CYP3A5*3 基因多态性对癫痫患者卡马西平及其主要代谢物浓度的影响

背景:卡马西平(carbamazepine,CMZP)主要由CYP3A酶家族代谢,其代谢酶主要包括CYP3A5。本研究探讨了CYP3A5基因多态性与卡马西平血清浓度(CMZP)之间的关系,对个体化药物治疗的开展具有十分积极的意义。目的:CYP3A5*3的基因型可以影响CYP3A药物的药代动力学。本研究旨在评估CYP3A基因多态性对癫痫患者血清卡马西平稳态浓度及其代谢物水平的影响。方法:研究共纳入278例患者,检测个体卡马西平的血清浓度及CYP3A5基因型,并探讨CYP3A5基因型对卡马西平稳态血药浓度的影响。结果:根据基因型分为成CYP3A5表达组(CYP3A5*1/*1和CYP3A5*1/*3)和非表达组(CYP3A5*3/*3)两组。278例患者中120例为CYP3A5表达组,158例患者为CYP3A5非表达组。CYP3A5非表达组的总卡马西平剂量和剂量标准化后的卡马西平血清浓度均高于CYP3A5表达组(P=0.608和P=0.000)。CYP3A5表达组中卡马西平环氧化物浓度更高(P=0.000),但这两组间的血清药物浓度无显著差异(P=0.090)。结论:CYP3A5*3基因多态性与卡马西平的血清浓度之间有密切的关系。CYP3A5基因影响了卡马西平血药浓度水平和代谢过程,其可能是导致卡马西平在癫痫患者中个体变异的一个重要因素。     

应用HRM技术对CYP2C19*2和CYP2C19*3进行双重SNP分型

氯吡格雷是一种广泛用于预防静脉血栓形成的抗血小板药物。研究表明, 携带有CYP2C19基因功能缺失型等位基因CYP2C19*2、CYP2C19*3的病人, 其体内代谢氯吡格雷成为其活性形式的能力降低, 导致氯吡格雷抑制血小板聚集功能减弱。文章旨在建立一种利用高分辨率熔解曲线分析(High-resolution melting curve analysis,HRM)技术在闭合单管中同时对CYP2C19*2、CYP2C19*3两个多态性位点进行简便、准确分型的方法。本实验针对两个SNP位点分别设计特异性的HRM引物, 并在两个位点引物的5′端分别加上富含AT和GC的序列, 保证两个位点的扩增产物熔解峰无重叠。利用HRM技术, 快速、灵敏地对64例随机DNA样本的CYP2C19*2 、CYP2C19*3两个多态性位点进行了基因分型, 且HRM方法的分型结果与测序验证结果完全一致。因此, 利用HRM技术可以实现在闭合单管中简便、准确地对CYP2C19*2 、CYP2C19*3两个多态性位点同时进行基因分型。该方法有望应用于临床, 指导氯吡格雷的个体化用药。     

小型猪CYP3A88基因克隆与其他动物的序列比较

目的克隆我国资源小型猪品系巴马香猪肝脏中的CYP3A88基因,并进行生物信息学分析。方法应用RACE（Rapid Amplification of cDNA Ends）技术对其全长进行扩增,测序,利用Internet和GenBank数据库对其序列进行生物信息学分析。结果首次克隆并鉴定了我国资源小型猪品系巴马香猪肝脏中CYP3A88（GenBank登录号：EF625347）的编码区,获得大小为1965bp的全长cDNA,编码区长为1512bp,编码503个氨基酸;比较核苷酸序列,与小型猪CYP3A39相似性高达94%,而与人等其它动物的CYP3A相似性则在86%以下;推导和分析氨基酸序列表明,与小型猪CYP3A其它成员（CYP3A39、CYP3A29、CYP3A22）进行对比,其相似性分别为92%,89%,80%,而将小型猪与人的CYP3A分别比对,小型猪CYP3A88与人CYP3A4相似性最高,为77%;对其二级结构预测,它可能含12个α螺旋,4个β折叠;经NCBI上的CDD程序分析可知,其39～491氨基酸区域为P4503A亚家族保守结构区域;经聚类分析,小型猪和狗的CYP3A与人有较近的进化关系;通过同源建模法对其在线建模,人CYP3A4晶体结构作为其模建模型,得到了其经典的三维结构。结论在猪CYP3A家族四个基因中,CYP3A88在序列和高级结构上均与人CYP3A4的最为相似。     

CYP2C8及CYP3A4细胞表达体系的构建及其在小分子激酶药物对紫衫醇代谢途径抑制研究中的应用

构建CYP2C8及其3种突变体细胞表达体系,以紫杉醇为底物研究CYP2C8基因多态性对其酶活性的影响,以及构建CYP2C8和CYP3A4共转染细胞体系研究小分子激酶抑制剂对紫杉醇代谢途径的抑制。根据基因文库分别合成CYP3A4以及CYP2C8及其3种突变体CYP2C8*2(805AT)、CYP2C8*3(416GA,1196AG)、CYP2C8*4(792CG)的基因编码片段,将其连接到PEGFP-N1表达质粒,测序验证。将CYP2C8野生型及其突变体分别转染HepG2细胞,24 h后加入紫杉醇进行孵育,通过建立好的LC-MS/MS方法对代谢物进行定量分析。同时,也将野生型CYP2C8和CYP3A4质粒按一定的浓度比转入Hep G2细胞构建共表达体系。并筛选出合适的质粒浓度比转染细胞,在加入紫杉醇孵育时,同时加入小分子激酶抑制剂,考察小分子激酶抑制剂对紫杉醇代谢途径的抑制作用。结果表明,CYP2C8*4代谢酶对紫杉醇的代谢能力存在明显差异,其中CYP2C8*2和CYP2C8*3代谢活性分别是野生型的81%(P0.05)和87%(P0.05),而CYP2C8*4则是野生型的65%(P0.01)。尼洛替尼完全抑制了紫杉醇的代谢,阿法替尼对紫杉醇的两条代谢途径抑制达30%,而伊马替尼选择性抑制了CYPD3A4的活性。不同基因型CYP2C8对紫杉醇的代谢存在差异,可能是导致临床疗效不同的原因。小分子激酶抑制剂在与紫杉醇联合使用时,对紫杉醇代谢的抑制各不相同。     

CYP2C8及CYP3A4细胞表达体系的构建及其在小分子激酶药物对紫衫醇代谢途径抑制研究中的应用北大核心CSCD

构建CYP2C8及其3种突变体细胞表达体系,以紫杉醇为底物研究CYP2C8基因多态性对其酶活性的影响,以及构建CYP2C8和CYP3A4共转染细胞体系研究小分子激酶抑制剂对紫杉醇代谢途径的抑制。根据基因文库分别合成CYP3A4以及CYP2C8及其3种突变体CYP2C8*2(805A>T)、CYP2C8*3(416G>A,1196A>G)、CYP2C8*4(792C>G)的基因编码片段,将其连接到PEGFP-N1表达质粒,测序验证。将CYP2C8野生型及其突变体分别转染HepG2细胞,24 h后加入紫杉醇进行孵育,通过建立好的LC-MS/MS方法对代谢物进行定量分析。同时,也将野生型CYP2C8和CYP3A4质粒按一定的浓度比转入Hep G2细胞构建共表达体系。并筛选出合适的质粒浓度比转染细胞,在加入紫杉醇孵育时,同时加入小分子激酶抑制剂,考察小分子激酶抑制剂对紫杉醇代谢途径的抑制作用。结果表明,CYP2C8*4代谢酶对紫杉醇的代谢能力存在明显差异,其中CYP2C8*2和CYP2C8*3代谢活性分别是野生型的81%(P<0.05)和87%(P<0.05),而CYP2C8*4则是野生型的65%(P<0.01)。尼洛替尼完全抑制了紫杉醇的代谢,阿法替尼对紫杉醇的两条代谢途径抑制达30%,而伊马替尼选择性抑制了CYPD3A4的活性。不同基因型CYP2C8对紫杉醇的代谢存在差异,可能是导致临床疗效不同的原因。小分子激酶抑制剂在与紫杉醇联合使用时,对紫杉醇代谢的抑制各不相同。     

氯吡格雷对大鼠肝药物酶CYP3A4 和CYP1A2 表达的影响

目的：氯吡格雷主要由CYP3A4 催化使其激活,CYP1A2 也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外 文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4 和CYP1A2 的表达,探讨氯吡格雷对大鼠肝药物酶的影 响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13.5,6.75mg/kg/d）,雄性健康大鼠连续灌胃给药7天,脱臼处 死,取肝组织,通过western blot法检测大鼠肝脏CYP3A4 和CYP1A2 蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4 蛋白 表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4 蛋白表达量降低（P<0.05）;氯吡格雷低中高剂量组间进行比较,大 鼠CYP3A4 蛋白表达量呈梯度减少（P<0.05）;2）、氯吡格雷抑制大鼠CYP1A2 蛋白表达,氯吡格雷高中低剂量组分别比生理盐水 组大鼠CYP1A2 蛋白表达量降低（P<0.05）,氯吡格雷低中高剂量组间进行比较,大鼠CYP1A2 蛋白表达量呈梯度减少（P<0.05）。 结论：氯吡格雷使肝细胞色素氧化酶CYP3A4 和CYP1A2 的表达量减少,因此氯吡格雷高、中、低3 个剂量组均不同程度的抑制 大鼠肝脏CYP3A4 和CYP1A2 的表达,提示当氯吡格雷与某些主要经CYP3A4 和CYP1A2 代谢的药物合用时,发生代谢性相关 作用的可能性大。     

实验用西藏小型猪CYP3A基因的克隆及序列分析

目的:克隆西藏小型猪CYP3A基因并与人及其它小型猪品种进行序列比对分析。方法:根据Genebank数据库设计引物,取西藏小型猪新鲜肝脏组织,Trizol法提取肝脏总RNA,应用RT-PCR反转获得肝脏cDNA。分别扩增基因CYP3A46、CYP3A22、CYP3A29及CYP3A39,PCR片段回收后分别连接pMD-18T载体,获得重组质粒pT-CYP3A,阳性重组克隆送测序。采用NCBI Blast及vector NTI软件进行序列比对分析。结果:CYP3A46、CYP3A22、CYP3A29及CYP3A39基因编码序列全长1512 bp,编码503个氨基酸,与人CYP3A4相同。其中CYP3A22和CYP3A39与NCBI记录巴马小型猪序列相同;CYP3A46与巴马小型猪CYP3A46(NM_001134824.1)有6个位点碱基不同。CYP3A29与巴马小型猪CYP3A46(EU918131.1)亦有6个位点碱基不同。对来自2个母本后代猪的基因经过PCR扩增后测序发现CYP3A46、CYP3A29序列完全一致。结论:成功克隆西藏小型猪CYP3A的4个基因,其中CYP3A46与人CYP3A4的序列相似性最高。所得CYP3A46与CYP3A29序列可能为西藏小型猪独有。     

CYP3A5基因单核苷酸多态性rs3800959与氯吡格雷抵抗的相关性研究

目的:探讨细胞色素P450 3A5基因(CYP3A5)单核苷酸多态位点rs3800959与氯吡格雷抵抗(Clopidogrel resistance,CR)发生的关系.方法:于2010年3月至2011年10月期间,连续入选在沈阳军区总医院心内科住院的接受标准双联抗血小板治疗(阿司匹林+氯吡格雷)的冠心病患者共800例.以光学比浊法测定20μmol/L浓度ADP诱导的残余血小板聚集率(Residual platelet agglutination,RPA),并定义RPA＞ 70％为CR,所有入选患者分为CR组和氯吡格雷非抵抗组(Non-clopidogrel resistance,NCR).所有入选病例提取血液白细胞基因组DNA后,采用直接测序的方法测定CYP3A5基因rs3800959单核苷酸多态位点的基因型及等位基因.结果:所入选的800例病人中,CR组为150例,NCR组为650例,CR发生率为18.75％.rs3800959基因型频率在CR组为TT型110例(73.3％)、CT型39例(26.0％)及CC型1例(0.7％);NCR组rs3800959基因型频率分别为477例、159例及14例(73.4％、24.5％及 2.1％).两组间各基因型频率分布无统计学差异(P=0.460,x2=1.554);T、C等位基因分布频率在两组间亦无明显差异(P=0.784,OR=0.942,95％CI=0.655～1.356).结论:CYP3A5基因单核苷酸多态位点rs3800959与冠心病人CR的发生无相关关系.     

实时荧光定量检测中国实验用小型猪肝脏CYP3A29 mRNA表达水平

CYP3A29是猪肝脏最重要的药物代谢关键酶。研究中国实验用小型猪肝脏CYP3A29 mRNA的表达特性对于评估其是否适宜于作为人CYP3A4介导的药理学研究动物模型具有一定意义。以b-actin作校正, 利用TaqMan定量技术对巴马香猪、贵州小型香猪肝脏CYP3A29 mRNA表达水平进行检测, 并以荣昌猪作为对照。结果表明, 巴马香猪、贵州小型香猪、荣昌猪肝脏CYP3A29 mRNA表达水平与报道的人肝脏CYP3A4相近; 三品系(种)猪间肝脏CYP3A29 mRNA表达水平较为接近, 但品系(种)内个体间变异较大。提示巴马香猪、贵州小型香猪作为药物评价的实验动物具有一定可行性。     

紫杉醇对食蟹猴和人CYP1A2、CYP3A4及CYP2A6酶代谢的影响

目的探讨紫杉醇对食蟹猴和人肝微粒体CYP1A2、CYP2A6和CYP3A4酶活性的影响。方法采用食蟹猴和人肝脏微粒体,分别以非那西汀、睾丸酮和香豆素分别作为CYP1A2、CYP2A6、CYP3A4的底物,建立CYP1A2、CYP2A6和CYP3A4体外代谢体系。采用不同浓度的紫杉醇分别与上述3种底物共同孵育于肝微粒体代谢体系中。用HPLC法分别测定各底物的代谢产物扑热息痛、6β-羟基睾丸酮、7-羟基香豆素的产生量,计算IC50值,以评估紫杉醇对CYP1A2、CYP2A6和CYP3A4代谢的影响。结果紫杉醇对食蟹猴肝微粒体3种酶的IC50值分别为570±5.9μmol/L、140±2.9μmol/L和无影响;紫杉醇对人肝微粒体3种酶的IC50值分别为193±6.6μmol/L、253±3.6μmol/L和24±1.6μmol/L。结论紫杉醇对食蟹猴肝微粒体CYP1A2和CYP3A4活性具有一定的抑制作用,但对CYP2A6酶的活性几乎没有影响。紫杉醇对人肝微粒体CYP1A2和CYP3A4活性的抑制作用较弱,但对CYP2A6酶的活性抑制作用较强,提示临床上紫杉醇与作为上述酶底物的药物联合用药时应慎重,以避免因中西药物相互作用所导致的不良反应发生。     

Hyaluronate degradation in 3T3 and simian virus-transformed 3T3 cells

The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.     

Cell density and amino acid transport in 3T3, SV3T3, and SV3T3 revertant cells

The transport of selected neutral and cationic amino acids has been studied in Balb/c 3T3, SV3T3, and SV3T3 revertant cell lines. After properly timed preincubations to control the size of internal amino acid pools, the activity of systems A, ASC, L, and Ly⁺ has been discriminated by measurements of amino acid uptake (initial entry rate) in the presence and absence of sodium and of transportspecific model substrates. L-Proline, 2-aminoisobutyric acid, and glycine were primarily taken up by system A; L-alanine and L-serine by system ASC; L-phenylalanine by system L; and L-lysine by system Ly⁺ in SV3T3 cells. L-Proline and L-serine were also preferential substrates of systems A and ASC, respectively, in 3T3 and SV3T3 revertant cells. Transport activity of the Na⁺-dependent systems A and ASC decreased markedly with the increase of cell density, whereas the activity of the Na⁺-independent systems L and Ly⁺remained substantially unchanged. The density-dependent change in activity of system A occurred through a mechanism affecting transport maximum (V_max) rather than substrate concentration for half-maximal velocity (K_m). Transport activity of systems A and ASC was severalfold higher in transformed SV3T3 cells than in 3T3 parental cells at all the culture densities that could be compared. In SV3T3 revertant cells, transport activity by these systems remained substantially similar to that observed in transformed SV3T3 cells. The results presented here add cell density as a regulatory factor of the activity of systems A and ASC, and show that this control mechanism of amino acid transport is maintained in SV40 virus-transformed 3T3 cells that have lost density-dependent inhibition of growth, as well as in SV3T3 revertant cells that have resumed it.     

3-Bromo-3-deazaneplanocin and 3-bromo-3-deazaaristeromycin: Synthesis and antiviral activity

As an outgrowth of our program to explore 3-deazaadenine carbocyclic nucleosides, 3-bromo-3-deazaneplanocin (5) and 3-bromo-3-deazaaristeromycin (6) have been synthesized from a readily available cyclopentenol and cyclopentanone and either 4-amino- or 4-chloro-1H-imidazo[4,5-c]pyridine (6-amino- or 6-chloro-3-deazaadenine) in 5 steps and 7 steps, respectively. Antiviral analysis found 5 to display significant activity towards a number of (-)-ssRNA and a few dsDNA viruses. Compound 6 was less active than 5 against selected examples of those viruses affected by 5.     

Ether-linked lipids of Balb/c3T3, SV3T3 and concanavalin A-selected SV3T3 revertant cells

Ether-linked lipids were analyzed in Balb/c3T3, SV3T3 and Concanavalin A-selected SV3T3 revertant cells. The three cell lines were found to contain significant quantities of alk-1-enyl- and alkyl-linked phosphatidylethanolamine (PE) and phosphatidylcholine (PC) and small amounts of alkyldiacylglycerols. Compared to 3T3 cells, SV3T3 cells contain a higher amount of alk-1-enyl-linked PC, while in SV3T3 revertant cells the concentrations of the various ether lipids are similar to those of 3T3 cells. The major difference in the composition of ether groups of SV3T3 cells, compared to 3T3 cells, is an increase of 18:0 accompanied by a decrease of 18:1 in the alk-1-enyl-linked PE and PC. Alk-1-enyl-linked PC of SV3T3 revertant cells also shows an increase of 18:0, while the decrease of 18:1 was not statistically significant.     

Protein degradation in 3T3 cells and tumorigenic transformed 3T3 cells

To study the relation of overall rates of protein degradation in the control of cell growth, we determined if transformation of fibroblasts to tumorigenicity affected their rates of degradation of short- and long-lived proteins. Rates of protein degradation were measured in nontumorigenic mouse Balb/c 3T3 fibroblasts, and in tumorigenic 3T3 cells transformed by different agents. Growing 3T3 cells, and cells transformed with Moloney sarcoma virus (MA-3T3) or Rous sarcoma virus (RS-3T3), degraded short- and long-lived proteins at similar rates. Simian virus 40 (SV-3T3)- and benzo(a)pyrene (BP-3T3)-transformed cells had slightly lower rates of degradation of both short- and long-lived proteins. Reducing the serum concentration in the culture medium from 10% to 0.5%, immediately caused about a twofold increase in the rate of degradation of long-lived proteins in 3T3 cells. Transformed lines increased their rates of degradation of long-lived proteins only by different amounts upon serum deprivation, but none of them to the same extent as did 3T3. Greater differences in the degradation rates of proteins were seen among the transformed cells than between 3T3 cells and some transformed cells. Thus, there was no consistent change in any rate of protein degradation in 3T3 cells due to transformation to tumorigenicity.     

Endogenous hyaluronate-cell surface interactions in 3T3 and simian virus-transformed 3T3 cells

3T3 cells have a large, pericellular coat which contains 30 times more hyaluronate than the amount of cell surface hyaluronate associated with simian virus 40-transformed 3T3 (SV-3T3) cells. On the other hand, SV-3T3 cells have high affinity binding sites for exogenously added hyaluronate, whereas 3T3 cells have much lower affinity sites. Removal of cell surface hyaluronate from SV-3T3 cells by treatment with hyaluronidase caused a reproducible increase in their maximum binding capacity for exogenous hyaluronate but no significant change in binding affinity or specificity. For 3T3 cells, however, the maximum amount of binding decreased and the affinity of binding increased after hyaluronidase treatment. When endogenous cell surface hyaluronate was labeled metabolically and then the cells incubated in the presence of exogenous unlabeled hyaluronate, the labeled cell surface hyaluronate was quantitatively displaced from the SV-3T3 cells but was not displaced from the 3T3 cells. Chondroitin sulfate and heparin did not displace cell surface hyaluronate from either cell type. Membranes isolated from SV-3T3 cells bound hyaluronate specifically and with high affinity, whereas membranes from 3T3 cells did not consistently bind a significant amount of hyaluronate. We conclude from these studies that the retention of endogenous hyaluronate on the surface of SV-3T3 cells is mediated by binding sites similar to those detected by the addition of exogenous hyaluronate, and the mechanism of retention of endogenous hyaluronate on the surface of 3T3 cells differs from SV-3T3 cells.     

Synthesis and biological activity of 3 beta-fluorovitamin D3: comparison of the biological activity of 3 beta-fluorovitamin D3 and 3-deoxyvitamin D3

The alteration in the biologic activity of the vitamin D3 molecule resulting from the replacement of a hydrogen atom with a fluorine atom is a subject of fundamental interest. To investigate this problem we synthesized 3 beta-fluorovitamin D3 6 and its hydrogen analog, 3-deoxyvitamin D3 7, and tested the biologic activity of each by in vitro and in vivo methods. Contrary to previous reports which showed that 3 beta-fluorovitamin D3 was as active as vitamin D3 in vivo, we found that the fluoro-analog was less active than vitamin D3. With regard to stimulation of intestinal calcium transport and bone calcium mobilization in the D-deficient hypocalcemic rat, 3 beta-fluorovitamin D3 showed significantly greater biologic activity than its hydrogen analog, 3-deoxyvitamin D3. In the organ-cultured, embryonic chick duodenum, 3 beta-fluorovitamin D3 was approx 1/1000th as active as the native hormone, 1,25-dihydroxyvitamin D3, while 3-deoxyvitamin D3 was inactive even at microM concentrations, in the induction of the vitamin D-dependent, calcium-binding protein. With regard to in vitro activity in displacing radiolabeled 25-hydroxyvitamin D3 from vitamin D binding protein and radiolabelled 1,25-dihydroxyvitamin D3 from a chick intestinal cytosol receptor, 3 beta-fluorovitamin D3 and 3 beta-deoxyvitamin D3 both showed very poor binding efficiencies when compared with vitamin D3. Our results show that the substitution of a fluorine atom for a hydrogen atom at the C-3 position of the vitamin D3 molecule results in a fluorovitamin 6 with significantly more biological activity than its hydrogen analog, 3-deoxyvitamin D3 7.