几种化学计量学方法在放线菌及其次生代谢产物抗肿瘤活性筛选中的应用

当今肿瘤的诊断与治疗对人类健康非常重要. 在放线菌次生代谢产物内寻找抗肿瘤抗生素的过程中, 如何从种类多、数量大的土壤放线菌株中确定目标菌株, 筛选是关键. 将化学计量学方法用于大批量的放线菌及其次生代谢产物的筛选数据分析, 首先通过主成分分析获得具有抗肿瘤活性菌株对6种肿瘤细胞抑制作用的一些关系与特异性, 其次在进行菌株次生代谢产物的分子模型筛选数据分析时, 利用比较分析发现有较好肿瘤抑制作用者, 基本与细胞筛选结果吻合. 二者结合建立了一种简便适当的分析模式, 能较快地从大批量放线菌株的筛选中找到具有抗肿瘤活性的有研究价值的目标放线菌株.     

中草药活性成分的高通量筛选技术研究进展

随着高通量筛选技术的不断发展,该技术已经成为发现新药物的重要途径之一。高通量筛选技术已大量应用于筛选药物活性成分的领域中,但是其中大部分为从化合物库中筛选活性成分,仅有十几篇文献应用于中药活性成分的筛选,而中国传统中草药却是探索和发展新药物的丰富来源。本文通过综述国内外2008年到2017年的相关文献,阐述了分子和细胞水平上的高通量筛选技术在中草药活性成分的筛选及其应用进展,为今后中草药新药研发提供参考。     

10-羟基喜树碱的光谱性质、抗肿瘤活性及应用于细胞标记研究

通过紫外-可见光谱、荧光光谱和红外光谱等方法研究了药物10-羟基喜树碱(HCPT)的光谱性质.采用溴化噻唑蓝四氮唑(MTT)法测定了HCPT对3种肿瘤细胞(HeLa,MCF-7和HT1080)的抗肿瘤活性,其IC50值分别为16.37,16.73和19.24μg/mL.测定了HCPT对正常细胞人胚肾细胞系HEK293T的生长抑制活性,最高抑制率达88.72%,表明正常细胞比3种肿瘤细胞对药物HCPT更敏感.以HeLa细胞为模型,利用Annexin V-FITC细胞凋亡检测试剂盒研究了HCPT的抗肿瘤作用机制,发现几乎所有细胞均同时被Annexin V-FITC和碘化丙啶(PI)染色,细胞膜为绿色,而细胞核为红色,表明HCPT诱导了HeLa细胞的晚期凋亡.通过荧光显微镜观察了HCPT在HeLa细胞中的分布,为其在细胞标记中的应用提供了科学依据.     

血脑屏障药物筛选模型的建立及丹参活性成分筛选分析

ECV304细胞在C6细胞的诱导下生长,通过细胞培养时间优化、渗透系数测定和细胞形态学观察等,建立ECV304/C6共培养血脑屏障(BBB)药物筛选模型.将该模型应用于从丹参提取液中筛选可能作用于中枢神经系统(CNS)的活性成分,结合液相色谱-质谱联用技术(LC-MS)分析,发现丹参提取液中至少有16种成分能够穿越BBB模型,其中4种成分被确认为隐丹参酮、丹参酮Ⅰ、丹参素和原儿茶酸.通过定量构效关系(QASR)分析,进一步从理论上证明所确认的4种化合物均符合CNS靶向药物的特征.研究结果表明,ECV304/C6共培养BBB模型能够在模拟生理状态下从中药复杂体系中筛选分离跨越BBB的活性成分组,可用于CNS药物开发的早期快速筛选,服务于中药现代化研究.     

钌配合物稳定端粒DNA的作用及其诱导细胞凋亡分子机制的研究

端粒酶在肿瘤细胞中高表达,已经成为抗肿瘤药物的重要靶点,由于很多肿瘤细胞中富含G4-DNA,通过稳定G-四链体DNA的形成来抑制端粒酶的活性已成为抗癌药物的一个新策略. 本文设计了两种钌配合物,调查了这两种钌配合物稳定G4-DNA的能力,发现配合物2稳定端粒 G4-DNA的能力强于配合物1,配合物2能够诱导端粒 G4-DNA发生构型的转化,而配合物1不能诱导G4-DNA发生构型的转化,这项研究结果证明,钌配合物与G4-DNA的作用能力与配体的平面性有关. 在抗肿瘤活性方面,配合物2表现出更强的抗肿瘤活性,尤其是对HepG2细胞具有较强的抑制作用,推测其是以端粒酶为靶点发挥的抗肿瘤作用. 配合物2能够诱导肿瘤细胞凋亡,能诱导G1期细胞阻滞和DNA碎片的形成（细胞凋亡的特征）. 据此推测本论文设计的钌配合物是一个潜在的抗肿瘤药物.     

聚异戊二烯基三胺的合成及生理活性评价

设计、合成了一系列聚异戊二烯基三胺化合物,目标化合物结构均经过核磁共振谱、质谱及元素分析确认;利用MTT法测试了目标化合物对人白血病细胞K562和人肝癌细胞Bel-7402的体外抗肿瘤活性.结果表明,目标化合物对两种肿瘤细胞的生长均有较强的抑制活性.     

微流控芯片－质谱联用技术在细胞代谢与药物代谢中的研究进展

细胞代谢与药物代谢是新药筛选和研发的关键环节,在推动人类大健康发展进程中具有重要意义。通常情况下,细胞代谢和药物筛选以传统细胞培养测定研究为主,多为静态培养条件,无法很好地模拟体内细胞动态微环境。微流控芯片－质谱联用是近年发展起来的一种新型高通量分析技术。微流控芯片模块可高度模拟细胞体内动态微环境,与质谱联用可实时在线检测样品物质,具有高效、快速、简便、样品和试剂消耗低等特点,广泛应用于细胞代谢和药物代谢分析,有利于加速药物筛选研发进程。该文重点综述了微流控芯片－质谱联用技术及其在细胞代谢和药物代谢方面的应用概况,并对目前存在的局限性进行了讨论和展望,以期为微流控芯片－质谱联用技术在新药研发与细胞分析领域的发展提供参考。     

3-氨基-4-氯-1H吲唑衍生物的合成及抗肿瘤活性测定

以2,6-二氯苯腈为起始原料通过5步反应合成了9个具有抗肿瘤活性的吲唑类化合物并对其抗肿瘤活性进行了初步筛选.目标产物结构经~1H-NMR和IR确证,体外细胞实验结果显示化合物2c的抗肿瘤活性最好,对K-562、SMMC7721肿瘤细胞有明显抑制作用.     

基于DNA纳米花的细胞自噬基因沉默用于增敏抗肿瘤化疗

自噬是真核细胞降解蛋白质的重要途径之一, 在细胞的更新代谢中起重要作用. 肿瘤细胞借助高水平的细胞自噬能够阻断细胞凋亡途径, 降低化疗药物的抗肿瘤效果. 本文通过设计编码有核酸适配体序列(Aptamer)和DNA酶序列(DNAzyme)的多功能DNA纳米花, 利用DNA序列可负载化疗药物阿霉素(Dox)的特性, 实现了对肿瘤细胞特异靶向的药物递送, 并高效沉默肿瘤细胞的自噬相关基因ATG5, 达到增敏抗肿瘤化疗的效果. 通过RT-PCR实验验证合成的DNA纳米花可以有效剪切肿瘤细胞中自噬相关基因ATG5的mRNA; 并通过DNA纳米花的细胞毒性和细胞凋亡实验研究了其对肿瘤细胞系MCF-7的靶向治疗作用, 结果显示该多功能DNA纳米花在增敏抗肿瘤化疗方面具有明显优势.     

快速线扫描拉曼成像技术在研究CaSki细胞凋亡过程中的应用

应用快速线扫描拉曼成像技术研究了在抗肿瘤药物顺铂诱导下宫颈癌Ca Ski细胞凋亡过程中细胞色素c、蛋白质和脂质等的时空变化规律.结果表明,细胞色素c与蛋白质的相对拉曼峰强可以反映细胞凋亡程度.与常用的表征细胞活性的噻唑蓝(MTT)实验的统计方法相比,利用拉曼成像技术可以更灵敏地观察到细胞凋亡早期生物活性分子的变化,从而在单细胞水平检测细胞凋亡过程,为解析药物与细胞的作用机制提供分子水平信息.     

Screening of cardioprotective diseases bioactive components from Danshen extracts and LC–MS analysis

A rapid and efficient analysis and screening method is adopted for cell affinity capture coupled with HPLC–MS (CAC–HPLC–MS) analysis of bioactive components that have possible efficiency against cardiovascular diseases. This method involves affinity capture, concentration, and separation of bioactive components from Danshen library using oxidatively damaged endothelial cells induced by H₂O₂, as well as analysis and identification of targeted compounds with HPLC and MS. It combines the specific interaction between cell membrane receptors and bioactive components with the powerful analysis and identification function of HPLC–MS. The CAC–HPLC–MS method was also used for analysis and screening of bioactive components from crude extracts of Danshen. A total of 19 components were found to be bound to oxidatively damaged endothelial cells with seven of these identified. Existing literature confirms that these seven components have many activities related to cardioprotective diseases. Therefore, the combination of biological affinity capture with HPLC–MS should be regarded as an attractive method with great potential for rapid and efficient screening of bioactive components related to anti-cardiovascular diseases from natural product libraries.     

用高表达EGFR细胞膜色谱-HPLC/MS联用快速筛选独活中抗EGFR活性成分

报道了用高表达表皮生长因子受体细胞膜色谱与高效液相色谱/质谱在线联用方法(EGFR/CMC-online-HPLC/MS)快速筛选发现中药独活中的活性成分.实验中,采用高表达EGFR的细胞膜制备色谱固定相,建立EGFR/细胞膜色谱(EGFR/CMC)模型,利用柱切换和固相萃取技术,将EGFR/CMC模型与高效液相色谱/质谱(HPLC/MS)在线联用,构成一种新的可同时"识别-鉴定"目标成分的二维色谱系统,并应用于快速筛选独活中具有抗EGFR活性的目标成分.结果发现独活中的蛇床子素具有与对照药物达沙替尼类似的色谱保留特性,能够作用于EGFR;同时MTT及Elisa分析实验证实蛇床子素对HEK293EGFR细胞增殖及EGFR表达均有抑制作用.本文建立的EGFR/CMC-online-HPLC/MS二维色谱方法,可以选择性地从中药复杂体系中快速"识别-鉴定"目标组分,且筛选结果与特定生物效应显著相关.     

Plasma pharmacochemistry combined with pharmacokinetics and pattern recognition analysis to screen potentially bioactive components from Daming capsule using ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry

Daming capsule is a traditional Chinese medicine for hyperlipidemia treatment. However, the vague understanding of the bioactive components of Daming capsule hampers its modernization and internationalization. This work first developed a high‐throughput, high‐resolution, and high‐sensitivity ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method for identifying the absorbed compounds and monitoring the pharmacokinetics of Daming capsule. A high‐throughput strategy integrating plasma pharmacochemistry, pharmacokinetics, and pattern recognition analysis was also established for screening the bioactive components of Daming capsule in vivo. The established strategy based on ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry was successfully applied to screen the bioactive components of Daming capsule. Up to 53 absorbed compounds were identified. Six anthraquinones with fast and high absorption, namely, emodin‐O‐glucoside, aurantio‐obtusin, aloe‐emodin, rhein, emodin, and chrysophanol, were screened as potentially bioactive components of Daming capsule. The plasma pharmacochemistry and pharmacokinetics of Daming capsule were reported for the first time. Notably, the high‐throughput and reliable strategy facilitated the screening and identification of bioactive components of traditional Chinese medicine, thereby providing novel insights into the research and development of new drugs.     

微量元素锌、血清胱抑素 C在糖尿病肾病早期诊断的意义

目的：探讨早期糖尿病肾病患者（ DN）的微量元素锌、血清胱抑素C（ CysC）、血清肌酐（ SCr）和血清尿素（ BUN）水平的变化程度及意义。方法将糖尿病肾病患者分为3组：正常蛋白尿组,微量蛋白尿组,临床蛋白尿组。所有患者和对照组均空腹抽取静脉血检测微量元素锌、血清胱抑素C、血清尿素、血清肌酐。结果正常蛋白尿组、微量蛋白尿组、临床蛋白尿组微量元素锌均明显低于正常对照组,差异有统计学意义（ P＜0.05）。正常蛋白尿组、微量蛋白尿组、临床蛋白尿组血清胱抑素C水平均明显高于正常对照组,差异有统计学意义（ P＜0.05）。微量蛋白尿组、临床蛋白尿组血清肌酐检测值和正常对照组有明显差异,差异有统计学意义（ P＜0.05）。结论检测微量元素锌、血清胱抑素C对糖尿病肾病的早期肾损害有较高的临床价值,可作为早期糖尿病肾病诊断的参考指标。     

体外细胞模型和高效液相质谱联用分析预测黄芪中的活性成分

利用体外细胞模型模拟体内细胞对中药有效成分的特异性吸收,结合高效液相色谱/质谱分析筛选中药黄芪中的生物活性成分。将中药黄芪提取液与Caco-2细胞及红细胞分别混合培养,破碎与药材结合后的细胞,使之释放出结合的药材中的成分。运用高效液相色谱/飞行时间质谱(HPLC-ESI/TOFMS)分析中药黄芪提取液与活性细胞有结合的成分,并对其进行结构鉴定。结果显示:黄芪中有10个化合物与Caco-2细胞结合,14个化合物与红细胞结合。本方法可用于预测口服药物在体内的吸收以及与特定靶细胞的结合情况,特异性地筛选中药复杂体系中的药效物质基础。     

儿童复发性口腔溃疡血清微量元素的分析

目的探讨了儿童复发性口腔溃疡血清微量元素的变化。方法分别收集实验组和对照组静脉血清样品各50例,应用等离子体发射光谱仪测定Zn、Cu、Fe、Mg含量。结果儿童血清微量元素含量测定,实验组Zn含量明显低于对照组（P〈0.05）,Cu、Fe、Mg含量与对照组比无显著性差异（P〉0.05）。结论儿童复发性口腔溃疡的发生与体内缺Zn有关,值得进一步探讨。     

Screening active anti‐breast cancer compounds from Cortex Magnolia officinalis by 2D LC–MS

Most of the anti‐breast cancer drugs are often limited owing to drug resistance and serious adverse reactions. Therefore, development of more targeted and low toxic drugs from traditional Chinese medicines for breast cancer are needed. At the same time, establishment of fast and effective drug screening methods are urgently required. We describe here a 2D LC method of MDA‐MB‐231 cell membrane chromatography combined with HPLC/MS for recognition, separation, and identification of target components from traditional Chinese medicine Cortex Magnolia officinalis. The MDA‐MB‐231 cells membrane was used to prepare the chromatographic stationary phase in the first dimension. The active compounds had a retention characteristic on the cell membrane chromatography model (10 × 2.0 mm, 5 μm). The retention fractions were enriched using an online C₁₈ column (10 × 1.0 mm, 5 μm) and were analyzed by the second dimension RP chromatography. Finally, the activity of the retention fractions was tested through in vitro experiments. Results showed that the retention fractions were honokiol and magnolol and the inhibition rate on MDA‐MB‐231 cell growth were 23 and 64 μM, respectively. These results support the conclusion that this coupled analytical technique could be an efficient method in drug discovery.     

In Vitro Tests for a Rapid Evaluation of Antidiabetic Potential of Plant Species Containing Caffeic Acid Derivatives: A Validation by Two Well-Known Antidiabetic Plants,Ocimum gratissimum L. Leaf and Musanga cecropioides R. Br. ex Tedlie (Mu) Stem Bark

Plant bioactive extracts represent a major resource for identifying drugs and adjuvant therapy for type 2 diabetes. To promote early screening of plants’ antidiabetic potential, we designed a four in vitro tests strategy to anticipate in vivo bioactivity. Two antidiabetic plants were studied: Ocimum gratissimum L. (Oc) leaf extract and Musanga cecropoides R. Br. ex Tedlie (Mu) stem bark extract. Chemical compositions were analyzed by LCMS and HPLC. Antidiabetic properties were measured based on (1) INS-1 cells for insulin secretion, (2) L6 myoblast cells for insulin sensitization (Glut-4 translocation), (3) L6 myoblast cells for protection against hydrogen peroxide (H₂O₂) oxidative stress (cell mortality), and (4) liver microsomial fraction for glucose-6-phosphastase activity (G6P). Oc extract increased insulin secretion and insulin sensitivity, whereas it decreased oxidative stress-induced cell mortality and G6P activity. Mu extract decreased insulin secretion and had no effect on insulin sensitivity or G6P activity, but it increased oxidative stress-induced cell mortality. Results were compared with NCRAE, an antidiabetic plant extract used as reference, previously characterized and reported with increased insulin secretion and insulin sensitivity, protection against oxidative stress, and decreased G6P activity. The proposed set of four in vitro tests combined with chemical analysis provided insight into the interest in rapid early screening of plant extract antidiabetic potential to anticipate pharmaco-toxicological in vivo effects.     

低血铅水平与免疫球蛋白和淋巴细胞相关性

目的：探讨低铅水平对IgA、 IgG 、 IgM、 C3、 C4、 B淋巴细胞的影响。方法用石墨炉原子吸收光谱法对39名印刷印染工龄5年以上工人进行血铅测定,另选择距污染源较远且无水系联系的,不受工业污染的,从未接触过重金属的为对照组,分别对两组用免疫比浊法进行（ IgA、IgG、 IgM、 C3和C4）5项测定,流式细胞仪检测血B淋巴细胞水平,全自动生化仪检测白细胞数、总淋巴细胞绝对数和百分比。结果高低铅两组IgA、 IgG 、 IgM、 C3水平比较差异有统计学意义（P＜0.05）,白细胞总数、淋巴细胞百分比均低于低铅组（P＜0.05）,总淋巴细胞百分比和NK淋巴细胞亚群的绝对数和百分比却低于低铅组（ P＜0.05）。结论高血铅对体液免疫功能有一定的影响。     

高效液相色谱/质谱联用技术在天然来源生物活性成分快速识别中的应用

天然来源生物活性成分是药物先导分子的重要源泉之一,它们种类繁多,结构复杂多样,逐一分离提取纯化然后进行结构分析鉴定的这一传统植物化学(天然药物化学)研究模式尽管已沿袭多年,但往往费时费力,目标性不强,且常常忽略含量甚少的微量成分.高效液相色谱及多级质谱联用技术(HPLC/MSn)兼顾了色谱的高效分离能力和质谱的强大定性功能,为天然生物活性成分的分析研究提供了新途径.本文总结了我们课题组近年来对糖苷、植物酚类以及酰胺类生物碱等几类天然有效成分的分析研究进展:应用HPLC/MSn联用技术在线分析鉴定多种天然来源的已知成分,快速筛选出多种未知成分,并运用多级质谱技术获得未知成分的结构信息,为快速准确发现新的目标结构提供靶向指导.这一研究方法的应用,突破了传统分离分析的束缚,为现代植物化学或天然药物化学研究提供了新思路.