铬、汞、镉、镍四种重金属化合物对FL/P4501A1细胞酶活性的影响

利用不同浓度的重金属化合物溴化汞、三氯化铬、硫酸镉、氯化镍处理人羊膜ＦＬ／Ｐ４５０１Ａ１细胞，其半数抑制浓度（ＩＣ５０，ｍｇ／ｍｌ）分别为６．０２６０，３７．５２１３，２４．５４９１和４５．３８４５。结果表明：不同的重金属化合物对ＦＬ／Ｐ４５０１Ａ１细胞的毒性相差很大，溴化汞和硫酸镉的细胞毒性较大。ＦＬ／Ｐ４５０１Ａ１细胞带有Ｐ４５０１Ａ１基因，该基因产物为乙氧基异吩恶唑Ｏ—去乙基酶（ｅｔｈｏｘｙｒｅｓｏｒｕｆｉｎＯ－ｄｅｅｔｈｙｌａｓｅ，ＥＲＯＤ）。酶活性测定结果表明溴化汞、硫酸镉能抑制ＦＬ／１Ａ１细胞自发产生的ＥＲＯＤ活性；三氯化铬、溴化汞、氯化镍、硫酸镉能抑制去甲肾上腺素诱导ＦＬ／１Ａ１细胞产生的ＥＲＯＤ活性。Ｐ４５０１Ａ１酶能激活多环芳烃，溴化汞和硫酸镉可通过降低Ｐ４５０１Ａ１酶活性干扰多环芳烃的致癌作用。

Homer1a reduces inflammatory response after retinal ischemia/reperfusion injury

Elevated intraocular pressure(IOP)is one of the causes of retinal ischemia/reperfusion injury,which results in NRP3 inflammasome activation and leads to visual damage.Homerla is repo rted to play a protective role in neuroinflammation in the cerebrum.However,the effects of Homerla on NLRP3inflammasomes in retinal ischemia/reperfusion injury caused by elevated IOP remain unknown.In our study,animal models we re constructed using C57BL/6J and Homer1^(flox/-)/Homerla^(+/-)/Nestin-Cre^(+/-)mice with elevated IOP-induced retinal ischemia/repe rfusion injury.For in vitro expe riments,the oxygen-glucose deprivation/repe rfusion injury model was constructed with M uller cells.We found that Homerla ove rexpression amelio rated the decreases in retinal thickness and Muller cell viability after ischemia/reperfusion injury.Furthermore,Homerla knockdown promoted NF-κB P65^(Ser536)activation via caspase-8,NF-κB P65 nuclear translocation,NLRP3 inflammasome formation,and the production and processing of interleukin-1βand inte rleukin-18.The opposite results we re observed with Homerla ove rexpression.Finally,the combined administration of Homerla protein and JSH-23 significantly inhibited the reduction in retinal thickness in Homer1^(flox/-)Homer1a^(+/-)/Nestin-Cre^(+/-)mice and apoptosis in M uller cells after ischemia/reperfusion injury.Taken together,these studies demonstrate that Homer1a exerts protective effects on retinal tissue and M uller cells via the caspase-8/NF-KB P65/NLRP3 pathway after I/R injury.

Bone marrow-derived mesenchymal stem cell-derived exosomeloaded miR-129-5p targets high-mobility group box 1 attenuates neurological-impairment after diabetic cerebral hemorrhage

BACKGROUND Diabetic intracerebral hemorrhage(ICH)is a serious complication of diabetes.The role and mechanism of bone marrow mesenchymal stem cell(BMSC)-derived exosomes(BMSC-exo)in neuroinflammation post-ICH in patients with diabetes are unknown.In this study,we investigated the regulation of BMSC-exo on hyperglycemia-induced neuroinflammation.AIM To study the mechanism of BMSC-exo on nerve function damage after diabetes complicated with cerebral hemorrhage.METHODS BMSC-exo were isolated from mouse BMSC media.This was followed by transfection with microRNA-129-5p(miR-129-5p).BMSC-exo or miR-129-5poverexpressing BMSC-exo were intravitreally injected into a diabetes mouse model with ICH for in vivo analyses and were cocultured with high glucoseaffected BV2 cells for in vitro analyses.The dual luciferase test and RNA immunoprecipitation test verified the targeted binding relationship between miR-129-5p and high-mobility group box 1(HMGB1).Quantitative polymerase chain reaction,western blotting,and enzyme-linked immunosorbent assay were conducted to assess the levels of some inflammation factors,such as HMGB1,interleukin 6,interleukin 1β,toll-like receptor 4,and tumor necrosis factorα.Brain water content,neural function deficit score,and Evans blue were used to measure the neural function of mice.RESULTS Our findings indicated that BMSC-exo can promote neuroinflammation and functional recovery.MicroRNA chip analysis of BMSC-exo identified miR-129-5p as the specific microRNA with a protective role in neuroinflammation.Overexpression of miR-129-5p in BMSC-exo reduced the inflammatory response and neurological impairment in comorbid diabetes and ICH cases.Furthermore,we found that miR-129-5p had a targeted binding relationship with HMGB1 mRNA.CONCLUSION We demonstrated that BMSC-exo can reduce the inflammatory response after ICH with diabetes,thereby improving the neurological function of the brain.

TAX1BP1 and FIP200 orchestrate non-canonical autophagy of p62 aggregates for mouse neural stem cell maintenance

Autophagy plays a pivotal role in diverse biological processes,including the maintenance and differentiation of neural stem cells(NSCs).Interestingly,while complete deletion of Fip200 severely impairs NSC maintenance and differentiation,inhibiting canonical autophagy via deletion of core genes,such as Atg5,Atg16l1,and Atg7,or blockade of canonical interactions between FIP200 and ATG13(designated as FIP200-4A mutant or FIP200 KI)does not produce comparable detrimental effects.This highlights the likely critical involvement of the non-canonical functions of FIP200,the mechanisms of which have remained elusive.Here,utilizing genetic mouse models,we demonstrated that FIP200 mediates non-canonical autophagic degradation of p62/sequestome1,primarily via TAX1BP1 in NSCs.Conditional deletion of Tax1bp1 in fip200hGFAP conditional knock-in(cKI)mice led to NSC deficiency,resembling the fip200hGFAP conditional knockout(cKO)mouse phenotype.Notably,reintroducing wild-type TAX1BP1 not only restored the maintenance of NSCs derived from tax1bp1-knockout fip200hGFAP cKI mice but also led to a marked reduction in p62 aggregate accumulation.Conversely,a TAX1BP1 mutant incapable of binding to FIP200 or NBR1/p62 failed to achieve this restoration.Furthermore,conditional deletion of Tax1bp1 in fip200hGFAP cKO mice exacerbated NSC deficiency and p62 aggregate accumulation compared to fip200hGFAP cKO mice.Collectively,these findings illustrate the essential role of the FIP200-TAX1BP1 axis in mediating the non-canonical autophagic degradation of p62 aggregates towards NSC maintenance and function,presenting novel therapeutic targets for neurodegenerative diseases.

Ghrelin regulates insulin resistance by targeting insulin-like growth factor-1 receptor via miR-455-5p in hepatic cells

Objective: To explore the mechanism by which ghrelin regulates insulin sensitivity through modulation of miR-455-5p in hepatic cells. Methods: HepG2 cells were treated with or without DAG (1 μM). Glucose consumption, intracellular glycogen content, phosphorylation of PI3K and Akt stimulated by insulin, expression of miR-455-5p, as well as IGF-1R protein level were analyzed. In addition, bioinformatic analysis, dual luciferase reporter assay, miR- 455-5p mimic or inhibitor treatment was conducted to investigate the molecular mechanisms. Results: High glucose treatment upregulated miR-455-5p expression but reduced glucose consumption and glycogen content. DAG reversed the effect of high glucose on glucose metabolism, increased protein level of IGF-1R and phosphorylation of PI3K/Akt stimulated by insulin, as well as downregulated miR-455-5p expression. Bioinformatic analysis indicated IGF-1R was the target of miR-455-5p. Dual luciferase reporter assay, as well as transfection with miR-455-5p mimic/inhibitor confirmed that DAG activated IGF-1R/PI3K/Akt signaling via inhibiting miR-455-5p. Conclusion: DAG improves insulin resistance via miR-455-5p- mediated activation of IGF-1R/PI3K/Akt system, suggesting that suppression of miR-455-5p or activation of DAG may be potential targets for T2DM therapy.

Genetic polymorphisms in cytochrome P4502E1, alcohol and aldehyde dehydrogenases and the risk of esophageal squamous cell carcinoma in Gansu Chinese males

AIM:To evaluate the association between genetic polymorphisms in CYP2E1, ALDH2 and ADH1B and the risk of esophageal squamous cell carcinoma (ESCC) in a high risk area of Gansu Province, in Chinese males. METHODS: A case-control study was conducted to investigate the genetic polymorphisms of these enzymes (CYP2E1 *c1/*c2, ALDH2 *1/*2 and ADH1B *1/*1 genotypes). A total of 80 esophageal cancer cases and 480 controls were recruited. RESULTS: Compared with controls, cases had a greater prevalence of heavier alcohol consumption (53.8% vs 16.2%) and a higher proportion of alcohol drinkers with > 30 drink-years (28.8% vs 13.5%). Heavier alcohol consumption and alcohol drinking with > 30 drink- years increased the risk of ESCC, with ORs (95% CI) of 3.20 (1.32-9.65) and 1.68 (0.96-3.21). CYP2E1 (*c1/*c1), ALDH2 (*1/*2) and ADH1B (*1/*1) genotype frequencies were higher among patients with squamous cell carcinomas, at a level close to statistical significance (P = 0.014; P = 0.094; P = 0.0001 respectively). There were synergistic interactions among alcohol drinking and ALDH2, ADH1B and CYP2E1 genotypes. The risk of the ESCC in moderate-to-heavy drinkers with an inactive ALDH2 encoded by ALDH2 *1/*2 as well as ADH1B encoded by ADH1B *1/*1 and CYP2E1 encoded by CYP2E1 *c1/*c1 was higher than that in the never/rare-to-light drinkers with an active ALDH2 (*1/*1 genotype) as well as ADH1B (*1/*2 + *2/*2) and CYP2E1 (*c1/*c2 + *c2/*c2) genotypes, with a statistically significant difference; ORs (95% CI) of 8.58 (3.28-22.68), 27.12 (8.52-70.19) and 7.64 (2.82-11.31) respectively. The risk of the ESCC in moderate-to-heavy drinkers with ALDH2 (*1/*2) combined the ADH1B (*1/*1) genotype or ALDH2 (*1/*2) combined the CYP2E1 (*c1/*c1) genotype leads to synergistic interactions, higher than drinkers with ALDH2 (*1/*1) + ADH1B (*1/*2 + *2/*2), ALDH2 (*1/*1) + CYP2E1 (*c1/*c2 + *c2/*c2) respectively , ORs (95% CI) of 7.46 (3.28-18.32) and 6.82 (1.44-9.76) respectively. Individuals with the ADH1B combined the CYP2E1 genotype showed no synergistic interaction. CONCLUSION: In our study, we found that alcohol consumption and polymorphisms in the CYP2E1, ADH1B and ALDH2 genes are important risk factors for ESCC, and that there was a synergistic interaction among polymorphisms in the CYP2E1, ALDH2 and ADH1B genes and heavy alcohol drinking, in Chinese males living in Gansu Province, China.

Cytotoxicity of Benzo(a)pyrene 7,8-dihydrodiol on Genetically Engineered V79 Chinese Hamster Cells Expressing Rat Liver Cytochrome P450 1A1

To develop well defined in vitro cell system to test cytotoxicity of a number of model toxins, genetically engineered V79 Chinese hamster fibroblasts expressing isoenzymes of cytochrome P4501A1 XEM2 cells and V79 cells (parental), which lack cytochrome P450 enzyme activities, were used as controls. The cytotoxic effect of trans 7,8 dihydrbenzo(a)pyrene (BP 7,8 diol) on the parental cells V79 and V79 derived XEM2 cells were evaluated by two methods for cell viability. The data obtained expressed that BP 7,8 diol ranging from 1.0 μmol·L 1 to 5.0 μmol·L 1 in concentrations incubated for 24 h showed a strong cytotoxic effect in XEM2 cells (expressing rat cytochrome P4501A1) in a concentration dependent manner. Time dependent decrease for survival of XEM2 cells was also observed at 2.5 μmol·L 1 concentration. Likewise, BP 7,8 diol did not alter the survival of the parental cells V79 under the same condition. This study also showed that α naphthoflavone (αNF), a well known inhibitor of cytochrome P4501A1 might alter BP 7,8 diol induced cytotoxicity in the XEM2 cells. Our results suggested that cytochrome P4501A1 is responsible for BP 7, 8 diol induced cytotoxicity.

Combined Antitumor Effect of Ursolic Acid And 5-Fluorouracil on Human Esophageal Carcinoma Cell Eca-109 In Vitro

Objective： To study the combined antitumor effect and possible mechanisms of ursolic acid with 5-fluorouracil （5-FU） on human esophageal carcinoma cell Eca-109 in vitro. Methods： Eca-109 cells were treated with ursolic acid （10-50 μmol/L） and/or 5-fluorouracil （48.0-768.8 μmol/L） for 48 h in vitro. And then cell proliferation was determined by MTT assay. Cell cycle and apoptosis rate were analyzed by flow cytometry （FCM）. The morphological changes of apoptosis were observed by fluorescent microscopy. At last the expression of P27kipl, bcl-2 and bax were detected by western blot. Results： Results： In comparison with single agent treatment, the combination of ursolic acid and 5-fluorouracil produced greater efficacy in growth inhibition, cell cycle arrest at G0/G1 phase, and apoptosis induction （P〈0.05）. Western blot analysis showed that the combination use of ursolic acid and 5-fluorouracil suppressed the expression of bcl-2 and increased the expressions of bax and P27kip1. Conclusion： Ursolic acid combined with 5-fluorouracil showed adjuvant antiproliferative effects on human esophageal carcinoma cell Eca-109 in vitro, which mainly due to the induction of cell cycle arrest as well as apoptosis.

细胞色素P4502E1稳定转染Flp-In^(TM)CHO细胞模型的建立

目的构建稳定表达细胞色素P450家族2亚家族E成员1(CYP2E1)的Flp-In^(TM)CHO细胞系,并进行药物相互作用筛选。方法利用Lipofectamine■2000转染试剂将pcDNA5/FRT-CYP2E1重组质粒和pcDNA5/FRT-空质粒-并转染Flp-In^(TM)CHO细胞,采用潮霉素B(500 mg/L)进行稳定性筛选、聚合酶链式反应(PCR)检测细胞中是否成功插入能够表达CYP2E1酶的目的基因、实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹法(Western blot)检测细胞中CYP2E1 mRNA和蛋白表达,采用对乙酰氨基酚(APAP)检测CYP2E1酶对APAP的毒性敏感性。结果与Flp-In^(TM)CHO空质粒组比较,PCR结果显示Flp-In^(TM)CHO-CYP2E1细胞分别在2000 bp、200 bp左右有CYP2E1酶目的基因的明显条带;qRT-PCR和Western blot结果显示,Flp-In^(TM)CHO-CYP2E1细胞的CYP2E1 mRNA和蛋白表达水平均明显增加(P<0.001);APAP检测结果显示CYP2E1酶对APAP的毒性敏感性也显著增强。结论成功构建了稳定表达CYP2E1的Flp-In^(TM)CHO细胞模型,且表达的CYP2E1酶对APAP的毒性敏感性增强。

牙鲆dmrt1基因的克隆及其与P450arom基因的组织表达分析

通过基因组步移和3’RACE获得了牙鲆(Paralichthys olivaceus)dmrt1的cDNA全序列,该基因开放阅读框全长909bp,其编码的蛋白具有高度保守的DM结构域,5’UTR区含有性别相关转录因子Sox9和Sox5的结合位点。牙鲆dmrt1基因只在牙鲆的性腺中表达,且在精巢中的表达明显高于卵巢,表明牙鲆的dmrt1可能是一种性别相关基因。同时,对牙鲆的另一性别相关基因P450arom在成体各组织的表达分析结果表明,该基因除在牙鲆的性腺中有表达外,在肾脏、脾脏、鳃和脑等其他组织中也有不同程度的表达。牙鲆P450arom基因在性腺中的表达也存在两性差异,其表达模式与dmrt1的正好相反,在卵巢中的表达量明显高于精巢。

中国人细胞色素P450IA1基因变异与吸烟相关性肺癌的风险

目的：探讨多环芳烃类致癌物代谢酶细胞色素Ｐ４５０ＩＡ１ 基因（ＣＹＰ１Ａ１） 多型性与中国人肺癌易感性的关系。方法：应用ＰＣＲＲＦＬＰ 方法，分析１５０ 例原发性肺癌和３９１ 例正常对照者ＣＹＰ１Ａ１ 基因的ｍ １、ｍ２ 和ｍ ４ 位点突变。以比值比（ＯＲ） 及其９５ ％ 可信区限（ＣＩ） 比较不同基因型与肺癌风险的关系，以及与吸烟的交互作用。结果：位于３＇端ＭｓｐＩ识别的位点（ ｍ１） 和位于第７ 外显子ＢｓｒＤＩ 识别的位点（ｍ２） 具多态性，而位于第７ 外显子由ＢｓａＩ识别的位点（ｍ ４） 未见有变异。ｍ１ 变异型等位频率在对照组中为０－３６，而在肺癌病例组中为０－４６ 。携带至少一个变异基因拷贝者发生肺癌的风险比携带野生基因型者高２ 倍（ＯＲ ２－３ ；９５％ ＣＩ１－９～２－９） 。分层分析发现，肺鳞癌患者中ｍ １ 变异基因型频率更高（０－４８） ，携带此种基因型者发生肺鳞癌的相对风险（ＯＲ） 为３－０（９５％ ＣＩ２－２ ～４－０） 。ｍ２ 变异与ｍ１变异密切关联，符合率达７６ ％ 。携带至少一个ｍ ２ 变异基因拷贝者的ＯＲ 为１－９ （９５％ ＣＩ１－６ ～２－４） 。此外，ＣＹＰ１Ａ１ｍ１ 变异与吸烟有明显的交互作用。

黄酮类化合物对细胞色素P450 CYP1A2的抑制作用及其构效关系研究

利用本实验室已建立的体外肝微粒体模型,测定36个黄酮类单体化合物对人细胞色素P450 CYP1A2的抑制活性,并使用三维定量构效关系方法研究化合物的分子结构参数与其抑制活性之间的关系。CoMSIA模型证实黄酮类化合物的结构参数与其CYP1A2抑制活性存在明显的相关性(模型的相关系数R2为0.948),且有良好的预测能力(交叉验证相关系数q2为0.630),同时使用"留五法"证实模型的稳定性和可靠性。结果表明,相对于黄酮,α-萘黄酮的π-π共轭体系更有利于提高化合物的抑制活性。根据获得的模型的三维等势图,在α-萘黄酮基础上,6、3′、4′位引入带正电基团或是疏水基团,同时5位引入带负电基团,能有效改善化合物的CYP1A2抑制活性。

大鼠酒精性肝病细胞凋亡与细胞色素P450 2E1和氧化应激的关系

目的 观察酒精性肝病（ALD）大鼠肝组织病理学改变,探讨细胞凋亡与细胞色素P4502E1（CYP2E1）的表达以及与氧化应激的关系。方法 用乙醇灌胃法制备ALD大鼠模型,模型组（37只）给予体积分数为40%的乙醇8g·kg^-1·d^-1分两次灌胃,连续8周;对照组（33只）给予等量生理盐水灌胃。实验第8周末,各组选30只大鼠观察肝组织的病理学改变;用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）检测肝细胞凋亡,用全自动生化仪检测丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）值,用聚合酶链反应（PCR）法测定肝CYP2E1的表达;分别用硫代巴比妥酸法和黄嘌呤氧化酶法测定血清丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性。结果 模型组凋亡的肝细胞明显增多,主要分布在中央静脉周围、点状和灶状坏死区。对照组CYP2E1的c1基因频率为91.65%、c2基因频率为8.35%;模型组c1基因频率为53.35%、c2基因频率为46.65%,差异均有显著性（P均〈0.05）。长期摄入乙醇的大鼠血清MDA含量增加,SOD活性下降,与ALD肝细胞凋亡程度有相关性（rMDA=0.644,rSOD=-0.511,P均〈0.05）,且MDA与SOD两指标间呈负相关（r=-0.582,P〈0.05）。结论 长期摄入乙醇可引起大鼠ALD及肝功能损伤,肝细胞凋亡明显增加。CYP2E1基因Pst及Rsa限制性片段长度多态性与ALD有关,其中c2基因可能与大鼠ALD的发生有关。MDA含量和SOD活性在ALD的肝细胞凋亡过程及脂质过氧化反应中发挥重要作用。

细胞色素P450 2E1基因多态与肝癌遗传易感性研究

目的：探讨细胞色素 P450 2E1基因多态与肝癌的关系。方法：应用 PCR RFLP方法对 84例肝癌患者和 144例健康对照的细胞色素 P450 2E1基因 Rsa I多态进行检测。结果：病例组基因型 A频率为 71.43％，等位基因 c1频率为 84.52％，对照组则分别为 55.56％和 75.35％，两组差别均有统计学意义 (P0.05)。结论：细胞色素 P450 2E1基因 Rsa I多态位点的基因型 A或等位基因 c1增加了个体患肝癌的危险性。

细胞色素 P450 2E1 的研究进展

ＣＹＰ２Ｅ１在药物和人们经常接触的溶剂与环境污染物的代谢中具有重要作用。本文综述ＣＹＰ２Ｅ１的结构特点、基因、底物和探药以及影响其活性的主要因素。

酒精性肝损伤大鼠细胞色素P450 CYP2E1和细胞色素P450 CYP3A的代谢活性

目的观察酒精性肝损伤对大鼠细胞色素P450CYP3A(CYP3A)和细胞色素P450CYP2E1(CYP2E1)代谢活性的影响。方法采用ig给予白酒制备大鼠酒精性肝损伤模型,检测血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性,采用HE染色法光镜下观测酒精对肝脏损伤程度。大鼠ip给予CYP3A探针药物咪达唑仑10mg·kg-1或ig给予CYP2E1探针药物氯唑沙宗50mg·kg-1后,采用高效液相色谱法测定不同时间点大鼠血浆中咪达唑仑和氯唑沙宗的血药浓度,并应用3P87软件计算其药代动力学参数,以考察CYP2E1和CYP3A的代谢活性的变化。大鼠ig给予氯唑沙宗80mg·kg-1后,热板方法测定大鼠添足次数和添足反射潜伏期。结果酒精性肝损伤可致大鼠肝小叶结构不清,肝索排列紊乱,肝细胞体积增大,呈弥漫性中度水变性,肝窦受压,大部分肝细胞胞浆内见大小不等的脂肪空泡;与正常对照组相比,酒精性肝损伤组大鼠GPT和GOT活性分别增加了16.0%和20.0%(P<0.05,P<0.01)。酒精性肝损伤致大鼠CYP2E1对探针药物氯唑沙宗的代谢活性增强,AUC,t1/2和cmax分别降低了38.0%,30.5%和35.0%(P<0.05);酒精肝损伤组大鼠氯唑沙宗镇痛效果明显降低;酒精性肝损伤致大鼠CYP3A对探针药物咪达唑仑的代谢活性增强,AUC,t1/2和cmax分别降低了122.6%,54.9%和56.9%(P<0.01,P<0.05)。结论酒精性肝损伤可使大鼠CYP2E1和CYP3A代谢活性增强。

人肝细胞色素P450含量及其同工酶1A1、2A6活性的测定

从成人肝细胞中提取微粒体，测定其蛋白浓度、细胞色素Ｐ４５０（ＣＹＰ４５０）总量，并建立了通过测定代谢产物异噁唑和香豆素生成量确定ＣＹＰ４５０ 同工酶ＣＹＰ１Ａ１、ＣＹＰ２Ａ６ 活性的测定方法。结果表明：该测定方法简单、稳定、重复性好。人肝微粒体于－ ８０°Ｃ保存６ 个月对其活性无明显影响。

酮康唑对健康成人肝细胞微粒体细胞色素P450同工酶3A4、1A2活性的作用

目的观察酮康唑对健康成人肝细胞微粒体细胞色素P450同工酶3A4、1A2活性的作用,为临床上安全有效地联合用药提供实验依据。方法采用健康成人肝细胞微粒体,分为对照组和处理组。酮康唑处理组分别加入不同浓度的酮康唑1 ml,对照组仅加入培养液,孵育15 min后再加入CYP450同工酶3A4和1A2的相应底物(分别为睾酮和非那西丁)再孵育20 min。反应终止后用高效液相色谱仪测量代谢产物(分别为6β-羟基睾酮与对乙酰氨基酚)的生成量,分别代表3A4和1A2的活性。结果细胞色素P450同工酶3A4的相对活性百分比减小到对照组50%时(IC50)酮康唑的质量浓度为0.16 mg/L。而同工酶3A4的相对活性百分比随质量浓度酮康唑增加逐渐减小,各处理组与对照组比较差异均有显著性意义(P<0.05)。低剂量细胞色素P450同工酶1A2的相对活性百分比处理组与对照组比较明显降低(P<0.05),高剂量细胞色素P450同工酶1A2的相对活性百分比处理组与对照组比较则明显升高(P<0.05)。结论酮康唑对健康成人肝细胞微粒体细胞色素P450同工酶3A4的活性有抑制作用,然而对细胞色素P450同工酶1A2的活性则是低剂量有抑制作用,而高剂量有诱导作用。

细胞色素P450新家族的第一个基因CYP337A1的分子克隆与序列分析

依据家蚕基因组数据,通过BLASTP比对,选择了主要与抗性相关的CYP3集团(clan)中具有完全编码框并含有P450基因特征结构域的1条序列为研究对象,用RT-PCR方法对其进行了克隆.结果表明,该基因ORF为1 467 bp,编码489个氨基酸,推定的蛋白质分子质量为56.60 KDa,等电点为8.64.只有1个内含子,外显子/内含子边界处符合GT-AG规则.与美国棉铃虫的CYP321A、邪恶按蚊的CYP6P9的相似性(identity)相对较高,分别为34%、32%,低于同一家族成员氨基酸相似性应高于40%的规定,从而被细胞色素P450委员会命名为新家族的第一个基因CYP337A1(GenBank登陆号:EF415297).

细胞色素P450 3A5和多药耐药基因1基因多态性在肾移植患者他克莫司血药浓度监测中的应用

目的 :研究细胞色素P4 5 0 3A5 (CYP3A5 )和多药耐药基因 1 (MDR1 )基因多态性对他克莫司 (FK5 0 6 )血药浓度的影响 ,探讨FK5 0 6在不同个体间吸收、代谢差异的基因背景 ,建立个体化用药的药物代谢遗传学监控体系。 方法 :观察 1 1 8例肾移植术后常规使用FK5 0 6 +霉酚酸酯 +强的松三联免疫抑制治疗的成年患者 ,记录体重 ,FK5 0 6剂量 ,术后第 7天、术后 1个月和术后 3个月的FK5 0 6全血谷浓度等指标。采用PCR 限制性长度多态性的方法检测CYP3A5 1 / 3和MDR1 3435位点C/T多态性 ,比较不同基因型之间FK5 0 6的浓度 /剂量比的差异。 结果 :CYP3A5基因为 1 / 1、 1 / 3和 3/ 3型的患者数分别有 1 2、36和 70例。 1 / 1型和 1 / 3型患者的FK5 0 6浓度 /剂量比明显低于 3/ 3型的患者 (术后第 7天分别为 32 8± 1 7 7,4 1 6± 1 5 8,1 0 2 3± 5 1 2 ;术后 1个月分别为 33 1± 7 5 ,4 6 4± 1 2 9,1 0 3± 4 7 5 ;术后 3个月分别为 35 3± 2 0 9,5 9 0± 2 0 6 ,1 5 0± 85 3;P均 <0 0 0 1 ) , 1 / 1纯合子患者的血药浓度略低于 1 / 3杂合子 ,具有统计学意义 (术后第 7天P >0 0 5 ,术后 1个月和 3个月P <0 0 1 )。动态观察MDR1基因型与FK5 0 6血药浓度之间无明显相关性。 结论 :?