同病情严重程度呈负相关关系．

AIM： To investigate the apoptosis of pancreatic acinar cells and the expression of apoptosisregulated genes Bax and Caspase-8 in experimental acute pancreatitis and their correlations with the severity of the disease in rats. METHODS： The models of acute edematous pancreatitis （AEP） and acute necrotizing pancreatitis （ANP） were established by retrograde injection of different concentrations of sodium deoxycholate into the common biliary and pancreatic duct, and then the rats were randomly divided into 3 groups （10 for each）： sham oper-ated group （control）, AEP group （treated with 7.5 g/L sodium deoxycholate） and ANP group （treated with 15 g/L sodium deoxycholat）. The apoptosis of acinar cells was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling （TUNEL） assay. The amounts of Bax and Caspase-8 mRNA expression in the pancreas were analyzed by semiquantitative reverse transcriptase polymerase chain reaction （RT-PCR）. The expression of Bax and Caspase-8 protein were detected by immunohistochemical method. The levels of serum amylase, tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） were also measured respectively. RESULTS： The levels of serum amylase （63 413 ± 6118, 1 532 134 ± 17 654 nkat/L vs 15 736 ± 483 nkat/L, P 〈 0.01）, TNF-α （1.86 ± 0.13, 2.97 ± 0.14 μg/L vs 0.95 ± 0.08 μg/L, P 〈 0.01） and IL-6 （47.10 ± 7.05, 170.10 ± 7.59 ng/L vs 29.20 ± 4.47 ng/L, P 〈 0.01） in AEP and ANP group were significantly higher than those in control group, and there were also significant differences between ANP and AEP group （P 〈 0.05）. The apoptotic index （AI） in both AEP and ANP group was obviously higher than that in control group （22.09 ± 3.71, 6.50 ± 1.58 vs 0.13 ± 0.05, P 〈 0.01）, and there was also marked difference between ANP and than AEP group （P 〈 0.05）. Meanwhile, in AEP and ANP group the expression of Bax （mRNA： 1.530 ± 0.501, 1.046 ± 0.337; protein： 453.7 ± 30.5, 339.4 ± 26.7） and Caspase-8 （mRNA： 0.595 ± 0.17, 0.505 ± 0.173; protein： 3606 ± 337, 3134 ± 231） were observably higher than those in control group （Bax mRNA： 0.613 _± 0.244, Bax protein： 245.2 ± 30.0; Caspase-8 mRNA： 0.357 ± 0.130, Caspase-8 protein： 2396 ± 266） （P 〈 0.01）. Bax expression was also significantly different between ANP and AEP group both at mRNA and protein level, but not Caspase-8 mRNA and protein. CONCLUSION： In acute pancreatitis, the apoptosis of pancreatic acinar cells is negatively correlated with the severity of pancreatitis. Bax and Caspase-8 may contribute to the acinar cell apoptosis and reduction of AP severity.