Application of Precision-Cut Rat Liver Slice to Study the Influence of Monocrotaline, Tussilago farfara Alkaloids on the Expression of Cytochrome P450 Enzymes

Precision-cut liver slice has been successfully used to study the mechanism of drug-induced hepatotoxicity, the prediction of liver toxicity, the discovery of early hepatic toxicity biomarker and the metabolism of drug in liver. We detected the expression of CYP3A4, CYP2B1 + CYP2B2 and CYP2E1 in precision-cut liver slice after co-cultured with monocrotaline or Tussilago farfara alkaloids to investigate the hepatotoxicity mechanism of those drugs. After co-culturing with monocrotaline or Tussilago farfara alkaloids for 6 hours, the expression of CYP3A4 in the microsome of precision-cut liver slices was detected by Western blot, and the expressions of CYP2B1 + CYP2B2 and CYP2E1 were detected by immunofluorescence. The results showed that monocrotaline induced the expression of CYP3A4 and CYP2B1 + CYP2B2, and Tussilago farfara alkaloids obviously up-regulated the expression of CYP2E1 and CYP3A4. Thus, we conclude that the up-regulation of CYP3A4, CYP2B1 + CYP2B2 and CYP2E1 may be one of the toxic mechanisms of liver injury of those drugs.

In vitro and in vivo cytochrome P450 3A enzyme inhibition by Aframomum melengueta and Dennettia tripetala extracts

Objective: To evaluate the in vitro and in vivo inhibitory effects of two commonly used herbs, Aframomum melengueta(A. melengueta) and Dennettia tripetala(D. tripetala) on CYP 3A enzymes. Methods: In vitro inhibition of the enzymes were assessed with microsomes extracted from female albino rats using erythromycin-N-demethylation assay(EMND) method while their in vivo effects were measured by estimating simvastatin plasma concentrations in rats. Pharmacokinetic parameters were determined using non-compartmental anaysis as implemented in Win Nonlin pharmacokinetic program. Results: EMND assay with intestinal microsomes indicated that aqueous extracts of D. tripetala and A. melengueta significantly(P < 0.05) inhibited intestinal CYP 3A activity at both 50 μg and 100 μg concentrations. Petroleum ether extract of D. tripetala and ethanol extracts of A. melengueta inhibited intestinal CYP3 A activity at 100 μg but not at 50 μg concentrations. All the extracts showed an in vitrodose dependent CYP 3A inhibition with liver microsomes. In vivo analysis showed that pretreatment with the extracts enhanced systemic absorption of simvastatin with reductions in metabolizing enzymes activity as indicated in significant increases in maximal concentration, area under curve, area under moment curve and mean resident time of simvastatin(P < 0.05). Conclusions: Herbal preparations containing these plants' extracts should be used with caution especially in patients on CYP450 3A substrate medications.

Cytochrome P450 Directed Prodrug Activation Therapy in Research of Cancer Enzymology

Cancer enzymology is a promising filiation of bio-medical sciences. In thepast decades, enzymes, such as GST(glutathione S-transferase) , PKC(protein kinase C) , Topo(DNAtopoisomerases), TK(tyrosine kinase), CD (bacterial cytosine deaminase), CPG2(carboxypeptidase G2) ,and PNP (purine nucleoside phosphorylase), have been known to bear close relations to cancer. Theirspecific expression and influence on the process of tumor initiation, promotion and progressionattract scientists to apply them as a biochemical marker of certain malignant tumor, a predictor ofresponse in cancer chemotherapy; to apply them to drug design, tumor prevention and as adjuvant toradiotherapy or surgery.

Self-sufficient Cytochrome P450s and their potential applications in biotechnology

Cytochrome P450s(CYPs)are ubiquitously found in all kingdoms of life,playing important role in various biosynthetic pathways as well as degradative pathways;accordingly find applications in a vast variety of areas from organic synthesis and drug metabolite production to modification of biomaterials and bioremediation.Significantly,CYPs catalyze chemically challenging CAH and CAC activation reactions using a reactive high-valent iron-oxo intermediate generated upon dioxygen activation at their heme center,while the other oxygen atom is reduced to the level of water by electrons provided through a reductase partner protein.Self-sufficient CYPs,encoding their heme domain and reductase protein in a single polypeptide,facilitate increased catalytic efficiency and render a less complicated system to work with.The self-sufficient CYP enzyme from CYP102A family(CYP102A1,BM3)is among the earliest and most-investigated model enzymes for mechanistic and structural studies as well as for biotechnological applications.An increasing number of self-sufficient CYPs from the same CYP102 family and from other families have also been reported in last decade.In this review,we introduce chemistry and biology of CYPs,followed by an overview of the characteristics of self-sufficient CYPs and representative reactions.Enzyme engineering efforts leading to novel self-sufficient CYP variants that can catalyze synthetically useful natural and non-natural(nature-mimicking)reactions are highlighted.Lastly,the strategy and efforts that aim to circumvent the challenges for improved thermostability,regio-and enantioselectivity,and total turnover number;associated with practical use of self-sufficient CYPs are reviewed.

Cytochrome P450 monooxygenase-mediated eicosanoid pathway:A potential mechanistic linkage between dietary fatty acid consumption and colon cancer risk

Human consumption of linoleic acid(LA,18:2ω-6,abundant in vegetable oils)is very high.Animal experiments showed that excessive LA intake increased azoxymethane-induced colon tumorigenesis,however,the impact of excessive LA on colon cancer in human is not conclusive,making it difficult to make dietary recommendations for optimal intake of LA.Understanding the molecular mechanisms of LA on colon tumorigenesis could help to clarify its health effect,and facilitate development of mechanismbased strategies for preventing colon cancer.Recent studies show that the previously unappreciated cytochrome P450 monooxygenase-mediated eicosanoid pathway is upregulated in colon cancer and plays critical roles in its pathogenesis,and could contribute to the effects of dietary LA,as well asω-3 fatty acids,on colon tumorigenesis.In this review,we will discuss recent studies about the roles of cytochrome P450 monooxygenases in fatty acid metabolism and its roles in colonic inflammation and colon cancer,and how this information could help us to clarify the health impacts of dietary fatty acids.

Functionalized selenium nanoparticles ameliorated acetaminophen-induced hepatotoxicity through synergistically triggering PKCδ/Nrf2 signaling pathway and inhibiting CYP 2E1

Selenium nanoparticles(SeNPs)have been demonstrated potential for use in diseases associated with oxidative stress.Functionalized SeNPs with lower toxicity and higher biocompatibility could bring better therapeutic activity and clinical application value.Herein,this work was conducted to investigate the protective effect of Pleurotus tuber-regium polysaccharide-protein complex funtionnalized SeNPs(PTR-SeNPs)against acetaminophen(APAP)-induced oxidative injure in HepG2 cells and C57BL/6J mouse liver.Further elucidation of the underlying molecular mechanism,in particular their modulation of Nrf2 signaling pathway was also performed.The results showed that PTR-SeNPs could significantly ameliorate APAP-induced oxidative injury as evidenced by a range of biochemical analysis,histopathological examination and immunoblotting study.PTR-SeNPs could hosphorylate and activate PKCδ,depress Keap1,and increase nuclear accumulation of Nrf2,resulting in upregulation of GCLC,GCLM,HO-1 and NQO-1 expression.Besides,PTR-SeNPs suppressed the biotransformation of APAP to generate intracellular ROS through CYP 2E1 inhibition,restoring the mitochondrial morphology.Furthermore,the protective effect of PTR-SeNPs against APAP induced hepatotoxicity was weakened as Nrf2 was depleted in vivo,indicating the pivotal role of Nrf2 signaling pathway in PTR-SeNPs mediated hepatoprotective efficacy.Being a potential hepatic protectant,PTR-SeNPs could serve as a new source of selenium supplement for health-promoting and biomedical applications.

Effects of Yanhusuo(Rhizoma Corydalis),Baizhi(Radix Angelicae Dahuricae)and Their Combination Extracts on Cytochrome P450 Activities in Rats

Background:Yuanhu Zhitong Prescription(元胡止痛方,YZP),a well-known herbal prescription for an analgesic effect,is recorded in the China Pharmacopoeia,consisting of Yanhusuo(Rhizoma Corydalis)and Baizhi(Radix Angelicae Dahuricae).Objective:To explore the influence of 70%EtOH extracts from Yanhusuo(Rhizoma Corydalis),Baizhi(Radix Angelicae Dahuricae)and YZP on the CYP450s,especially the differences between the single drug and prescription.Materials and methods:Cocktail probe drugs method was used to evaluate Cytochrome P450 activities in rat liver microsomes,including CYP1A2,CYP2D1,CYP2C11,CYP2C6 and CYP3A1,after rats repeatedly administrated with the extracts of Yanhusuo(Rhizoma Corydalis),Baizhi(Radix Angelicae Dahuricae)and YZP for 7 days.Results:Yanhusuo(Rhizoma Corydalis)extracts significantly increased the activities of CYP1A2,2C6 and 3A1,and inhibited that of 2D1.Baizhi(Radix Angelicae Dahuricae)extracts significantly increased the activities of CYP1A2 and inhibited that of 2D1 and 2C11.YZP extracts exhibited the same effect with single drugs.Conclusion:These results might partly interpret the TCM compatibility.Moreover,co-administration of prescriptions containing Yanhusuo(Rhizoma Corydalis),Baizhi(Radix Angelicae Dahuricae)or YZP should consider a potential herb(drug)-drug interaction medicated by the induction of CYP1A2,2C6 and 3A1 and inhibition of CYP2D1 and 2C11 enzymes.

丹参酮对大鼠细胞色素P-450酶系和谷胱甘肽转移酶的作用

目的探讨丹参有效成分丹参酮 (Tan)对大鼠肝、肾组织内的细胞色素P 450酶 (cytochromeP 450,CYP)系和谷胱甘肽转移酶(GT)的影响。方法给予SD雄性大鼠丹参酮 (100mg·kg-1 )灌胃,每日 1次,连续10天后,选用CYP1A1、1A2、2B1、2E1及 3A特异性代谢底物,检测它们在肝和肾微粒体中的活性,同时检测肝、肾微粒体中的GT水平变化。结果实验表明,丹参酮可诱导肝微粒体内的CYP1A1、CYP1A2和CYP2B1活性显著升高(均P<0. 01),同时显著抑制CYP2E1的活性 (P<0. 01),也可诱导肝微粒体中GT的活性升高 (P<0.05)。结论丹参酮对CYP亚型的不同调节作用可能会影响与其合用药物在体内的代谢消除,丹参酮对GT的影响也具有一定的临床意义。

表没食子儿茶素-3-没食子酸酯对细胞色素P450酶活性的影响

目的研究表没食子儿茶素-3-没食子酸酯(EGCG)对人肝微粒体中细胞色素P450(CYP450)酶活性的影响。方法以未处理的肝微粒体作为阴性对照组,CYP450酶各亚型的特异性抑制剂作为阳性对照组,通过EGCG或特异性抑制剂与探针底物共同孵育,高效液相色谱法定量分析特异性底物的代谢产物,分析EGCG对CYP450酶各亚型活性的影响,并通过统计学分析拟合获得相应的动力学参数。结果与阴性对照组相比,EGCG显著抑制了CYP1A2酶和CYP3A4酶的活性,但抑制作用远小于阳性抑制剂的抑制作用。EGCG对CYP1A2酶和CYP3A4酶的抑制作用受EGCG浓度的影响,IC50值分别为8.69和14.07μmol/L。EGCG能够竞争性抑制CYP1A2酶的活性,而对CYP3A4酶为非竞争性抑制作用。此外,EGCG对CYP3A4酶的抑制作用具有时间依赖性,随着培养时间的延长,抑制作用逐渐增强。结论EGCG可显著抑制CYP1A2酶及CYP3A4酶的活性。因此,在EGCG的应用中应充分考虑可能出现的药物相互作用及潜在风险。

A Bacterial Cytochrome P450 Enzyme Catalyzes Multistep Oxidation Reactions in Pyrroindomycin Biosynthesis

Cytochrome P450 enzymes (P450s) belong to a large family of oxidative hemeproteins and catalyze highly diverse oxygenation reactions that are involved in the biosynthesis of various natural products. Here, we report a multifunctional cytochrome P450 enzyme, PyrE2, which catalyzes the regioselective, successive 6-electron oxidation of an inert methyl group to produce a carboxyl product through formation of the hydroxyl and aldehyde intermediates in pyrroindomycin biosynthesis. The time-course biotransformation was characterized by the presence of the hydroxyl and aldehyde intermediates, the lag of the formation of the carboxyl product, and the subsequent loss of both intermediates, indicating that each 2-electron oxidation exhibits the distributive mechanism that requires substrate binding and product releasing. Bioinformatics analysis shows that the homologs of pyrE2 are common in the gene clusters of the spirotetronates varying in the oxidative state of the corresponding exocyclic carbon, indicating the generality and diversity of P450-catalyzed oxygenation in related biosynthetic pathways.

Tobacco smoking and its drug interactions with comedications involving CYP and UGT enzymes and nicotine

Tobacco smoking is a global public health threat causing several illnesses including cardiovascular disease(Myocardial infarction), cerebrovascular disease(Stroke), peripheral vascular disease(Claudication), chronic obstructive pulmonary disease, asthma, reduced female infertility, sexual dysfunction in men, different types of cancer and many other diseases. It has been estimated in 2015 that approximately 1.3 billion people smoke, around the globe. Use of medications among smokers is more common, nowadays. This review is aimed to identify the medications affected by smoking, involving Cytochrome P450(CYP)and uridine diphosphate-glucuronosyltransferases(UGTs) enzymes and Nicotine. Polycyclic aromatic hydrocarbons(PAHs) of tobacco smoke have been associated with the induction of CYP enzymes such as CYP1A1, CYP1A2 and possibly CYP2E1 and UGT enzymes. The drugs metabolized by CYP1A1,CYP1A2, CYP2E1 and UGT enzymes might be affected by tobacco smoking and the smokers taking medications metabolized by those enzymes, may need higher doses due to decreased plasma concentrations through enhanced induction by PAHs of tobacco smoke. The prescribers and the pharmacists are required to be aware of medications affected by tobacco smoking to prevent the toxicityassociated complications during smoking cessation.

Cytochrome P450 Enzyme-Copper Phosphate Hybrid Nano-Flowers with Superior Catalytic Performances for Selective Oxidation of Sulfides

Cytochrome P450 enzyme-copper phosphate hybrid materials with flower-like shape were prepared with a simple but efficient coprecipitation method.The growth process of the hybrid flowers can be divided into three successive steps：coordination/nucleation,growth,and further ripen.The concentration of enzymes in the mother liquor exerted great influence on the morphology and surface enzyme content of the nano-composites.The catalytic performance in the reaction of selective oxidation of sulfide to sulfoxide was also investigated.The hybrid flowers exhibited superior catalytic performance：satisfied thioanisole conversion and selectivity to methyl phenyl sulfoxide （both above 97%） with H2O2 as oxidant under mild reaction conditions,excellent stability and recyclability,and wide scope of substrates.Such results indicate that the hybrid materials are potentially good candidates in the industrial enzyme catalysis.

复方丹参滴丸对大鼠肝细胞色素P450酶的影响

目的观察复方丹参滴丸及其单味药对大鼠肝细胞色素P450酶(CYP)主要亚型的影响。方法 SD大鼠分别ig给予复方丹参滴丸0.3258 g.kg-1,丹参0.27 g.kg-1,三七0.0528 g.kg-1和冰片0.003 g.kg-1,每天1次,连续28 d,取大鼠肝微粒体,与CYP1A2,CYP2B6,CYP2C12,CYP2C13,CYP2D2和CYP3A1特异性底物探针共孵育,用高效液相色谱-质谱联用仪(HPLC-MS/MS)测定底物的代谢产物,分析CYP1A2,CYP2B6,CYP2C12,CYP2C13,CYP2D2和CYP3A1酶活性,同时用PCR方法检测cyp1a2,cyp2b1/2,cyp2c11,cyp2e1和cyp3a1 mRNA表达的变化。结果与正常对照组相比,苯巴比妥(阳性对照)对CYP2D2和CYP3A1活性有明显抑制作用,对CYP1A2,CYP2B6,CYP2C12和CYP2C13有明显诱导作用(P<0.05,P<0.01);复方丹参滴丸对CYP1A2和CYP2B6活性有明显抑制作用,对CYP2D2有诱导作用(P<0.05);丹参对CYP1A2和CYP2B6活性有明显抑制作用(P<0.05);三七对CYP1A2,CYP2B6,CYP2C13和CYP2D2活性有明显抑制作用(P<0.05);冰片明显抑制CYP1A2,CYP2B6,CYP2C12,CYP2C13和CYP2D2活性(P<0.01),且抑制强度高于复方丹参滴丸。与正常对照组相比,苯巴比妥对cyp1a2和cyp2b1/2 mRNA水平有明显诱导作用(P<0.05);复方丹参滴丸、丹参和三七对cyp1a2,cyp2b1/2,cyp2c11,cyp2e1和cyp3a1 mRNA水平无明显影响;冰片对cyp1a2,cyp2b1/2和cyp2c11 mRNA水平有明显抑制作用(P<0.05,P<0.01)。结论复方丹参滴丸中各单味药对CYP酶的影响强于全方对CYP酶的影响,其中以冰片对药物代谢酶的影响最为显著。

细胞色素P450介导的补骨脂酚代谢减毒

目的:分析补骨脂酚代谢的细胞色素P450(cytochrome P450,CYP)表型,并在体外研究人肝微粒体(hu-man liver microsomes,HLM)对补骨脂酚代谢减毒的机制。方法:用HLM与化学抑制剂合用法以及人源重组CYP酶法分析代谢补骨脂酚的CYP酶亚型。用HPLC-MS法分析CYP亚型酶特异底物的代谢产物生成量,研究CYP酶广谱抑制剂1-氨基苯并三唑(1-aminobenzotriazole,ABT)对HLM中CYP亚型酶活性的抑制作用。用HPLC法分析补骨脂酚在HLM孵育液中的浓度,研究ABT对其代谢的影响。应用MTT法检测人肾近曲小管上皮细胞(human kid-ney-2,HK-2)的存活率来评价补骨脂酚对HK-2的毒性作用以及HLM中CYP酶和ABT对其毒性的影响。结果:HLM中的CYP1A2、CYP2C9、CYP2C19和CYP3A4参与了补骨脂酚的代谢,其中以CYP2C19的代谢转化率最高;2.5 mmol/L ABT能明显抑制0.5 g/L HLM中上述4种CYP同工酶活性,抑制率达到90%以上;2.5 mmol/L ABT对补骨脂酚在HLM的代谢抑制率为83.24%±2.13%。在HK-2细胞存活率实验中,ABT能显著降低HLM对30～90μmol/L补骨脂酚的代谢减毒作用(P<0.05)。结论:HLM减低补骨脂酚HK-2细胞毒性的作用与HLM中CYP酶将补骨脂酚代谢转化为无毒或毒性较小的代谢产物有关,应用CYP酶广谱抑制剂能逆转HLM对补骨脂酚的代谢解毒作用。

氟喹诺酮类药物对大鼠肝微粒体细胞色素P450酶系的影响

目的比较4种氟喹诺酮类药物[左氧氟沙星(LVFX)、加替沙星(GTFX)、莫西沙星(MXFX)、帕珠沙星(PZFX)]对大鼠肝微粒体细胞色素P450(CYP450)酶系的影响。方法 30只雄性Wistar大鼠随机分为空白对照组、LVFX组(LV组)、GTFX组(GT组)、MXFX组(MX组)、PZFX组(PZ组),每组6只,给药方案为120mg/(kg.d),尾静脉注射给药,连续7d。末次给药后24h处死动物,差速离心法制备肝微粒体混悬液,Lowry法测定肝微粒体蛋白浓度,分光光度法检测肝微粒体CYP450酶系的含量及活性,并采用单因素方差分析进行统计。结果与空白对照组比较,MX组和GT组大鼠肝重明显降低(P<0.01,P<0.05),LV组、GT组和MX组大鼠肝微粒体蛋白浓度明显增加(P<0.01),LV组和GT组CYP450含量增加(P<0.05,P<0.01),GT组细胞色素b5(Cytb5)含量增加(P<0.05)。肝微粒体NADPH-CytC还原酶活性测定结果显示,给药组与空白对照组差异无统计学意义(P>0.05)。氨基吡啉-N-脱甲基酶活性测定结果显示,LV组、GT组和MX组酶活性与空白对照组比较差异均有统计学意义(P<0.01)。红霉素-N-脱甲基酶活性测定结果显示,与空白对照组比较,GT组酶活性降低,MX组酶活性升高,差异有统计学意义(P<0.01)。大鼠肝微粒体CYP450酶系亚家族活性检测结果显示,与空白对照组比较,LV组、MX组和PZ组7-苄基香豆素脱烃酶(BROD)活性升高(P<0.01),GT组7-甲氧基香豆素脱烃酶(MROD)活性降低(P<0.05)、7-苯基香豆素脱烃酶(PROD)活性增加,PZ组PROD活性降低(P<0.01),4种药物均可使乙氧基香豆素脱烃酶(EROD)活性增加(P<0.01)。结论 4种氟喹诺酮类药物对CYP450酶系均有肯定作用,对不同的酶其作用效果不同,从影响范围来看,GTFX、MXFX、LVFX和PZFX的作用依次减小。

“cocktail”探针药物法及其在研究中药对细胞色素P450影响中的应用进展

"cocktail"(鸡尾酒)探针药物法作为一种快速、高通量的研究方法,目前已广泛应用于药物对细胞色素P450(CYP450)活性影响评估、药物代谢途径确认、药物-药物相互作用预测、药物代谢表型分析、临床用药方案优化等诸多研究方向。此方法具有独特的优势和广阔的应用前景。本文主要从CYP450同工酶的特性、"cocktail"探针药物法的特点、探针药物选择依据、"cocktail"探针药物法在中药对CYP450代谢酶影响中的应用进行综述。旨在较为系统地梳理相关研究进展,并为此方面的深入研究工作提供参考。

冠心病血瘀证患者细胞色素P4502C19*17基因型分布与氯吡格雷治疗后血小板聚集率及出血风险的关系(英文)

目的:探讨细胞色素P450(cytochrome,CYP)2C19*17等位基因变异对中国冠心病血瘀证患者经皮冠状动脉介入术(percutaneous coronary intervention,PCI)后应用氯吡格雷治疗的血小板聚集率及出血事件的影响。方法:以限制性片段长度多态性聚合酶链反应基因分析方法检测CYP2C19*17基因多态性,研究2009年7月至2011年4月福建省立医院心内科择期进行PCI成功的冠心病血瘀证患者520例。PCI术前常规服用阿司匹林300mg、氯吡格雷300mg。术前采血,制备富血小板血浆、贫血小板血浆及二磷酸腺苷(adenosine diphos phate,ADP)诱导剂,采用比浊法,以最终浓度为5μmol/LADP为诱导剂,在氯吡格雷治疗前及治疗后10d,检测血小板聚集率。分析最大血小板聚集率(maximalag gregation,Aggmax)和残余血小板聚集率(5-min aggregation,Agglate)。结果:试验发现有5.96%的患者发生心肌梗死血栓溶解术出血事件,而本试验中病人的CYP2C19*17等位基因频率为7.98%。对于CYP2C19*17等位基因携带者,其出血事件发生率远高于野生型(P<0.01);在基线水平,5μmol/LADP诱导的Aggmax和Agglate以及血小板聚集率在CYP2C19*17各基因型之间并没有显著区别;然而经氯吡格雷治疗10d后,CYP2C19*17等位基因携带者与野生型相比,上述3项指标均明显降低(P<0.01或P<0.05),血小板聚集抑制率显著高于野生型患者(P<0.01);携带有CYP2C19*17等位基因的患者与野生型相比,具有更高的出血风险(P<0.01)。结论:冠心病血瘀证患者CYP2C19*17等位基因携带者有着显著的氯吡格雷反应性且其出血风险明显增加。

二苯乙烯苷对小鼠肝细胞色素P450的影响

目的观察二苯乙烯苷(TSG)对小鼠肝微粒体细胞色素P450(CYP)的影响。方法将昆明种雄性小鼠分为空白组、TSG低剂量组和TSG高剂量组,TSG灌胃3、5、7d后分别麻醉处死小鼠,取肝脏通过荧光实时逆转录聚合酶链反应(real time RT-PCR)检测小鼠肝脏组织CYP相关基因mRNA的表达。结果 TSG作用第3、5、7天时均能够抑制CYP1A2和CYP3A4mRNA表达;TSG呈时间依赖性的增加CYP2E1mRNA表达;TSG作用7d能够显著抑制CYP4A14mRNA表达。此外,TSG对CYP2B10、3A11和3A25mRNA表达无显著性影响。结论 TSG对CYP1A2、CYP2E1、CYP3A4和CYP4A14有显著影响,对CYP2B10、3A11和3A25无明显作用。

银杏内酯对大鼠肝细胞色素P450基因表达的影响

目的:观察银杏内酯对大鼠肝细胞色素P-450基因表达的影响。方法:银杏内酯100mg·kg-1,大鼠灌胃给药,1日1次,连续4d,取肝组织,用竞争法反转录-聚合酶链式反应(competitiveRT-PCR)检测给药后细胞色素P-450基因表达的变化。结果:反转录反应体系不含目的RNA时,用参考模板竞争RNA反转录后用CYP和看家基因Cyclophilin引物进行PCR,CYP1A1,CYP1A2,CYP281/B2,CYP2C11,CYP2E1,CYP3A1,CYP4A1,Cyclophilin均出现了其预期的特异性片段,无其他非特异性带出现,表明参考模板竞争RNA对CYP和看家基因Cyclophilin引物具有特异性竞争能力。给银杏内酯后,对照组和给药组大鼠肝组织的CYP1A1mRNA均检测不到。CYP2C11和CYP2E1的基因表达水平没有变化;CYP1A2和CYP2B1/B2的基因表达水平下降;CYP3A1和CYP4A1的基因表达水平则明显增加。结论:银杏内酯对大鼠肝组织的细胞色素P-450基因表达水平的影响表现出特异性,对不同的细胞色素P-450成员的作用不同。

pcDNA3.1(+)-CYP19-GFP真核表达质粒构建

目的:构建pcDNA3.1(+)-CYP19-GFP、pcDNA3.1(+)-CYP19W39R-GFP、pcDNA3.1(+)-CYP19R264C-GFP与pcDNA3.1(+)-CYP19W39R-R264C-GFP真核表达质粒,并观察pcDNA3.1(+)-CYP19-GFP质粒在MCF-7和Bcap-37细胞中的表达。方法:①采用RT-PCR技术扩增CYP19 cDNA,插入pcDNA3.1(+)载体中,构建pcDNA3.1(+)-CYP19表达质粒;酶切pcDNA3.1(+)-CYP19(304bp BamHⅠ)-GFP和pcDNA3.1(+)-CYP19质粒,构建pcDNA3.1(+)-CYP19-GFP表达质粒;②以pcDNA3.1(+)-CYP19-GFP质粒为模板,采用定点突变技术,构建pcDNA3.1(+)-CYP19W39R-GFP和pcDNA3.1(+)-CYP19R264C-GFP及pcDNA3.1(+)-CYP19W39R-R264C-GFP表达质粒;③pcDNA3.1(+)-CYP19-GFP质粒通过脂质体介导转染MCF-7和Bcap-37细胞,荧光显微镜观察其在细胞中的表达。结果:①酶切鉴定及测序验证pcDNA3.1(+)-CYP19-GFP构建成功;②测序验证pcDNA3.1(+)-CYP19W39R-GFP和pcDNA3.1(+)-CYP19R264C-GFP及pcDNA3.1(+)-CYP19W39R-R264C-GFP构建成功;③在经转染的MCF-7和Bcap-37细胞中观察到较强的绿色荧光。结论:pcDNA3.1(+)-CYP19-GFP、pcDNA3.1(+)-CYP19W39R-GFP和pcDNA3.1(+)-CYP19R264C-GFP及pcDNA3.1(+)-CYP19W39R-R264C-GFP真核表达质粒已成功构建,并证实pcDNA3.1(+)-CYP19-GFP质粒能在MCF-7和Bcap-37细胞中表达,为进一步研究CYP19基因单核苷酸多态性的确切功能奠定基础。