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1、佐米曲坦对大鼠原代肝细胞 PXR 的激活作用 摘摘 要要 细胞色素 P450 3A (cytochrome P450 3A,CYP3A)是细胞色素 P450 超基因家族中最重要的一类代谢酶，体内含量很高，在肝脏和小肠中都有分布。CYP 3A是催化很多内源性和外源性化学物质代谢的主要酶系,其底物相当广泛，一半以上的临床用药由其参与代谢，在药物相互作用中有很重要的作用。CYP 介导的解毒作用是机体保护自身不受外来物质损伤的重要防御机制。孕甾烷 X 受体（PXR）是孤儿核受体家族成员之一，近年来一系列研究发现，PXR 作为关键的转录调控因子参与 CYP3A 基因的诱导表达。 PXR 是个泛宿主性的受
2、体，目前研究已经表明许多结构不同的化合物都能通过激活 PXR 而诱导 CYP3A 的表达，包括外源性物质和内源性甾体物质，如糖皮质激素地塞米松、PCN 和抗菌药利福平等。PXR 的激活可保护机体对抗外来毒物的侵害。 佐米曲坦 （zolmitriptan, C16H21N3O2） 是一种高选择性 5HT1B/1D受体激动剂，临床上用于偏头痛的治疗。本品通过中枢作用，抑制脑干中疼痛的传递；通过作用于外周神经，本品收缩颅血管，并抑制血管活性肽的释放和神经原炎症。 本实验研究佐米曲坦对大鼠 PXR 受体的激活作用，对阐明佐米曲坦对大鼠CYP450 的诱导作用的机制具有重要意义。本文通过构建大鼠 CYP
3、3A1 和CYP3A2 启动子基因（含 PXR 的 DNA 结合区域（DBD）的 PGL3 载体，然后将其和对照质粒共转染至大鼠原代培养肝细胞中，6 h 后换液，在细胞培养基中加入佐米曲坦，孵育 24 h 后通过检测荧光强度获得结果，实验中以地塞米松和PCN 作为阳性对照，溶剂 DMSO 作为阴性。 目的：目的：本实验通过 PCR 扩增得到 CYP3A1 和 CYP3A2 启动子基因序列（含PXR DNA 结合区域（DBD） ，重组至含荧光素报告基因的 PGL3-Basic 载体上，构建 PGL3-3A1/2 P 质粒，建立大鼠原代培养肝细胞重组 PGL3-Basic 质粒模型，从而研究佐米曲
4、坦对大鼠 CYP3A 诱导作用的机制及佐米曲坦性别差异性诱导大鼠 CYP3A 表达的作用机制。 方法：方法： 本实验以大鼠肝脏总 DNA 为模板， 通过 PCR 法扩增 CYP3A1/2 启动子基因序列。通过 TA 克隆，连接 PMD18 载体和 CYP3A1/2 启动子基因片段构建克隆载体，转化大肠杆菌 DH 5 细胞，通过酶切鉴定和 DNA 测序鉴定确定CYP3A1/2 启动子基因序列。 将 CYP3A1/2 启动子基因片段插入 PGL3-Basic 载体的多克隆位点( Xho I，Mlu I)，通过酶切鉴定连接。PGL3-3A1/2 P 质粒与对照质粒 pRL-TK,大鼠 PXR 表达质
5、粒 rPXR-pcDNA 共转染至新鲜制备的大鼠原代肝细胞，转染 6h 后换液给药，24h 后检测荧光强度。 结果结果：DNA 测序鉴定正确，PGL3-3A1/2 P 质粒（多克隆位点 Xho I，Mlu I）和 rPXR-PCDNA 质粒（多克隆位点 EcoR I，Hind III）构建成功。Dex、PCN 的阳性对照明显，且 Dex 的诱导作用比 PCN 稍强，中浓度的佐米曲坦对大鼠CYP3A1 和 CYP3A2 的诱导作用比低浓度和高浓度的明显，但实验的重现性不佳。佐米曲坦大鼠 CYP3A 诱导作用的机制及性别差异性由于时间关系还有待继续研究。 结论：结论： CYP3A1/2 启动子基因
6、片段插入 PGL3-Basic 载体的多克隆位点 Xho I、Mlu I，PGL3-3A1/2 P 质粒构建成功，可用于转染大鼠原代培养肝细胞。研究佐米曲坦对大鼠 PXR 的激活作用，从而阐明佐米曲坦诱导大鼠 CYP3A 的作用机制。 【关键词】【关键词】佐米曲坦；PXR；PGL3-3A1/2 P；质粒构建；原代肝细胞；共转染 Induction of PXR in primary cultured rat hepato- cytes by zolmitriptan Abstract Cytochrome P450 3A enzyme （CYP3A） ,the most abundant CY
7、P isoform,is the most important metabolic enzyme in Cytochrome P450 superfamily and mainly localized on the liver and intestine. CYP3A is the main enzyme system catalyzing the metabolism of many endogenous and xenobiotics.The substrates of CYP3A are widespread and over half of drugs used in clinic a
8、re metabolized by CYP3A.CYP3A also appears to be a key enzyme in drug-drug interactions. The deintoxication regulated by CYP is an important defense mechanism by which organism defends itself from the damage of extraneous materials. Pregnane X receptor （PXR） is one member of the orphan nuclear recep
9、tor family. For the past few years, some researches discovered that PXR played a key transcriptional control factor in induction of CYP3A.This elucidates why PXR can reduce the accumulation of extraneous materials in vivo on the molecular level and meanwhile reveals a molecule mechanism of drug-drug
10、 interactions. PXR is a promiscuous receptor. Recent studies have indicated many compounds including exogenous materials and endogenous steroids which are different in structure, such as glucocorticoid-dexamethasone, pregnenolone-16-car bonitrile, antibacterial rifampicin, can activate PXR, and then
11、 induce the gene transport of CYP3A. The activation of PXR can defend organism from the damage of foreign poison . Zolmitriptan is used to cure migrainous clinically and it is a hypso-selective receptor agonist of 5-HT1B/1D.Zolmitriptan inhibites the transmit of algesic by central action, which can
12、also anastole cranium blood vessel and inhibite the release of vasoactive peptide and nerve inflammation by peripheral nerve action. The Paper study the activation of rats PXR by zolmitriptan, and has an important sense to illuminate the mechanism of induction of rat CYP by zolmitriptan. The study c
13、onstructs a PGL3-3A1P and PGL3-3A2P plasmid which includes CYP 3A1 and CYP 3A2 promoter gene（contains PXR DNA binding domain）.Then PGL3-3A1/2P plasmid and control plasmid are cotransfected into rat primary culture hepatocyte, after 6 hours of cotransfection the culture medium is replaced, then zolmi
14、triptan is added into the culture medium. After 24 hours of incubation, fluorescence of lysate of hepatocyte is detected. The results are compared to that of Dex, PCN and solvent DMSO control groups. Objective: CYP3A1/2 promoter gene fragment（contains DNA binding domain of PXR）is cloned by PCR. PGL3
15、-3A1/2P plasmid which contains PGL3-Basic （including a luciferase report gene）and CYP3A1/2 promoter gene is to be constructed.Then, rat primary culture hepatocytes model recombinated PGL3-3A1/2P plasmid was established. Finally, using this model the mechanism of sex difference induction of CYP3A by
16、zolmitriptan was studied. Methods: using the DNA of rats liver as mould,we amplified CYP3A1/2 promoter gene of rat by PCR. Then, connect PMD18 plasmid and CYP3A1/2 promoter gene fragment to construct clone carrier, the follow step was to transform it into the E.coli DH 5 cell, after that determined the CYP3A1/2 promoter gene sequence by restriction enzyme and DNA sequencing. CYP3A1/2 promoter gene fragment is inserted into the multiple clone site （Xho I， Mlu I） of PGL3-Basic plasmid, th
