發(fā)布時(shí)間：2018-01-24 06:23

  本文關(guān)鍵詞： 肝損傷 肝毒性 適應(yīng) 診斷 利福平 膽汁淤積 肝膽汁酸轉(zhuǎn)運(yùn)體 膽汁酸　出處：《安徽醫(yī)科大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：背景藥物性肝損傷是涉及臨床用藥安全的重大問題,但存在藥物性肝損傷適應(yīng)現(xiàn)象。藥物性肝損傷適應(yīng)現(xiàn)象含義是,一些能夠誘發(fā)嚴(yán)重特異質(zhì)性藥物性肝損傷的藥物,可引起短暫的,無癥狀的肝臟生化指標(biāo)升高,繼續(xù)使用相同藥物和劑量時(shí),升高的血清肝臟生化指標(biāo)會(huì)恢復(fù)正常,稱之為對(duì)肝損傷的適應(yīng)現(xiàn)象。認(rèn)識(shí)藥物性肝損傷適應(yīng)現(xiàn)象的意義在于有可能減少或避免不必要的停藥,以免失去控制重要臨床疾病的手段。美國FDA藥物試驗(yàn)報(bào)告以及國外研究已有諸多報(bào)道,但我國尚缺乏類似研究報(bào)道。因此有必要規(guī)范性收集藥物性肝損傷適應(yīng)現(xiàn)象的病例,總結(jié)分析其臨床特點(diǎn),為我國藥物性肝損傷適應(yīng)現(xiàn)象研究提供較為可靠的臨床資料�？菇Y(jié)核藥物是我國急性藥物性肝損傷的主要病因。我們前期動(dòng)物試驗(yàn)發(fā)現(xiàn),利福平可導(dǎo)致小鼠膽汁淤積典型生化指標(biāo)的變化,繼續(xù)用藥后其生化指標(biāo)堿性磷酸酶、總膽紅素和結(jié)合膽紅素較前下降。需要進(jìn)一步采用在同只動(dòng)物連續(xù)抽血檢測(cè)的動(dòng)態(tài)觀察方法,證實(shí)與建立利福平致膽汁淤積適應(yīng)現(xiàn)象的動(dòng)物模型,同時(shí)探討其膽汁淤積適應(yīng)過程的分子機(jī)制。目的1.調(diào)查分析近10年安徽省多家醫(yī)院住院期間存在藥物性肝損傷適應(yīng)性現(xiàn)象患者的臨床資料,分析患者臨床生化指標(biāo)水平及其時(shí)序關(guān)系,總結(jié)藥物性肝損傷適應(yīng)現(xiàn)象的臨床特點(diǎn)。2.建立穩(wěn)定的利福平肝內(nèi)膽汁淤積適應(yīng)現(xiàn)象的大鼠模型,明確血清和膽汁生化標(biāo)志物適應(yīng)過程的動(dòng)態(tài)變化。在此基礎(chǔ)上初步探討膽汁淤積適應(yīng)過程中肝膽汁酸轉(zhuǎn)運(yùn)體的適應(yīng)性變化。方法1.藥物性肝損傷適應(yīng)現(xiàn)象病例的臨床分析制定統(tǒng)一的病案調(diào)查表,采用病例登記和隨訪的方式。在全省多家醫(yī)院所提交的藥物性肝損傷適應(yīng)現(xiàn)象的病例中,根據(jù)數(shù)據(jù)完整性情況,從中篩選出數(shù)據(jù)較為完整,可供分析的病例。采用RUCAM評(píng)分系統(tǒng),對(duì)適應(yīng)現(xiàn)象病例的藥物與肝損傷的相關(guān)性做出評(píng)價(jià)。每例病例在專家討論會(huì)上作出診斷和排除。按藥物性肝損傷后是否繼續(xù)用藥情況,分為持續(xù)用藥和停藥后再用藥兩組,依據(jù)生化指標(biāo)和臨床癥狀對(duì)肝損傷嚴(yán)重程度分級(jí),探討適應(yīng)患者肝損傷類型、肝損傷與用藥間的時(shí)間關(guān)系,肝臟生化指標(biāo)變化的特點(diǎn)。2.利福平致大鼠膽汁淤積適應(yīng)現(xiàn)象的動(dòng)物模型建立和分子機(jī)制初步探討36只大鼠隨機(jī)分為利福平組(27只)和對(duì)照組(9只),分別給予利福平混懸液100mg/(kg?d)和0.9%氯化鈉注射液10ml/(kg?d)連續(xù)7周每日上午一次定時(shí)空腹灌胃,在用藥的7周過程中每周經(jīng)大鼠眶底靜脈叢抽血,動(dòng)態(tài)監(jiān)測(cè)兩組大鼠治療前以及7周治療期間每周末的血谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)活性以及堿性磷酸酶(ALP),總膽紅素(TB),直接膽紅素(DB)水平變化。分批處死大鼠,觀察各組大鼠肝組織病理學(xué)及肝細(xì)胞超微結(jié)構(gòu)改變。檢測(cè)肝勻漿總膽汁酸(TBA)水平和血清TBA水平。采用RT-PCR技術(shù)檢測(cè)肝膽轉(zhuǎn)運(yùn)體Mrp2,Bsep,Ntcp和Mrp4m RNA表達(dá)水平,Western Blot技術(shù)檢測(cè)肝膽轉(zhuǎn)運(yùn)體Mrp2,Bsep,Ntcp和Mrp4蛋白表達(dá)。結(jié)果1.藥物性肝損傷適應(yīng)現(xiàn)象病例的臨床分析(1)共收集來自全省7家醫(yī)院共50例藥物性肝損傷適應(yīng)病例,剔除資料不完整病例7例。采用DILI國際共識(shí)意見的RUCAM評(píng)分系統(tǒng)評(píng)價(jià)43例資料完整病例,其肝損傷與藥物之間的因果關(guān)聯(lián)性依次為:非常可能0例(0%),很可能相關(guān)16例(37.2%),可能相關(guān)27例(62.8%),不大可能0例(0%),無關(guān)0例(0%)。持續(xù)用藥組RUCAM評(píng)分中位數(shù)4分(3~6分)明顯低于停藥后再用藥組RUCAM評(píng)分中位數(shù)6分(5~7)(P0.05)。繼而在專家討論會(huì)上排除3例,確定具有適應(yīng)現(xiàn)象定義的病例共40例。男性25例,女性15例,年齡20歲~74歲,平均年齡39.22歲,持續(xù)用藥組24例,停藥后再用藥組16例。導(dǎo)致肝損傷適應(yīng)現(xiàn)象的藥物分別為抗結(jié)核藥物39例,抗腫瘤藥物1例。(2)在40例藥物性肝損傷適應(yīng)現(xiàn)象患者中,ALT升高程度在0級(jí)11例,1級(jí)12例,2級(jí)13例,3級(jí)2例,4級(jí)2例;AST升高程度在0級(jí)11例,1級(jí)9例,2級(jí)17例,3級(jí)2例,4級(jí)1例;TBIL在正常范圍內(nèi)即0級(jí)36例,1例為3級(jí),3例為2級(jí)。80%的患者的肝臟生化指標(biāo)峰值水平為輕度到中度升高(2級(jí)以內(nèi)),8例為3級(jí)以上肝生化升高。(3)在40例藥物性肝損傷適應(yīng)現(xiàn)象患者中,按照肝損傷生化分型為肝細(xì)胞型36例,膽汁淤積型3例,混合型1例。根據(jù)美國DILIN藥物性肝損傷嚴(yán)重程度分級(jí)標(biāo)準(zhǔn),1級(jí)38例,2級(jí)2例。(4)藥物性肝損傷適應(yīng)現(xiàn)象的病例潛伏期中位數(shù)為10.5天,范圍在2天~138天,肝生化恢復(fù)正常的時(shí)間中位數(shù)為8天,范圍在5天~67天。停藥后至再用藥時(shí)間中位數(shù)為12.5天,范圍在7~68天。持續(xù)用藥組和停藥后再用藥組的潛伏期和肝生化恢復(fù)時(shí)間無明顯差異(P0.05)。2.利福平致大鼠膽汁淤積適應(yīng)現(xiàn)象的動(dòng)物模型建立和分子機(jī)制機(jī)制初步探討(1)在利福平致大鼠膽汁淤積適應(yīng)現(xiàn)象的動(dòng)物實(shí)驗(yàn)中,正常對(duì)照組血清肝生化ALT,AST,ALP,TBIL,DBIL水平在動(dòng)態(tài)觀察7周時(shí)間內(nèi)沒有明顯變化。利福平組大鼠血清ALT,AST有升高趨勢(shì),差異無統(tǒng)計(jì)學(xué)意義,但ALP,TBIL,DBIL水平升高,與對(duì)照組比較差異有顯著性(P0.01),持續(xù)給藥后約在第7天至第14天達(dá)到高峰,之后呈現(xiàn)緩慢下降趨勢(shì)。用藥后7天,利福平組肝組織和血清膽汁酸水平也明顯升高達(dá)到高峰(P0.01),至7周末時(shí)呈下降趨勢(shì)(P0.05)。肝組織病理學(xué)顯示利福平組大鼠部分肝細(xì)胞僅呈輕度脂肪變性,匯管區(qū)輕微炎癥;在實(shí)驗(yàn)第7天,14天,49天三個(gè)階段,利福平組病理變化無明顯差異。電鏡下可見部分毛細(xì)膽管擴(kuò)張及膽汁淤積,線粒體形態(tài)改變。符合大鼠膽汁淤積適應(yīng)現(xiàn)象的肝臟生化和病理的動(dòng)態(tài)變化征象。(2)在分子機(jī)制研究方面發(fā)現(xiàn),雖然與正常對(duì)照組相比,利福平組各時(shí)間點(diǎn)肝膽轉(zhuǎn)運(yùn)體Mrp2,Bsep,Ntcp m RNA表達(dá)水平無明顯改變,但49天利福平組的Mrp4m RNA表達(dá)水平明顯升高,差異具有顯著性(P0.05)。此外,利福平組7天,14天,49天Mrp4蛋白水平表達(dá)較正常對(duì)照組明顯增加,在14天達(dá)到高峰,Bsep蛋白表達(dá)在利福平組7天,14天,49天逐漸增加,在14天和49天增加具有顯著性(P0.05)。利福平組49天的Ntcp蛋白水平明顯下降(P0.01)。初步發(fā)現(xiàn)利福平大鼠膽汁淤積適應(yīng)現(xiàn)象的分子機(jī)制與轉(zhuǎn)運(yùn)體的適應(yīng)性調(diào)節(jié)有關(guān)。結(jié)論1.藥物性肝損傷的適應(yīng)性變化的特點(diǎn)是肝臟生化指標(biāo)暫時(shí)性輕至中度升高,在繼續(xù)用藥的情況下,異常的肝生化恢復(fù)正常,并且不再出現(xiàn)肝損傷。藥物性肝損傷適應(yīng)現(xiàn)象病例以肝細(xì)胞型肝損傷為主,膽汁淤積型和混合型肝損傷少見。具有適應(yīng)現(xiàn)象病例的肝損傷嚴(yán)重程度均在2級(jí)以下。2.通過動(dòng)態(tài)觀察實(shí)驗(yàn),建立與驗(yàn)證了利福平導(dǎo)致大鼠肝內(nèi)膽汁淤積型肝損傷適應(yīng)現(xiàn)象模型。發(fā)現(xiàn)利福平導(dǎo)致膽汁淤積適應(yīng)性下降與轉(zhuǎn)運(yùn)體的適應(yīng)性調(diào)節(jié)有關(guān),包括通過上調(diào)Mrp4和Bsep表達(dá)增加膽汁酸的排泄,抑制Ntcp表達(dá)以減少膽汁酸的攝取。
[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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