细胞色素P4501A1基因mRNA的化学诱导及在人肝中的基…

利用Ｎｏｒｔｈｅｒｎ印迹杂交及逆转录聚合酶链式反应技术研究了几种传代培养细胞和成人肝细胞及人胚胎肝组织细胞色素Ｐ４５０１Ａ１基础表达水平并观察了几种化学诱导对人羊膜细胞ＦＬ系和原代胚肝细胞该基因ｍＲＮＡ水平的诱导。     

人细胞色素P4502B6在哺乳类细胞中的稳定表达

〗在诱变试验中广泛应用人羊膜ＦＬ细胞，中国仓鼠ＣＨＬ和Ｖ７９细胞，但由于它们不能表达内源性细胞色素Ｐ４５０（ＣＹＰ）活化前致突变物，而严重限制了它们的使用．本工作构建了携有编码ＣＹＰ２Ｂ６单胺氧化酶的ｃＤＮＡ重组表达体并分别导入上述三种细胞中表达，经Ｇ４１８抗性筛选，建立了ＦＬ－２Ｂ６，ＣＨＬ－２Ｂ６和Ｖ７９－２Ｂ６三种转基因细胞系，用Ｎｏｒｔｈｅｒｎ印迹杂交证明它们都有ＣＹＰ２Ｂ６的表达．在ＣＨＬ－２Ｂ６细胞中还测得较高水平的７－乙氧基－４－三氟甲基香豆素去乙基酶活性，它对前致突变物甲基亚硝胺吡啶酮的致微核形成作用有很高敏感性，并呈剂量效应关系，但其母系细胞皆为阴性．     

芳基烃受体及其转录调控的细胞色素P4501A在环境污染物所致毒性中的作用

芳基烃受体属于碱性螺旋-环-螺旋蛋白超家族的转录因子。环境污染物激活芳基烃受体,上调cyp1a的表达,诱导自身的Ⅰ相代谢,增加代谢中间体的生成,代谢中间体与生物大分子结合,造成机体损伤。环境污染物同时可激活芳基烃受体并改变一系列与细胞增殖、细胞周期进程和凋亡相关基因的表达,导致机体损伤并诱发肿瘤。     

细胞色素P4501A2活性与奥氮平代谢的相关性研究

目的 研究奥氮平(抗精神病药)体内代谢与细胞色素P450酶亚型1A2(CYP1A2)活性的相关性.方法 15例男性健康志愿者单次口服咖啡因150mg,第5 h末采血;2天后单次口服奥氮平10 mg,收集血样;用高效液相色谱电化学法测定奥氮平血浆浓度,紫外法测定咖啡因及其代谢产物次黄嘌呤的血浆浓度.结果 次黄嘌呤与咖啡因的比值与奥氮平清除的奥氮平AUC0.96的倒数不相关(γ=0.057,P=0.84).结论 CYP1A2酶活性与奥氮平体内代谢不相关.     

抗肿瘤活性物SYUIQ-5对大鼠肝组织细胞色素P450基因表达的影响

目的研究抗肿瘤活性物SYUIQ-5对大鼠肝组织细胞色素P（CYP）450基因表达的影响.方法抗肿瘤活性物SYUIQ-5,0.1 mg/kg、5 mg/kg、10 mg/kg三个剂量,雄性SD大鼠经腹腔注射连续处理5 d后,取肝组织,提取总RNA,应用反转录-聚合酶链反应（RT-PCR）检测CYP 3A1、2B1/2、1A1、1A2和2E1信使核糖核酸（mRNA）的表达.结果与空白对照组相比,抗肿瘤活性物SYUIQ-5可显著诱导CYP 1A1 mRNA的表达,对CYP 3A1、2B1/2、1A2和2E1 mRNA表达无影响.结论抗肿瘤活性物SYUIQ-5可显著诱导大鼠肝组织CYP 1A1基因表达.对CYP 3A1、2B1/2、1A2和2E1的基因表达无影响.     

重组人胸腺肽α1对大鼠肝组织细胞色素P450基因表达的影响

目的评价重组人胸腺肽α₁（rh-Tα₁）对大鼠肝组织细胞色素P450（CYP450）基因表达的影响。方法SD大鼠经腹腔注射不同剂量的rh-Tα₁（150、300和600μg/kg）,14 d后从肝组织中提取总RNA,应用逆转录-聚合酶链反应（RT-PCR）检测CYP3A1、CYP1A2和CYP2E1 mRNA的表达。结果与空白对照组相比,rh-Tα₁可显著诱导CYP3A1、CYP 1A2和CYP 2E1的基因表达。结论rh-Tα₁通过诱导CYP450酶系的mRNA表达影响其活性。     

吸烟人群细胞色素P4501A1和谷胱甘肽-S-转移酶M1的基因型

采用分子流行病学的方法 ,研究吸烟人群细胞色素 P450 1 A1基因 (CYP1 A1 )和谷胱甘肽 - S-转移酶 M1基因 (GST M1 )的多态性特征 .吸烟人群与同性别及同年龄 (年龄 <2周岁 )健康人群一对一配对 .提取血液基因组 DNA. PCR扩增 CYP 1 A1Exon7位点片段和 GST M1部分片段 .限制性酶切片段长度多态性 (RELP)分析 P450 1 A1片段和GST M1片段多态性特点 .结果显示吸烟组人群中CYP 1 A1 Exon 7Val/Val型 (mm型 )人群数增高 ,并有统计学差异 .吸烟组人群中 :GST M1缺失人群数略有升高 ;CYP1 A1 wm型人群数略有升高 ;CYP 1 A1 wm型 +mm型人群数略有升高 ,但这些变化均无统计学意义     

细胞色素P450酶在癌症研究中的应用

细胞色素P450酶是体内代谢转换的重要酶系.现从细胞色素P450酶在活性调节、代谢表型、致癌 物活化、抗癌药物代谢、肿瘤组织中特异表达和肿瘤基因治疗等方面综述细胞色素P450酶的特点及其在癌症研究中的应用潜力.     

Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes

Freshly prepared human hepatocytes are considered as the 'gold standard' for in vitro testing of drug candidates. However, several disadvantages are associated with the use of this model system. The availability of hepatocytes is often low and consequently the planning of the experiments rendered difficult. In addition, the quality of the available cells is in some cases poor. As an alternative, cryopreserved human hepatocytes were validated as a model to study cytochrome P450 1A2 (CYP1A2) and cytochrome P450 3A4 (CYP3A4) induction. In a single blinded experiment, hepatocytes from three separate lots were incubated with three concentrations of different compounds, and compared to non-treated cells and cells incubated with omeprazole or rifampicin. CYP1A2 and CYP3A4 induction was determined by measuring 7-ethoxyresorufin-O-deethylation activity and 6beta-hydroxytestosterone formation, respectively. CYP1A2 and CYP3A4 mRNA and protein expression were analyzed by TaqMan QRT-PCR and immunodetection. Cells responded well to the prototypical inducers with a mean 38.8- and 6.2-fold induction of CYP1A2 and CYP3A4 activity, respectively. Similar as with fresh human hepatocytes, high batch-to-batch variation of CYP1A2 and CYP3A4 induction was observed. Except for 1 and 10 microM rosiglitazone, the glitazones did not significantly affect CYP1A2. A similar result was observed for CYP3A4 activity although CYP3A4 mRNA and protein expression were dose-dependently upregulated. In conclusion, cryopreserved human hepatocytes may be a good alternative to fresh hepatocytes to study CYP1A and 3A induction.     

肝细胞微粒体的制备和细胞色素P450氧化酶活性测定

目的：为测定人肝细胞微粒体细胞色素Ｐ４５０氧化酶的活性。方法：用差速离心法制备３例人肝细胞微粒体。结果：细胞色素Ｐ４５０的含量为０．５２３±０．００５ｎｍｏｌ·ｍｇ－１；细胞色素ｂ５为０．２８５±０．０２５ｎｍｏｌ·ｍｇ－１；氨基比林Ｎ－脱甲基酶的活力为０．５±０．６ｎｍｏｌ·ｍｇ－１；乙基吗啡Ｎ－脱甲基酶活力为０．９８±０．０８ｎｍｏｌ·ｍｇ－１。结论：Ｐ４５０酶活性影响因素较多，个体差异大。临床用药时应考虑患者的个体情况。     

Regulation of cytochrome P450 gene expression by ketamine: a review

Introduction: Although used as an anesthetic drug for decades, ketamine appears to have garnered renewed interest due to its potential therapeutic uses in pain therapy, neurology, and psychiatry. Ketamine undergoes extensive oxidative metabolism by cytochrome P450 (CYP) enzymes. Considerable efforts have been expended to elucidate the ketamine-induced regulation of CYP gene expression. The safety profile of chronic ketamine administration is still unclear. Understanding how ketamine regulates CYP gene expression is clinically meaningful.

Areas covered: In this article, the authors provide a brief review of clinical applications of ketamine and its metabolism by CYP enzymes. We discuss the effects of ketamine on the regulation of CYP gene expression, exploring aspects of cytoskeletal remodeling, mitochondrial functions, and calcium homeostasis.

Expert opinion: Ketamine may inhibit CYP gene expression through inhibiting calcium signaling, decreasing ATP levels, producing excessive reactive oxygen species, and subsequently perturbing cytoskeletal dynamics. Further research is still needed to avoid possible ketamine–drug interactions during long-term use in the clinic.     

Characterization of triptolide hydroxylation by cytochrome P450 in human and rat liver microsomes

Triptolide, the primary active component of a traditional Chinese medicine Tripterygium wilfordii Hook F, has a wide range of pharmacological activities. In the present study, the metabolism of triptolide by cytochrome P450s was investigated in human and rat liver microsomes. Triptolide was converted to four metabolites (M-1, M-2, M-3, and M-4) in rat liver microsomes and three (M-2, M-3, and M-4) in human liver microsomes. All the products were identified as mono-hydroxylated triptolides by liquid chromatography-mass spectrometry (LC-MS). The studies with chemical selective inhibitors, complementary DNA-expressed human cytochrome P450s, correlation analysis, and enzyme kinetics were also conducted. The results demonstrate that CYP3A4 and CYP2C19 could be involved in the metabolism of triptolide in human liver, and that CYP3A4 was the primary isoform responsible for its hydroxylation.     

吸烟人群细胞色素P4501A1和谷胱甘肽-S-转移酶M1的基因型

采用分子流行病学的方法,研究吸烟人群细胞色素P450 1A1基因(CYP 1A1)和谷胱甘肽-S-转移酶M1基因（GST M1）的多态性特征.吸烟人群与同性别及同年龄（年龄<2周岁）健康人群一对一配对. 提取血液基因组DNA. PCR扩增CYP 1A1 Exon 7位点片段和GST M1部分片段. 限制性酶切片段长度多态性（RELP）分析P450 1A1片段和GST M1片段多态性特点. 结果显示吸烟组人群中CYP 1A1 Exon 7 Val/Val型（mm型）人群数增高,并有统计学差异. 吸烟组人群中：GST M1缺失人群数略有升高;CYP 1A1 wm型人群数略有升高;CYP 1A1 wm型＋mm型人群数略有升高,但这些变化均无统计学意义.     

酮康唑对大鼠肝脏CYP450酶系的影响*

目的:观察酮康唑对大鼠肝细胞色素P450及其主要亚型的影响。方法:Sprague-Dawley大鼠用140,280,420μmol.kg-1.d-1酮康唑连续灌胃7 d,测定肝脏微粒体中总CYP450含量和CYP1A1,1A2,1B1,2B1/2,2E1和3A亚型活性。结果:不同剂量酮康唑给药后大鼠肝脏脏器系数、CYP1A1和1B1亚型活性明显增高(P<0.05,P<0.01);总CYP450含量和CYP3A活性显著降低(P<0.01);低剂量的酮康唑抑制CYP1A2和CYP2B1/2亚型的活性,高剂量却出现了诱导作用(P<0.05,P<0.01)。各剂量组对CYP2E1均无明显影响。结论:酮康唑对大鼠肝脏CYP450及主要药物代谢亚型CYP1A1,1A2,1B1,2B1/2和3A有影响,临床长期用药或与经肝脏CYP450代谢的药物联合应用时要注意监测血药浓度和肝脏功能,防止药物代谢减缓出现蓄积中毒或药物代谢加快而降低药效。     

细胞色素P450 CYP4A的组织和细胞表达特异性

目的确定细胞色素P450 CYP4A在组织和细胞水平表达是否具有特异性。方法采用Western印迹法分析细胞色素P450 CYP4A蛋白在培养的牛肺动脉内皮细胞、平滑肌细胞及牛肾动脉内皮细胞的表达;采用原位杂交法检测细胞色素P450 CYP4A mRNA在大鼠肺支气管内皮和血管内皮细胞的表达;以14C标记的花生四烯酸(AA)作为反应底物,用荧光高效液相色谱法(HPLC)通过检测牛肺动脉内皮细胞催化外源性AA生成20-羟基廿碳四烯酸(20-HETE)的反应,确定肺动脉内皮细胞是否存在细胞色素P450 CYP4A;将肺动脉内皮细胞破碎并用乙酸乙酯萃取,用荧光物质标记萃取物,用荧光HPLC检测内源性20-HETE,从代谢产物水平确定细胞色素P450 CYP4A的功能蛋白质表达。结果①Western印迹法分析结果表明,细胞色素P450 CYP4A蛋白在牛肺动脉内皮细胞和平滑肌细胞中均有表达,在肺动脉内皮细胞中的表达明显高于平滑肌细胞(积分吸光度值分别为10182±279,5249±167);在牛肾动脉内皮细胞中也有表达,但明显低于肺动脉内皮细胞的表达(积分吸光度值分别为12173±171,17863±207)。②原位杂交结果显示,细胞色素P450 CYP4A mRNA可在大鼠肺支气管内皮细胞和大鼠肺血管内皮均有表达。③用HPLC在牛肺动脉内皮细胞中检测到20-HETE,表明细胞色素P450 CYP4A在肺动脉内皮细胞存在。结论细胞色素P450 CYP4A有其特定的组织和细胞分布,在牛和大鼠肺脏动脉内皮细胞有高表达。