药物性肝损伤适应现象的临床和基础研究

发布时间：2018-01-24 06:23

  本文关键词： 肝损伤 肝毒性 适应 诊断 利福平 胆汁淤积 肝胆汁酸转运体 胆汁酸　出处：《安徽医科大学》2015年博士论文　论文类型：学位论文

【摘要】：背景药物性肝损伤是涉及临床用药安全的重大问题,但存在药物性肝损伤适应现象。药物性肝损伤适应现象含义是,一些能够诱发严重特异质性药物性肝损伤的药物,可引起短暂的,无症状的肝脏生化指标升高,继续使用相同药物和剂量时,升高的血清肝脏生化指标会恢复正常,称之为对肝损伤的适应现象。认识药物性肝损伤适应现象的意义在于有可能减少或避免不必要的停药,以免失去控制重要临床疾病的手段。美国FDA药物试验报告以及国外研究已有诸多报道,但我国尚缺乏类似研究报道。因此有必要规范性收集药物性肝损伤适应现象的病例,总结分析其临床特点,为我国药物性肝损伤适应现象研究提供较为可靠的临床资料。抗结核药物是我国急性药物性肝损伤的主要病因。我们前期动物试验发现,利福平可导致小鼠胆汁淤积典型生化指标的变化,继续用药后其生化指标碱性磷酸酶、总胆红素和结合胆红素较前下降。需要进一步采用在同只动物连续抽血检测的动态观察方法,证实与建立利福平致胆汁淤积适应现象的动物模型,同时探讨其胆汁淤积适应过程的分子机制。目的1.调查分析近10年安徽省多家医院住院期间存在药物性肝损伤适应性现象患者的临床资料,分析患者临床生化指标水平及其时序关系,总结药物性肝损伤适应现象的临床特点。2.建立稳定的利福平肝内胆汁淤积适应现象的大鼠模型,明确血清和胆汁生化标志物适应过程的动态变化。在此基础上初步探讨胆汁淤积适应过程中肝胆汁酸转运体的适应性变化。方法1.药物性肝损伤适应现象病例的临床分析制定统一的病案调查表,采用病例登记和随访的方式。在全省多家医院所提交的药物性肝损伤适应现象的病例中,根据数据完整性情况,从中筛选出数据较为完整,可供分析的病例。采用RUCAM评分系统,对适应现象病例的药物与肝损伤的相关性做出评价。每例病例在专家讨论会上作出诊断和排除。按药物性肝损伤后是否继续用药情况,分为持续用药和停药后再用药两组,依据生化指标和临床症状对肝损伤严重程度分级,探讨适应患者肝损伤类型、肝损伤与用药间的时间关系,肝脏生化指标变化的特点。2.利福平致大鼠胆汁淤积适应现象的动物模型建立和分子机制初步探讨36只大鼠随机分为利福平组(27只)和对照组(9只),分别给予利福平混悬液100mg/(kg?d)和0.9%氯化钠注射液10ml/(kg?d)连续7周每日上午一次定时空腹灌胃,在用药的7周过程中每周经大鼠眶底静脉丛抽血,动态监测两组大鼠治疗前以及7周治疗期间每周末的血谷丙转氨酶(ALT)、谷草转氨酶(AST)活性以及碱性磷酸酶(ALP),总胆红素(TB),直接胆红素(DB)水平变化。分批处死大鼠,观察各组大鼠肝组织病理学及肝细胞超微结构改变。检测肝匀浆总胆汁酸(TBA)水平和血清TBA水平。采用RT-PCR技术检测肝胆转运体Mrp2,Bsep,Ntcp和Mrp4m RNA表达水平,Western Blot技术检测肝胆转运体Mrp2,Bsep,Ntcp和Mrp4蛋白表达。结果1.药物性肝损伤适应现象病例的临床分析(1)共收集来自全省7家医院共50例药物性肝损伤适应病例,剔除资料不完整病例7例。采用DILI国际共识意见的RUCAM评分系统评价43例资料完整病例,其肝损伤与药物之间的因果关联性依次为:非常可能0例(0%),很可能相关16例(37.2%),可能相关27例(62.8%),不大可能0例(0%),无关0例(0%)。持续用药组RUCAM评分中位数4分(3~6分)明显低于停药后再用药组RUCAM评分中位数6分(5~7)(P0.05)。继而在专家讨论会上排除3例,确定具有适应现象定义的病例共40例。男性25例,女性15例,年龄20岁~74岁,平均年龄39.22岁,持续用药组24例,停药后再用药组16例。导致肝损伤适应现象的药物分别为抗结核药物39例,抗肿瘤药物1例。(2)在40例药物性肝损伤适应现象患者中,ALT升高程度在0级11例,1级12例,2级13例,3级2例,4级2例;AST升高程度在0级11例,1级9例,2级17例,3级2例,4级1例;TBIL在正常范围内即0级36例,1例为3级,3例为2级。80%的患者的肝脏生化指标峰值水平为轻度到中度升高(2级以内),8例为3级以上肝生化升高。(3)在40例药物性肝损伤适应现象患者中,按照肝损伤生化分型为肝细胞型36例,胆汁淤积型3例,混合型1例。根据美国DILIN药物性肝损伤严重程度分级标准,1级38例,2级2例。(4)药物性肝损伤适应现象的病例潜伏期中位数为10.5天,范围在2天~138天,肝生化恢复正常的时间中位数为8天,范围在5天~67天。停药后至再用药时间中位数为12.5天,范围在7~68天。持续用药组和停药后再用药组的潜伏期和肝生化恢复时间无明显差异(P0.05)。2.利福平致大鼠胆汁淤积适应现象的动物模型建立和分子机制机制初步探讨(1)在利福平致大鼠胆汁淤积适应现象的动物实验中,正常对照组血清肝生化ALT,AST,ALP,TBIL,DBIL水平在动态观察7周时间内没有明显变化。利福平组大鼠血清ALT,AST有升高趋势,差异无统计学意义,但ALP,TBIL,DBIL水平升高,与对照组比较差异有显著性(P0.01),持续给药后约在第7天至第14天达到高峰,之后呈现缓慢下降趋势。用药后7天,利福平组肝组织和血清胆汁酸水平也明显升高达到高峰(P0.01),至7周末时呈下降趋势(P0.05)。肝组织病理学显示利福平组大鼠部分肝细胞仅呈轻度脂肪变性,汇管区轻微炎症;在实验第7天,14天,49天三个阶段,利福平组病理变化无明显差异。电镜下可见部分毛细胆管扩张及胆汁淤积,线粒体形态改变。符合大鼠胆汁淤积适应现象的肝脏生化和病理的动态变化征象。(2)在分子机制研究方面发现,虽然与正常对照组相比,利福平组各时间点肝胆转运体Mrp2,Bsep,Ntcp m RNA表达水平无明显改变,但49天利福平组的Mrp4m RNA表达水平明显升高,差异具有显著性(P0.05)。此外,利福平组7天,14天,49天Mrp4蛋白水平表达较正常对照组明显增加,在14天达到高峰,Bsep蛋白表达在利福平组7天,14天,49天逐渐增加,在14天和49天增加具有显著性(P0.05)。利福平组49天的Ntcp蛋白水平明显下降(P0.01)。初步发现利福平大鼠胆汁淤积适应现象的分子机制与转运体的适应性调节有关。结论1.药物性肝损伤的适应性变化的特点是肝脏生化指标暂时性轻至中度升高,在继续用药的情况下,异常的肝生化恢复正常,并且不再出现肝损伤。药物性肝损伤适应现象病例以肝细胞型肝损伤为主,胆汁淤积型和混合型肝损伤少见。具有适应现象病例的肝损伤严重程度均在2级以下。2.通过动态观察实验,建立与验证了利福平导致大鼠肝内胆汁淤积型肝损伤适应现象模型。发现利福平导致胆汁淤积适应性下降与转运体的适应性调节有关,包括通过上调Mrp4和Bsep表达增加胆汁酸的排泄,抑制Ntcp表达以减少胆汁酸的摄取。
[Abstract]:The background of drug-induced liver injury is a major problem related to the safety of clinical medication, but there are adapt to the phenomenon of drug-induced liver injury. The phenomenon is to adapt to the meaning of drug-induced liver injury, some drugs can severity of drug-induced liver injury induced by idiosyncratic, can cause transient, elevated liver biochemical indexes of asymptomatic, continue to use the same drug and when the dose of serum, liver biochemical indexes increased will be normal, called on to damage liver. Understanding damage adaptation significance is likely to reduce or avoid unnecessary discontinuation of drug induced liver disease, so as not to lose control of important clinical means. The report from the American FDA test drug and study abroad there have been many reports however, China is still a lack of similar reports. So it is necessary to regulate collection of drug-induced liver injury to patients, summarize its clinical features and drug in China Liver injury to provide clinical data more reliable. Research on the phenomenon of anti tuberculosis drugs is a major cause of acute liver injury induced by drugs in China. We found the animal test of rifampicin in mice may lead to cholestasis change of typical biochemical indexes, biochemical indexes after treatment to alkaline phosphatase, total bilirubin and direct bilirubin decreased. The dynamic observation method of continuous blood in the same animal testing, animal model establishment of rifampicin induced cholestasis and that adapt to the phenomenon, and to explore the molecular mechanism of cholestasis adaptation process. The purpose of the 1. survey of nearly 10 years of Anhui province hospital during the clinical data of patients with drug-induced liver injury adaptive phenomenon analysis of biochemical indexes, clinical level and timing of.2. to summarize the clinical features of drug-induced liver injury to establish stability Rifampicin intrahepatic cholestasis in rats model of adaptation, the dynamic process of adaptation to clear bile and serum biochemical markers. Based on the preliminary study of cholestasis bile acid transporter adaptive changes in the process of formulating a unified questionnaire to medical records. Clinical analysis of 1. cases of drug-induced liver injury by the method. The case registration and follow-up. Submitted by many hospitals in the province to adapt to the phenomenon of drug-induced liver injury cases, according to data integrity, from the selected data is complete, can be used for analysis cases. Using the RUCAM scoring system, to make the evaluation of drug cases and related to the phenomenon of liver damage. Each of the cases discussed will make diagnosis and exclusion in the experts. According to whether or not to continue the medication of drug-induced liver injury, divided into continuous medication and after discontinuation of medication two group, On the basis of biochemical indexes and clinical symptoms of liver injury severity, to study the type of patients with liver injury, liver injury and medication time relationship between the deposition of animal model establishment and molecular mechanism of adaptation: a study of 36 rats were randomly divided into Li Fuping rats bile induced changes in liver biochemistry characteristics of.2. Li Fuping (27 only) and control group (9 rats), were given Li Fuping suspension 100mg/ (kg? D) and 0.9% Sodium Chloride Injection 10ml/ (kg? D) morning once daily for 7 weeks fasting intragastric administration in the timing, medication for 7 weeks during the week after the rat orbital venous blood, serum alanine aminotransferase every weekend, the dynamic monitoring of rats in the two groups before treatment and 7 week treatment period (ALT), aspartate aminotransferase (AST) and the activity of alkaline phosphatase (ALP), total bilirubin (TB), direct bilirubin (DB) levels. The rats were killed, the liver tissue of rats were observed. The pathology of liver cell and liver homogenate. The ultrastructural changes of total bile acid detection (TBA) level and the level of serum TBA. RT-PCR was used to detect the hepatobiliary transporter Mrp2, Bsep, Ntcp and Mrp4m RNA expression level detection, hepatobiliary transporter Mrp2, Western Blot Bsep, Ntcp and Mrp4 protein expression. The clinical analysis of the phenomenon of injury cases to 1. drug-induced liver (1) were collected from 7 hospitals in the province a total of 50 cases of drug-induced liver injury to patients, excluding incomplete cases in 7 cases. RUCAM score system adopted DILI international consensus evaluation data of 43 cases of complete case, causal relationship between the liver injury and drugs were very likely 0 cases (0%), probably related to 16 cases (37.2%), 27 cases (62.8%) may be unlikely, 0 cases (0%), 0 cases (0%). Independent continuous medication group the RUCAM score was 4 points (3~6 points) was significantly lower than that after discontinuation of medication scores in RUCAM group 浣嶆暟6鍒，

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