胆红素尿苷二磷酸葡糖醛酸基转移酶分子生物学研究进展

胆红素尿苷二磷酸葡糖醛酸基转移酶（B—UGT）是一种主要存在于肝细胞内质网，对胆红素进行生物转化的重要代谢酶，它是一个超基因家族，该酶含量下降或缺乏将导致胆红素葡萄糖醛酸化修饰障碍，引起血浆胆红索升高，导致相关疾病发生。     

胆红素和胆汁酸调节尿苷二磷酸葡萄糖醛酸基转移酶的表达

目的 探讨胆红素和胆汁酸是否可以通过调节尿苷二磷酸葡萄糖醛酸基转移酶(UGT)的表达来加速它们自身的代谢。方法 应用原始鼠肝细胞培养和Northen斑迹杂交技术。观察胆红素和胆汁酸处理肝细胞前后UGT同工酶mRNA的变化。结果 用胆红素处理肝细胞24h后两个负责胆红素代谢的UGT同工酶。UGT1A1和UGT1A5mRNA的表达显著增加。当肝细胞暴露于胆酸、鹅脱氧胆酸、脱氧胆酸、猪脱氧胆酸和石胆酸时。负责胆汁酸代谢的UGT281 mRNA的表达水平明显增高。猪脱氧胆酸亦增加UGT283 mRNA的表达。结论 胆红素和胆汁酸可调节UGT的表达，这种调节可能会在清除病理性增高的胆红素和胆汁酸中起代偿作用。     

慢性肝炎和肝硬化患者肝内葡萄糖醛酸基转移酶mRNA变化及影响因素

实验旨在探讨慢性肝炎和肝硬化是否影响肝内葡萄糖醛酸基转移酶(UGT)的表达,以及UGT同工酶是否受到肝功能状态不同程度的影响。 一、资料与方法     

尿苷二磷酸葡萄糖醛酸转移酶在肝脏疾病中的作用及其转录调控机制

尿苷二磷酸葡萄糖醛酸转移酶（UDP-glucuronosyltransferases,UGTs）是机体重要的Ⅱ相代谢酶,在肝脏中高表达,在物质代谢和清除中发挥重要作用。肝脏疾病状态下机体对于UGTs的转录调控在疾病的发生发展中具有重要作用。本文对UGTs的一般家族及其与代谢的关系进行综述,探讨了UGTs在肝脏疾病中的表达和转录调控,UGTs在疾病机制中的研究以及UGTs在肝脏疾病临床治疗中的意义。     

胆红素-尿苷二磷酸葡萄糖醛酸转移酶A1基因多态性在Gilbert综合征发病机制中的作用

Gilbert综合征属于先天性非溶血性黄疸,是一种胆红素代谢障碍性疾病。胆红素-尿苷二磷酸葡萄糖醛酸转移酶(UGT)缺乏或活性降低是Gilbert综合征发病的重要原因。UGT1A1是UGT的同工酶,也是肝脏结合胆红素的关键酶。UGT1A1基因突变,导致UGT结构异常,从而导致胆红素结合功能减弱或丧失。介绍了UGT1A1及其基因多态性在Gilbert综合征发病机制及诊断价值等方面的进展。     

葡萄糖醛酸转移酶1A基因的组织差异性表达及多态调节

目的从mRNA、蛋白质及细胞水平分析尿苷二磷酸葡萄糖醛酸转移酶（UGT）1A基因的组织差异性表达及多态凋节。方法采用逆转录聚合酶链反应分析结直肠癌组40例、正常人群肠道黏膜20例、正常肝组织10例的UGT 1A mRNA表达；免疫印迹检测各组UGT 1A蛋白表达；高效液相色谱法测定各组UGT 1A酶的催化活性。结果①结直肠癌组织中UGT 1A mRNA表达量明显低于其周围正常黏膜，而后者低于正常人群肠黏膜组织，正常人群结直肠黏膜中UGT 1A mRNA表达量总体低于肝组织，P〈0．01。结直肠癌组、正常人群结直肠黏膜组织呈现个体差异表达，而肝组织的表达较均质。②癌组织、癌周正常黏膜及正常人群肠道黏膜中UGT 1A各同工酶的表达例数均不相同。③UGT 1A蛋白表达在癌组织显著低于癌周正常组织，后者又低于正常人肠黏膜（P〈0．01），各组蛋白表达量亦各不相同。而肝组织呈均质表达。④癌组织的UGT 1A酶活性低于癌周正常黏膜．而后者的微粒体酶活性低于正常人群肠黏膜微粒体蛋白，P〈0．01。结论①UGT 1A基因位点呈组织差异性表达。②结直肠黏膜上皮UGT 1A基因位点在转录水平及功能水平均存在多态调节，不同个体对致癌物的易感性不同可能是这种差异表达的结果。     

尿苷二磷酸葡萄糖醛酸转移酶1A6基因多态性与抗结核药致肝损害的相关性

目的 探讨尿苷二磷酸葡萄糖醛酸转移酶1A6(UGT1A6)基因多态性与中国唐山地区结核患者抗结核药致肝损害的相关性.方法采用病例对照研究,以抗结核治疗导致肝损害患者202例为病例组,无肝损害者239例为对照组,收集环境因素暴露情况及静脉血.UGT1A6基因多态性分析采用聚合酶链反应限制性片段长度多态方法,利用Hha Ⅰ、Dpn Ⅱ和NsiⅠ内切酶分析其基因多态性,以SPSS13.0软件对各危险因素进行单因素和多因素非条件Logistic回归分析.结果 UGT1A6的19T/G、308C/A和541A/G多态性位点均存在3种基因型,UGT1A6-19T/T、UGT1A6-19T/G和UGT1A6-19G/G在病例组和对照组的频率分别为51.5%、39.6%、8.9%和71.1%、25.5%、3.3%;UGT1A6-308C/C、UGT1A6-308C/A和UGT1A6-308A/A在病例组和对照组的频率分别为52.0%、40.6%、7.4%和79.1%、19.2%、1.7%; UGT1A6-541AA、UGT1A6-541A/G和UGT1A6-541G/G在病例组和对照组的频率分别为57.9%、33.7%、8.4%和79.5%、19.2%、1.3%,两组间差异均有统计学意义(x2值分别为17.956、37.385和24.095,P值均＜0.01).单因素和多因素分析均显示UGT1A6-19T/G、-308C/A、-541A/G 3个位点基因多态性与抗结核药致肝损害的发生有关(P值均＜0.05).结论 UGT1A6基因多态性可能与汉族人抗结核药致肝损害的发病有关,且各基因型存在相互作用.

Abstract：

Objective To investigate the relationship between the polymorphisms of UGT1A6 genes and anti-tuberculosis drug induced hepatic-injury (ADIH). Methods 202 cases and 239 controls were collected and a case-control study was conducted. Information on related risk factors of tuberculosis was collected. The genotypes of UGT1A6-19T/G, UGT1A6-308C/A and UGT1A6-541A/G genetic polymorphisms were detected by polymerase chain reaction and restriction fragment length polymorphism technique (PCRRFLP) in patients received anti-tuberculosis therapy. The Hha Ⅰ, Dpn Ⅱ and NsiⅠenzyme were employed.Univariate and multivariate conditional logistic analyses were conducted using SPSS13.0 for windows software.Results The allele frequency of gene UGT1A6-19T/T, UGT1A6-19T/G, UGT1A6-19G/G, GT1A6-308C/C, UGT1A6-308C/A, UGT1A6-308A/A, UGT1A6-541AA, UGT1A6-541A/G and UGT1A6-541G/G in ADIH group were 51.5%, 39.6%, 8.9%, 52.0%, 40.6%, 7.4%, 57.9%, 33.7%, 8.4% and 71.1%, 25.5%, 3.3%,79.1%, 19.2%, 1.7%, 79.5%, 19.2%, 1.3% in control group, respectively. Univariate analysis demonstrated that the frequency of UGT1A6-19T/G, UGT1A6-308C/A and UGT1A6-541A/G genotype in cases were significantly higher than that in controls (P ＜ 0.05). Conclusion A positive association is found between UGT1A6 genotype and the occurrence of ADIH. The synergetic effect is proved on susceptibility to pulmonary tuberculosis between UGT1A6 mutant genotypes.     

β葡萄糖醛酸苷酶与原发性胆总管结石的关系

目的β葡萄糖醛酸苷酶（βGlucuronidase,βG）与原发性胆总管结石的关系。方法对原发性胆总管结石患者（实验组）91例和（对照组）84例行内镜下逆行胰胆管造影术（endoscopicretrogradecholangiopancreatography,ERCP）,采用酶学比色法检测胆汁内、外源性βG含量。结果（1）原发性胆总管结石与对照组患者外源性13G比较无明显差异,然而内源性9G明显升高,具有统计学意义（P〈0．01）;（2）不同类型原发性胆总管结石患者中,胆红素结石及混合性结石患者胆汁中内源性9G表达明显高于胆固醇结石组,具有统计学意义（P〈0．01）,而外源性βG表达三组间无显著差异（P〉0．05）。结论内源性βG的表达上调可能在原发性胆总管结石的发生、发展中起着重要作用。     

糖尿病鼠葡萄糖转运蛋白基因mRNA表达的研究

采用分子生物学斑点杂交分析技术,对四氧嘧啶所致糖尿病鼠的肌肉及脂肪组织葡萄糖转运蛋白GLUT_1、GLUT_4基因mRNA表达情况进行了研究,结果:糖尿病鼠脂肪组织GLUT_4 mRNA表达减少(P<0.05),且胰岛素治疗可恢复其表达(P<0.05)。提示细胞GLUT_4基因表达的减少进而引起相应蛋白的减少可能是糖尿病胰岛素抵抗的原因之一。     

长胞内域瘦素受体全长mRNA表达增加对骨胳肌细胞葡萄糖氧化的影响

目的 观察增加骨胳肌细胞全长长胞内域瘦素受体(OBRb)mRNA表达以后,瘦素对原代培养的骨胳肌细胞葡萄糖氧化代谢的影响。方法 分离SD乳鼠的骨胳肌细胞,分4组进行细胞培养,其中转染重组质粒、转染空载质粒和非转染3组(分别加瘦素10ng/ml和胰岛素10mU/ml),另外1组为非转染未加瘦素和胰岛素的对照组。将载有全长OBRb cDNA的真核表达质粒或空载质粒在脂质体的介导下导入骨胳肌细胞。48h后加入瘦素和胰岛素,孵育1h,再加入D-[U-~(14)C]-葡萄糖,继续培养2h后终止反应,收集~(14)CO_2,液体闪烁计数。同时以RT-PCR检测转染细胞的OBRb mRNA表达水平。结果 重组质粒转染、空载质粒转染和非转染3组,特异性条带和β-actin条带积分光密度比值分别为1.22±0.10、0.41±0.08和0.49±0.09,前者较其它两组明显升高(均P<0.001)。葡萄糖氧化前者明显高于其他3组(均P<0.005)。结论 增加SD乳鼠骨胳肌细胞OBRb mRNA的表达,明显改善细胞葡萄糖氧化,提示OBRbmRNA的高度表达能改善瘦素的作用。     

Regulation of uridine diphosphate glucuronosyltransferase during the acute-phase response

The acute-phase response is associated with profound effects on oxidative drug metabolism. However, the effects on glucuronidation are poorly characterized. The aim of the present study was to determine the role of mediators of the acute-phase response in the regulation of hepatic uridine diphosphate glucuronosyltransferase (UGT) expression. Family 1 and family 2 UGT isoforms were studied in turpentine-injected rats and in primary hepatocyte cultures exposed to cytokines and/or dexamethasone. In the in vivo model, glucuronidation of p-nitrophenol was unaffected, while testosterone glucuronidation was reduced to 65% of control (P < 0.01). In contrast, the mRNA level of UGT1*1 (which metabolizes bilirubin, not phenols) was depressed to 16% of control (P < 0.002), while the mRNA level of UGT2B3 (which metabolizes testosterone) was reduced to 53% (P < 0.05). In primary hepatocyte culture, dexamethasone treatment resulted in a 3.4-fold induction of UGT1*1 mRNA levels (P < 0.001) but only a 1.5-fold induction of UGT2B3 (P=0.1). Interleukin-6 in the presence of dexamethasone resulted in a marked dose-dependent suppression of both UGT1*1 and UGT2B3, although to different degrees. Interleukin-1 had no effect on UGT mRNA levels. Thus, inflammatory mediators, such as cytokines and glucocorticoids, may be important determinants of both oxidative and conjugative drug metabolism by the liver.     

分子遗传学检查诊断Crigler-Najjar综合征Ⅱ型3例

目的 探讨采用分子遗传学检查诊断Crigler-Najjar综合征Ⅱ型的方法。方法 在本科收治的3例高间接胆红素血症患者,抽取外周静脉血,提取基因组DNA,应用PCR法扩增尿苷二磷酸葡萄糖醛酸转移酶1A1基因（UGT1A1）所含5个外显子及其侧翼序列,进行DNA直接测序。结果 3例患者均检出UGT1A1基因5号外显子存在c. 1456 T>G（p.Y486D）纯合突变;Y486D位于第5外显子上,使1456位胸腺嘧啶（T）突变为鸟嘌呤（G）,导致486位氨基酸由酪氨酸（Tyr）变为天冬氨酸（Asp）。结论 当临床上高度怀疑Crigler-Najjar综合征Ⅱ型时,应尽早行分子遗传学检查,确定其基因突变位点,以明确诊断。     

不同价态染砷大鼠肝脏MRP2表达水平与砷甲基化产物关联性

目的 分析亚砷酸钠(iAs3+)和砷酸氢钠(iAs5+)染毒后,对大鼠多药耐药相关蛋白2(MRP2)表达的影响,及其与肝脏砷及甲基化产物的关联性,寻找不同价态砷体内代谢转运间的差异,为进一步阐明砷毒作用机制提供依据.方法 70只Wistar大鼠分成7组,第1组为正常对照组,给予饮用去离子水;第2～4组为染毒iAs3+高、中、低剂量组,分别给予20.0、6.7、2.2 mg/kg体质量亚砷酸钠溶液;第5～7组为染毒iAs5+高、中、低剂量组,分别给予20.0、6.7、2.2 mg/kg体质量砷酸氢钠溶液.各组大鼠于染毒90 d后处死,取肝脏组织,用蛋白印迹法(Western-blotting)测定MRP2表达的变化.采用高效液相-氢化物发生原子荧光光谱分析法(HPLC-HGAFS)分析各组肝脏中砷及甲基化产物含量,并运用Pearson相关法分析MRP2表达与砷及甲基化产物的相关性.结果 对照组、iAs3+和iAs5+高、中、低剂量组MRP2蛋白表达分别为1.21±0.13、1.85±0.09、1.65±0.19、1.61±0.18、1.69±0.04、1.42±0.19、1.27±0.10,iAs3+的高、中、低剂量组和iAs5+高、中剂量组MRP2蛋白表达均高于对照组(P均＜0.05);相同受试物比较,iAs3+和iA83+的高剂量组MRP2蛋白表达均高于低剂量组(P均＜ 0.05);不同受试物同一剂量组比较,iAs3+高、中、低剂量组的MRP2蛋白表达均强于iAs5+高、中、低剂量组(P均＜0.05).同时,iAs3+染毒组肝脏中MRP2蛋白表达量与iAs3+和砷甲基化代谢产物MMA和DMA的含量均呈正相关关系(r值分别为0.575、0.678、0.395,P均＜0.05);iAs5+染毒组肝脏中MRP2表达量与MMA和DMA的含量均呈正相关关系(r值分别为0.593、0.643,P均＜0.05),与iAs3+的含量无相关关系(P＞0.05).结论 随着砷染毒剂量的增加,MRP2表达逐渐上调,从而致使肝细胞膜上MRP2表达代偿性改变.MRP2外排通路可能在iAs3+代谢径路中发挥重要作用.肝脏MRP2与不同价态砷甲基化产物的负荷或水平密切相关.     

Effects of uridine diphosphate glucuronosyltransferase activity on the maximal secretion rate of bilirubin conjugates in the rat

It is generally accepted that biliary secretion is rate-limiting for the plasma-to-bile transport of a saturating load of bilirubin. Previous studies from this laboratory have suggested a relationship between the hepatic bilirubin uridine diphosphate glucuronosyltransferase activity and the apparent maximal rate of bilirubin secretion (excretory transport maximum). The present study was undertaken to further investigate this relationship in rats with a wide range of transferase levels and to analyze the effects of transferase activity on the formation of bilirubin monoglucuronides and diglucuronides in vivo and in vitro. Animals with moderately decreased enzyme activity still showed a near-normal excretory transport maximum but decreased fractional amounts of conjugated bilirubins in plasma and liver and a decreased ratio of bilirubins diconjugates to monoconjugates in bile, liver, and plasma. A more pronounced decrease in the level of transferase activity yielded lower excretory transport maximum values as well. Enhanced transferase activities were obtained by pretreatment with a series of enzyme-inducing agents, usually at dosages not influencing bile flow. An enhanced transferase activity produced significant increases in bilirubin excretory transport maximum, decreased concentrations of both unconjugated and conjugated bilirubins in plasma, and an increased ratio of diconjugates to monoconjugates in bile and in liver. In all animals, the ratio of disconjugates to monoconjugates observed in bile was correlated with the ratio found after incubation of liver homogenates in vitro at low concentrations of bilirubin substrate. Overall, the relationship between bilirubin excretory transport maximum and uridine diphosphate glucuronosyltransferase activity can best be described by a hyperbolic curve. At "saturation" of the excretory transport maximum values, no increase of conjugates in liver or in plasma could be observed, suggesting that not the biliary secretion, per se, but rather the conjugation rate in vivo is rate-limiting. The latter seems lower than the in vitro assayed enzyme activity. The present investigations point to an equilibration of bilirubin pigments between plasma, liver, and bile. The in vivo conjugation rate can be estimated from the in vitro assay (but not at high enzyme activities) and determines the maximal biliary secretion rate, the ratio of diconjugates to monoconjugates in the three compartments studied, and the relative amount of conjugates in plasma. Our studies suggest that the conjugation rate in vivo rather than the biliary secretion step is the major determinant of the maximal bilirubin secretion rate.     

Genetic polymorphisms of the uridine diphosphate glucuronosyltransferase 1A7 and colorectal cancer risk in relation to cigarette smoking and alcohol drinking in a Chinese population

BACKGROUND: The impact of uridine diphosphate glucuronosyltransferase 1A7 (UGT1A7) polymorphisms on genetic susceptibility to digestive system cancer has received close attention since the discovery by Guillemette, the polymorphisms of which may alter enzyme activity. To clarify the allele frequency distribution and its association with risk of colorectal cancer, a population-based case-control study was carried out in Chinese population. METHODS: A total of 140 patients with colorectal cancer and 280 cancer-free frequency-matched controls from a follow-up cohort population established in 1989, were enrolled. For the UGT1A7 polymorphisms analysis, polymerase chain reaction (PCR)-based genotyping techniques including semi-nested PCR, allele-specific PCR and PCR-restriction fragment length polymorphism (RFLP) were developed. RESULTS: The variant allele frequencies in patients and controls were 50.0% and 38.6%, respectively, which were significantly associated with risk of colorectal cancer (odds ratio [OR]: 1.59; 95% confidence interval [CI]: 1.19-2.13). For the variant genotypes analysis, *2/*2 and *3/*3 exhibited a significant association with risk of colorectal cancer (OR: 7.80, 95%CI: 2.66-22.87; OR: 3.47, 95%CI: 1.51-7.97, respectively). Stratification analysis indicated that in previous-current cigarette smoking (cigarette smoking history), current cigarette smoking (current cigarette smoking status), previous-current alcohol drinking (alcohol drinking history) or current alcohol drinking individuals (current alcohol drinking status), the risk developing colorectal cancer increased: OR (95%CI), 2.81 (0.97-8.11), 3.39 (1.19-9.67), 2.89 (0.99-8.46) and 3.14 (1.09-9.09), respectively. CONCLUSIONS: UGT1A7 polymorphisms may have a significant modifying effect on colorectal cancer risk, which may interact with environmental factors, cigarette smoking and alcohol drinking in colorectal carcinogenesis.     

不同剂量亚砷酸钠染毒大鼠多药耐药相关蛋白2表达水平的变化

目的观察不同剂量亚砷酸钠染毒大鼠肝细胞膜转运蛋白——多药耐药相关蛋白2(multidrugresistance-associatedprotein2,MRP2)表达水平的变化及与砷代谢的关系。方法24只健康雄性Wistar大鼠随机分为4组:第1组为对照组,给予生理盐水;第2～4组为染砷组,分别给予4、10和20mg/kg体质量的亚砷酸钠溶液,隔天灌胃染毒1次,2周后将大鼠处死。原子吸收分光光度法测定胆汁、全血和肝组织中的总砷含量。蛋白印记法测定肝细胞膜上MRP2的改变。结果经方差分析检验,胆汁和肝脏中含砷量随着染砷剂量的增加而逐渐增加(P<0.05)。两两比较发现,3个染毒组的血砷与对照组之间差异有统计学意义,而3个染毒组间差异无统计学意义。随着染砷剂量增加,MRP2表达有增加的趋势,且MRP2表达量与胆汁含砷量呈正相关(r=0.986,P<0.05)。结论亚砷酸钠可以诱导MRP2的表达,随染砷剂量增加,MRP2表达量增加。MRP2在砷及其代谢产物的胆汁排泄过程中发挥了重要作用。     

The expression of uridine diphosphate glucuronosyltransferase gene is a major determinant of bilirubin level in heterozygous β-thalassaemia and in glucose-6-phosphate dehydrogenase deficiency

All- trans retinoic acid (ATRA) is currently recommended as standard treatment for acute promyelocytic leukaemia (APL). However, there has been increasing concern that ATRA is associated with unusual sites of relapse. We present three cases of APL previously treated with ATRA who ultimately relapsed within the central nervous system (CNS) and hypothesize that, by up-regulating intercellular adhesion molecules, ATRA may facilitate the passage of malignant promyelocytes across the blood–brain barrier.     

Gilbert综合征和Crigler-Najjar综合征相关尿苷二磷酸葡糖醛酸转移酶A1基因突变位点特征分析

目的探讨Gilbert综合征(GS)和Crigler-Najjar综合征(CNS)相关尿苷二磷酸葡糖醛酸转移酶A1(UGT1A1)基因的突变特征及与临床的相关性。方法通过检索PubMed和人类基因突变数据库归纳UGT1A1基因突变位点的特征并分析其临床相关性。结果截至2018年11月16日,共发现UGT1A1基因163个突变位点,上述位点存在以下规律:(1)GS或CNS表型相关的UGT1A1的不同外显子发生的基因突变个数,均与外显子长度呈正相关;(2)无义点突变主要发生在CNS I型;(3)GS、CNS II型的复合杂合突变位点的组合和分布存在一定规律,其中GS的4种复合杂合组成中,均有-3279T>G突变;(4)亚洲地区报道的UGT1A1基因突变位点在c.211-c.558有明显的聚集性。结论UGT1A1基因突变位点不同、报道地区不同及人群不同,其突变特征及临床相关性也不同。本研究对GS和CNS的基础研究和临床诊疗有参考价值。