以形态与组织学为基础筛选诱导胎鼠腭裂的四氯二苯二噁英最适剂量

目的观察在不同四氯二苯二噁英(TCDD)剂量的作用下,胎鼠腭裂形成过程中的形态学、组织学变化,以筛选TCDD诱导腭裂模型的最适剂量。方法C57BL/6J孕鼠于妊娠第10天随机分为对照组和实验Ⅰ～Ⅳ组,每组6只;对照组以玉米油0.1ml灌胃,实验Ⅰ、Ⅱ、Ⅲ、Ⅳ组分别以TCDD32、28、24、20μg/kg灌胃。妊娠第17.5天处死孕鼠,称量孕鼠及胚胎体重,记录活胎鼠数、腭裂数、吸收胎及死胎鼠数。另取15只C57BL/6J孕鼠随机分组同前,处理方法同前;各组分别于妊娠第13.5、14.5和15.5天剪取胎鼠头部,解剖显微镜下观察;并行苏木精-伊红染色(HE染色),观察。结果各组孕鼠增加的体重及活胎鼠的体重差异均无显著性。Ⅰ、Ⅱ、Ⅲ组较对照组双侧腭突发育瘦小,上抬延迟;Ⅳ组双侧腭突发育及上抬与对照组相比无明显差异。Ⅰ～Ⅳ组胎鼠腭裂发生率分别为97.37%、93.02%、65.12%、56.82%,对照组未见胎鼠腭裂形成。结论 TCDD28μg/kg是建立C57BL/6J胎鼠腭裂模型的最适剂量。     

药物预防腭裂发生的动物实验研究进展

腭裂是一种常见的先天性发育畸形,是遗传和环境因素相互作用所致的多因素、多基因遗传病,其病因仍未完全明确.尽管手术方法不断改进,技术手段不断更新,但治疗效果仍难以尽善尽美,因此,最好的方法是预防和阻止腭裂的发生.目前国内外学者进行了大量的动物实验研究,期望通过在动物妊娠期内,补充适宜的药物或化合物,以预防或减少其子代腭裂的发生.笔者就近年来选用药物阻断动物腭裂发生的主要实验研究进展进行综述,为在实验和临床上开展预防腭裂发生的研究工作提供参考.     

四氯二苯二噁英致胎鼠腭裂作用机制的初步探讨

目的 探讨四氯二苯二噁英(2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD)致胎鼠先天性腭裂的可能作用机制.方法 12只C57BL/6J孕鼠于妊娠第10天随机分为实验组和对照组,每组6只,实验组以TCDD 64 μg/kg灌胃,对照组以等量玉米油灌胃,于妊娠第18.5天在解剖显微镜下观察胎鼠腭裂的发生率.另取18只C57BL/6J孕鼠,于妊娠第10天随机分为实验组和对照组,每组9只,处理方法同前,根据标本采集时间不同,每组又分为妊娠第13.5、14.5和15.5天3个亚组,每个亚组3只,分别于妊娠第13.5、14.5和15.5天剪取胎鼠腭突组织提取RNA和DNA,采用RTPCR检测Smad 2～4及Smad 7 mRNA的表达情况、甲基化特异性PCR(methylmion specific PCR,MSP)检测转化生长因子-β3(transforming growth factor-β3,TGF-β3)基因启动子甲基化水平.两样本间均数比较采用t检验.结果 TCDD成功诱导建立C57BL/6J胎鼠先天性腭裂模型,实验组腭裂发生率为100％,对照组无腭裂发生.在妊娠第13.5、14.5和15.5天,RT-PCR显示实验组Smad 2mRNA的相对表达值分别为0.263±0.088、0.296±0.016和0.159±0.027,对照组为0.180±0.042、0.282±0.029和0.165±0.018,差异无统计学意义(t=- 1.474、- 0.762、0.321,P＞0.05);实验组Smad 3 mRNA的相对表达值分别为0.453±0.153、0.551±0.160和0.328±0.049,对照组为0.375±0.126、0.510±0.145和0.259±0.035,差异无统计学意义(t=-0.678、- 0.336、-2.005,P＞0.05);实验组Smad 4 mRNA的相对表达值分别为0.675±0.174、0.577±0.070和0.396±0.066,对照组为0.557±0.138、0.587±0.080和0.441±0.054,差异无统计学意义(t=- 0.926、0.161、0.927,P＞0.05);实验组Smad 7 mRNA的相对表达值分别为0.283±0.050、0.320±0.068和0.169±0.045,对照组为0.207±0.043、0.288±0.051和0.155±0.040,差异无统计学意义(t=-2.003、-0.651、-0.391,P＞0.05).MSP显示实验组和对照组TGF-β3基因启动子均处于非甲基化状态.结论 TCDD可致C57BL/6J胎鼠发生先天性腭裂,其机制不是通过经典的TGF-β/Smad信号通路和TGF-β3基因甲基化修饰而发生作用.     

Pharmacokinetics and Tissue Distribution of Clevidipine and Its Metabolite in Dogs and Rats

The purpose of the current study was to examine the pharmacokinetic profiles and tissue distribution of clevidipine, an ultra-short-acting calcium antagonist in Beagle dogs and Sprague-Dawley rats, respectively. The pharmacokinetics and biodistribution of its primary metabolite H 152/81 were also evaluated. Dogs received intravenous infusion of clevidipine at a dose rate of 17 μg/（kg·min）, and rats were given intravenous administration of clevidipine at a dose of 5 mg/kg. Dog plasma and rat tissues were collected and assayed by HPLC-MS/MS. It was found that plasma clevidipine quickly reached the steady state concentration. The terminal half-life was short （16.8 min）, pointing out a rapid elimination after the end of the infusion. The total clearance was 5 mL/（min·kg）. In comparison, plasma concentra- tion of H152/81 was increased more slowly and was significantly higher than that of clevidipine. After intravenous administration, clevidipine was distributed rapidly into all tissues examined, with the high- est concentrations found in the brain, heart and liver. Maximal concentrations of clevidipine were found in most tissues at 10 min post-dosing. However, the proportion of clevidipine distributed in all tissues was quite small （0.042‰） compared to the total administration dose. It was suggested that clevidipine was mainly distributed in blood and it transformed to inactive metabolite raoidlv.     

Development of a liquid chromatography-tandem mass spectrometry method for determination of butoconazole nitrate in human plasma and its application to a pharmacokinetic study

Summary： A liquid chromatography-tandem mass spectrometry （LC-MS/MS） method was developed and validated for the determination of butoconazole in human plasma. Human plasma samples of 0.2 μL were pretreated by a single step protein precipitation procedure and analyzed using a high performance liquid chromatography （HPLC） electrospray tandem mass spectrometer system. The compounds were eluted isocratically on an Inertsil ODS-SP column （100 min×2.1 mm, 3 μm）, ionized using a positive ion atmospheric pressure electrospray ionization source and analyzed using multiple reaction monitoring （MRM） mode. The ion transitions monitored were m/z 412.8→q65.1 for butoconazole and m/z 453.4→230.3 for the internal standard. The chromatographic run time was 3.5 min per injection, with retention time of 2.47 rain and 2.15 min for butoconazole and repaglinide, respectively. The method was validated to be linear over the range of 20 to 8000 pg/mL （r〉0.999） by using a weighted （1/x2） quadratic regression. The mean recovery rate was more than 86.7%, and the intra- and inter-day precision of the quality control samples （QCs） was less than 8.3% and the accuracy ranged from 96.0% to 110.2%, which indicated that the quantitative method was reliable and accurate. The method is simple, rapid, and has been applied successfully to a pharmacokinetics study of butoconazole nitrate suppositories in healthy Chinese females.     

左舒必利注射液在中国健康受试者的药代动力学

目的 研究左舒必利注射液在中国健康受试者单次及多次给药的药代动力学。方法 用开放、随机、平行的试验设计。30名受试者,男女各半,分为3个剂量组,分别接受单次及多次肌内注射不同剂量左舒必利,采集不同时间的血样,用高效液相色谱-串联质谱法(HPLC/MS/MS)测定血浆中左舒必利的浓度。用DAS 3.0软件计算药代动力学参数。结果 单次肌内注射25,50,75 mg左舒必利的主要药代动力学参数:cmax分别为(724.70±248.91),(949.60±234.80),(1619.00±366.80)μg·L-1;t1/2分别为(7.65±1.32),(7.58±0.89),(8.01±0.88)h,AUC0-t分别为(2874.17±1093.71),(4481.75±913.09),(7559.33±1428.87)μg·L-1·h。连续给药组t1/2为(7.41±0.79)h;AUC0-t为(4658.33±909.51)μg·L-1·h。结论 中国健康受试者单次肌内注射给药左舒必利在25~75 mg内呈线性药代动力学特征,多次给药没有蓄积倾向,男、女间比较,差异无统计学意义。     

Pharmacokinetic and pharmacodynamic properties of batifiban coadministered with antithrombin agents in Chinese healthy volunteers

The combined use of batifiban, a synthetic platelet GP II b/IIIa receptor antagonist, and an- tithrombin agents is an attractive option for the treatment of patients with non-ST-segment elevation （NSTE） acute coronary syndrome （ACS） and those scheduled for percutaneous coronary intervention. To observe whether antithrombin agents affect the pharmacokinetic and pharmacodynamic properties of batifiban in combination therapy and optimize clinical administration dosage of batifiban, an open-label and parallel study was conducted. Thirty healthy subjects were randomly divided into three groups, which were sequentially treated with batifiban alone, or oral coadministration of clopidogrel, aspirin and UFH, or batifiban coadministered with these antithrombin agents. Blood samples were collected at pre-specified time points. The evaluation index included the inhibition of platelet aggregation and pharmacokinetic parameters. The pharmacokinetic parameters of batifiban and batifiban coadministered with antithrombin agents showed no significant differences. The mean inhibition rate of platelet aggre- gation （%） suggested that neither batifiban alone nor antithrombin agents alone could provide such po- tent inhibition rate （〉80%） to obtain the best clinical efficacy, but they had a synergistic effect on plate- let inhibition. No serious adverse effects were observed. The results in these healthy subjects suggest that batifiban coadministrated with antithrombin agents could achieve optimum clinical treatment effect for patients with NSTE ACS, and also those scheduled for percutaneous coronary intervention.     

叶酸和白藜芦醇对TCDD致胎鼠腭裂的拮抗作用

目的 探讨叶酸和白藜芦醇是否具有预防动物模型腭裂发生的作用,并对比两者的效果.方法 将72只孕鼠随机分为9组,每组8只,2,3,7,8-四氯代二苯并二噁英(2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD)组于受孕后第10天(GD10)以TCDD 28 μg/kg灌胃；叶酸组分别于GD10以15、10、5 mg/kg叶酸+TCDD 28 μg/kg灌胃,对照组以玉米油0.1 ml灌胃；白藜芦醇处理孕鼠分3组,GD8～ 13组以50 rng/kg白藜芦醇于GD8 ～ 13灌胃6次,白藜芦醇GD8～ 13+ TCDD组以白藜芦醇50 mg/kg于GD8 ～ 13灌胃6次及TCDD 28 μg/kg于GD10灌胃1次,白藜芦醇GD10+TCDD组以自藜芦醇50 mg,/kg及TCDD 28 μg/kg于GD10灌胃,对照组以等量羧甲基纤维素钠溶液于GD8～13灌胃及玉米油0.1 ml于GD10灌胃.GD17.5处死孕鼠,称量孕鼠及胚胎体重,记录活胎鼠数、腭裂数、吸收胎及死胎鼠数；并剪取胎鼠头部,解剖显微镜下观察.结果 TCDD28 μg/kg所致胎鼠腭裂发生率为92.86％,对照组未见胎鼠腭裂形成.TCCD+叶酸15、10、5 mg/kg 3组致胎鼠腭裂发生率分别为84.00％、73.08％、86.00％.白藜芦醇GD8～ 13、GD8～ 13+ TCDD组、GD10+TCDD 3组致胎鼠腭裂发生率分别为0％、57.78％、74.51％.白藜芦醇GD8 ～ 13+ TCDD组孕鼠每窝活胎数、死胎和吸收胎数,与对照组及TCDD组相比,差异均有统计学意义(P＜0.05)；其余各实验组孕鼠体重增加量、活胎鼠体重及每窝平均胎数、每窝活胎数与对照组相比,差异均无统计学意义(P＞0.05).结论 试验剂量的叶酸与白藜芦醇均有一定的拮抗TCDD诱导胎鼠腭裂发生的作用,以叶酸10 mg/kg、白藜芦醇50 mg/kg GD8 ～ 13的剂量的拮抗作用较强.两者作用效果无明显差异,但白藜芦醇50 mg/kg(GD8-13)明显影响TCDD作用下胎鼠的生长发育.     

3种药物对饲服TCDD的胎鼠腭突超微结构的影响

目的：观察叶酸、白藜芦醇和α-萘黄酮拮抗四氯二苯二噁英（2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD）诱导胎鼠腭裂发生的效应,及对饲服TCDD的胎鼠腭突表面超微结构的影响。方法：妊娠第10天（gestation day 10,GD10）孕鼠随机分组,每组8只。TCDD组以TCDD 28 μg/kg灌胃,对照组以玉米油0.1 ml灌胃,叶酸组以叶酸10 mg/kg + TCDD 28 μg/kg灌胃、白藜芦醇组以白藜芦醇50 mg/kg+TCDD 28 μg/kg灌胃、α-萘黄酮组以α-萘黄酮5 mg/kg+TCDD 28 μg/kg灌胃,于GD17.5在解剖显微镜下观察胎鼠腭裂的发生率。另取15只孕鼠随机分组,每组3只,处理方法同前,分别于GD13.5、14.5、15.5剪取胎鼠头部,扫描电镜观察腭突表面的超微结构。结果：TCDD组胎鼠腭裂发生率为92.86%,对照组未见胎鼠腭裂形成。叶酸组、白藜芦醇组和α-萘黄酮组胎鼠腭裂发生率分别为73.08%、74.51%、65.52%,与TCDD组相比,差异有统计学意义（P<0.05）。TCDD组腭中嵴上皮细胞表面逐渐破裂、皱缩,伪足结构逐渐减少、消失,最终形成腭裂;对照组、叶酸组、白藜芦醇组和α-萘黄酮组腭中嵴上皮细胞表面光滑整齐、饱满膨胀,表面丝状伪足逐渐增多、延长,一直持续到腭突融合结束。结论：叶酸、白藜芦醇和α-萘黄酮均能恢复小鼠腭中嵴上皮细胞表面的超微结构,从而拮抗TCDD的致腭裂效应。