髓样相关蛋白14真核表达载体的构建及其细胞内定位研究

目的:构建小鼠髓样相关蛋白14(MRP14)的真核表达载体,观察其在NIH3T3细胞中的表达定位情况。方法:提取BALB/c小鼠肝脏组织的总RNA,通过逆转录-聚合酶链反应(RT-PCR)扩增得到MRP14编码序列。然后将该编码序列克隆到带有血凝素(HA)标记的载体pcDNA3-HA上,随后转染NIH3T3细胞,在荧光显微镜下观察结果。结果:重组质粒经聚合酶链反应(PCR)、酶切和测序鉴定证明构建正确。转染实验发现,该质粒能够在NIH3T3细胞中表达,表达产物主要定位在细胞质中。结论:成功构建带HA标签的MRP14真核表达载体。该载体能在哺乳动物细胞中有效表达并正确定位,为下一步深入研究MRP14作用细胞的信号通路提供了一个重要工具。     

葛根素的多晶型研究

目的：解析不同晶态葛根素的理化性质变化。方法：采用4种不同溶剂进行结晶，应用显微放大、溶解度、红外、X射线衍射、热分析。结果：不同晶态的晶体在水中的溶解度随着晶型的增大而减小；热分析显示4种晶体的熔点存在差异；X射线衍射测得4种晶体的厚度不一样，特别是用乙醇水结晶的样品结晶度最低；红外谱图显示4种晶体有差异，并且超临界流体结晶所得样品和标准红外谱图一致。结论：不同晶态的葛根素其理化性质有明显差异。     

A Case of Secondary Glaucoma due to Bilateral Anterior Chamber Shallowing Caused by Marfan′s Syndrome

Purpose:To report the clinical manifestations and treatment of a case of secondary glaucoma evolving from bilateral anterior chamber flattening caused by Marfan′s syndrome. Methods:The ophthalmic and systemic features,B-scan and UBM characteristics were recorded.Therapy and efficacy were analyzed. Results:Marfans syndrome,in this case,caused bilateral iris-lens diaphragm anterior dislocation,anterior chamber flattening, pupillary block, angle closure,and finally resulted in persistent increased intraocular pressure (IOP). After undergoing pars plana vitrectomy and lensectomy combined with anterior chamber reformation, the visual acuity of the patient’s right eye increased from 6/150 to 6/7.5.(best-corrected) and that of the left eye was improved from 6/100 to 6/10 (best-corrected). The IOP of the right eye fell to 18 mm Hg, and the left eye to 12 mm Hg. Conclusion:Marfan′s syndrome can cause bilateral anterior chambers flattening,and induce secondary angle closure glaucoma.Combined pars plana vitrectomy, lensectomy and anterior chamberplasty can re-form the anterior chamber, control IOP and maintain visual function.     

二氢吡啶类钙拮抗剂结构与药物动力学的定量关系分析

目的：研究二氢吡啶类钙拮抗剂的化学结构与药物动力学的关系，为新药设计中早期筛选提供理论方法。方法：采用Inisht Ⅱ软件的分子力学和分子动力学程序首先将钙拮抗剂的结构式转化为三维结构，并进行能量优化，使得结构趋于合理。HyperChem软件计算优化好的结构的理化性质参数(Log P，Surface Area，Volume，Hydration Energy，Refractivity，Polarizability)。对钙拮抗剂药物的理化参数与其药物动力学参数之间关系进行统计分析研究。结果：钙拮抗剂的疏水性常数(Log P)值与血管外给药后的达峰时间(Tmax)存在二次方程关系，二者的相关性达到0．9以上。结论：二氢吡啶类钙拮抗剂药物的疏水性常数(Log P)值与给药后的达峰时间(Tmax)存在的关系对该类药物的进一步结构改造，设计新药具有重要的指导意义。     

葛根芩连煎剂与颗粒剂的抗腹泻、抑菌药效研究

目的:比较葛根芩连汤与颗粒剂的抗腹泻、抑菌药效。方法:抗腹泻实验中将小鼠分为9组,即空白(等容生理盐水)、模型(等容生理盐水)、葛根芩连微丸(1g.kg-1)、葛根芩连煎剂高、中、低剂量(2、1、0.5g.kg-1)和葛根芩连颗粒高、中、低剂量(0.54、0.27、0.135g.kg-1)组。灌胃给药,每天1次,连续3d,末次给药30min后灌胃蓖麻油10mL.kg-1,测定小鼠稀便率。肠推进机能实验中将小鼠分为8组,即空白(等容生理盐水)、葛根芩连微丸(1g.kg-1)、葛根芩连煎剂高、中、低剂量(2、1、0.5g.kg-1)和葛根芩连颗粒高、中、低剂量(0.54、0.27、0.135g.kg-1)组。灌胃给药,每天1次,连续2d,末次给药20min后灌胃碳末,测定小鼠肠推进率。体外抑菌试验中测定葛根芩连煎剂与颗粒剂对5种菌的最低抑菌浓度。结果:与模型组比较,葛根芩连煎剂高、中剂量组与葛根芩连颗粒高、中、低剂量组稀便率显著减少(P<0.01);与空白组比较,葛根芩连煎剂高、中、低剂量与葛根芩连颗粒高、中、低剂量组小鼠肠推进率无显著改变(P>0.05)。葛根芩连颗粒对4种菌的最低抑菌浓度均低于葛根芩连煎剂。结论:葛根芩连颗粒比葛根芩连煎剂抗腹泻、抑菌作用更佳。     

小鼠急性肺损伤后糖皮质激素受体的活性改变与热休克蛋白的关系

目的 研究急性肺损伤(ALI)后机体内糖皮质激素受体(GR)的配体结合活性显著下降与其分子伴侣热休克蛋白(Hsps)改变的关系.方法 以小鼠油酸急性肺损伤为模型,用Western blotting和受体的放射配体结合试验动态观察伤后GR、Hsp90、Hsp70和GR的结合容量和亲和力变化.结果 伤后小鼠肺组织水肿、肺体指数和肺含水量显著升高;GR在伤后1 h升高,然后显著下降,在12 h达最低点;Hsp90和Hsp70在伤后的表达逐渐升高,在12 h达最高点.配体结合试验显示在伤后GR Bmax逐渐下降,Kd值升高,与Hsp90/GR的变化趋势一致.结论 急性肺损伤后的GR配体结合活性的改变可能与Hsp90的变化有关.     

A phase I randomized,double‐blind,single subcutaneous dose escalation study to determine the safety,tolerability, and pharmacokinetics of rezafungin in healthy adult subjects

Rezafungin is a novel echinocandin being developed for the treatment and prevention of invasive fungal infections. The objectives of this randomized, double‐blind study in healthy adults were to determine the safety, tolerability, and pharmacokinetics of rezafungin after subcutaneous (s.c.) administration. The study design consisted of six sequential cohorts of eight subjects, except for the first cohort with four subjects. The subjects were randomized in a 3:1 ratio of rezafungin to placebo and were to receive a single dose of 1, 10, 30, 60, 100, or 200 mg. The most common adverse events (AEs) were increased alanine aminotransferase and sinus bradycardia (unsolicited) and erythema at the injection site (solicited). Unsolicited AEs were generally mild to moderate and not rezafungin‐related. Although the study was terminated after the 10 mg dose cohort due to concerns of potential increased severity of injection site reactions, no predetermined dose escalation halting criteria were met. Following the 10 mg single s.c. dose of rezafungin (n = 6), the geometric mean (GM) maximum concentration (C _max) was 105.0 ng/ml and the median time to C _max was 144 h. The GM area under the concentration‐time curve was 32,770 ng*h/ml. The median estimated terminal half‐life was 193 h. The GM apparent oral clearance was 0.255 L/h and the GM apparent volume of distribution was 68.5 L. This study demonstrates that a single s.c. dose of rezafungin in healthy adult subjects: (1) did not result in serious AEs, death, or withdrawal from the study due to an AE; and (2) produced a pharmacokinetic profile with long exposure period postadministration. In an effort to reduce the occurrence of injection site reactions, a re‐evaluation of the rezafungin s.c. formulation could be considered in the future.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

Currently marketed antifungal agents of the echinocandin class are dosed by i.v. once daily and are therefore not practical for prolonged or outpatient prophylaxis. Rezafungin is an investigational echinocandin (currently in phase III trials studying once‐weekly i.v. administration) that has excellent activity against clinically relevant Candida and Aspergillus spp. and Pneumocystis jirovecii, a prolonged half‐life allowing for longer dosing intervals, and a stability/solubility profile that allows for the possibility of subcutaneous (s.c.) dosing.

• WHAT QUESTION DID THE STUDY ADDRESS?

The objectives of this randomized, double‐blind study in healthy adults were to determine the safety, tolerability, and pharmacokinetics (PKs) of rezafungin after s.c. administration.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

This study provided safety data regarding s.c. administration of rezafungin. Despite common solicited injection site reactions, s.c. administration of 10 mg of rezafungin did not result in serious adverse event (AEs), death, or withdrawals due to an AE in healthy adult subjects. The study also showed that rezafungin had a PK profile with a long exposure period.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

In an effort to reduce the occurrence of injection site reactions, these findings may lead to a future re‐evaluation of the s.c. formulation of rezafungin.     

活细胞内筛选地塞米松衍生物作用靶蛋白的研究

背景与目的：地塞米松衍生物（9-氟-16α-甲基11β,17-二羟基-3-氧-1,4-雄二烯-17β-羧酸）具有优于地塞米松的抗肿瘤活性,为探讨其抗肿瘤机制,本研究利用酵母三杂交技术在活细胞内筛选与之相互作用的靶蛋白。方法：构建诱饵质粒pGBKT7-GRα-LBD,利用酵母三杂交技术从人K562细胞cDNA文库中筛选与地塞米松衍生物相互作用的靶蛋白。结果：诱饵质粒成功构建,经Western blot分析可表达约31ku的诱饵蛋白,且诱饵蛋白没有毒性、渗漏和自激活现象。利用酵母三杂交技术从人K562细胞cDNA文库中筛选到37个能与地塞米松衍生物相互作用的蛋白质,并经酵母回转实验验证,得到20个真阳性克隆。结论：通过酵母三杂交技术在活细胞内筛选到20个与地塞米松衍生物有相互作用的蛋白。