重型胰腺炎大鼠血浆线粒体DNA变化与器官损伤的关系

摘要

目的:检测重型急性胰腺炎(SAP)模型大鼠血浆线粒体DNA(mtDNA)浓度,探讨循环血中mtDNA水平与SAP器官损伤的关系.方法:健康成年雄性Wistar大鼠30只,随机分为假手术组和SAP模型组,以5％牛磺胆酸钠胆胰管逆行注射法制备SAP大鼠模型,24 h后麻醉,取全血,留取血浆,测定淀粉酶和脂肪酶水平;提取血浆中游离DNA,采用线粒体细胞色素B和细胞色素C氧化酶亚基Ⅲ引物,进行荧光定量PCR扩增,以其扩增产物量代表循环血中mtDNA水平;取肺脏、肝脏、肾脏组织,一部分行HE染色、病理组织学检查及评分,一部分匀浆提取组织上清液,ELISA法检测TNF-α、IL-1及IL-6水平,两组间各指标的比较采用单因素方差分析;mtDNA水平与病理组织评分相关性分析采用Spearman秩相关分析.结果:与假手术组比较,SAP模型组大鼠血中淀粉酶活性[(10 444.5±1863.2) U/L]和脂肪酶活性[(1751.3±121.3) U/L]显著升高(P＜0.01);各脏器损伤评分和TNF-α[肺脏(88.4±15.4) pg/mg;肝脏(79.3±10.3) pg/mg;肾脏(69.5±9.6)pg/mg]、IL-1[肺脏(0.68±0.11)μg/g;肝脏(0.72±0.17)μg/g;肾脏(0.85±0.17) μg/g]、IL-6[肺脏(252.1±18.7) pg/mg;肝脏(107.5±12.3) pg/mg;肾脏(118.5±15.2)pg/mg]水平显著升高(P＜0.01);模型组循环血中线粒体细胞色素B(0.4891±0.0921)及线粒体细胞色素C氧化酶亚基Ⅲ相对表达量(0.6082±0.1142)均较假手术组[细胞色素B(0.2856±0.0878);细胞色素C氧化酶亚基Ⅲ(0.1985±0.0854)]显著升高(P＜0.01),且二者水平与脏器损伤评分显著正相关(P＜0.05).结论:SAP大鼠循环血中mtDNA水平升高与器官损伤程度相关.%Objective To detect the concentration of plasma mitochondrial DNA (mtDNA) in rat model of severe acute pancreatitis (SAP) and investigate the relationship between mtDNA level and organ damage.Methods Thirty healthy adult male Wistar rats were randomly divided into Sham operation group and SAP model group with 15 rats in each group.The rat SAP model was induced by retrogradely injection of 5 ％sodium taurocholate through the biliopancreatic duct.After 24 h,the whole blood was taken and the plasma was separated to measure the levels of amylase and lipase.The dissociative DNA was extracted from plasma and amplified by fluorescent quantitative PCR amplification using mitochondrial cytochrome B and cytochrome oxidase subunit Ⅲ primers.The level of mtDNA in circulating blood was represented by the PCR amplification product.The lung,liver and kidney tissues were collected.One part was used for HE staining,histopathological examination and scoring,and the other part was homogenized to extract the supematant and used to measure the levels of TNF-α,IL-1 and IL-6 by ELISA method.The two groups were compared using the index single factor variance analysis.Correlation analysis between the mtDNA level and pathological score was performed by Spearman rank correlation analysis.Results Compared with Sham operation group,the levels of amylase [(10444.5 ± 1863.2)U/L] and lipase [(1751.3 ± 121.3)U/L] in SAP model rats were significantly increased (P ＜ 0.01).The pathological scores and levels of TNF-α [(88.4 ± 15.4) pg/mg in lung,(79.3 ± 10.3) pg/mg in liver,(69.5 ± 9.6) pg/mg in kidney],IL-1 [(0.68 ± 0.11) μg/g in lung,(0.72 ± 0.17) μg/g in liver,(0.85 ± 0.17) μg/g in kidney] and IL-6 [(252.1 ± 18.7) pg/mg in lung,(107.5 ± 12.3) pg/mg in liver,(118.5 ± 15.2) pg/mg in kidney] were significantly increased (P ＜ 0.01).The circulating mtDNA level was also increased significantly and had a significant positive correlation with organ injury score (P ＜ 0.05).The relative expression of cytochrome B (0.4891 ± 0.0921) and cytochrome oxidase subunit Ⅲ (0.6082 ± 0.1142)in mitochondria of model group was increased significantly compared with that in Sham operation group (0.2856 ± 0.0878) and (0.1985 ± 0.0854),respectively) (P ＜ 0.01).The expression levels were positively correlated with the organ damage score.Conclusion The increase of mtDNA concentration in circulating blood of SAP model rats is related to the degree of organ damage.