高内涵肝毒筛选的方法学考察及在中药注射液中的应用

发布时间：2018-05-25 15:45

本文选题：方法学 + 高内涵分析技术　；参考：《北京中医药大学》2017年硕士论文

【摘要】：所谓药物毒性反应是由于用药剂量过大、用药时间过长或机体对药物敏感性过高时产生的危害性反应。肝脏是外源物质包括药物在体内代谢和转化的最重要器官,特别容易遭受药物损伤,是药物产生毒性的重要靶器官。因此,药物肝毒性的评价在新药研发及药物安全风险评估的毒理学评价中占重要地位。然而,实验动物在体肝毒性评价周期长,受试药品用量大,耗费的研究成木较大,实验结果也不能完全反应人体肝毒性的特点。因此,近年来体外肝细胞在药物安全性评价中逐渐成为通用的肝细胞毒性评价的体外试验工具,并广泛应用于药物细胞毒性的研究。HCA(High Content Analysis,HCA)是一种基于细胞表型分析的高效新药筛选技术,能够在保持细胞结构和功能完整的前提下,同时检测待测样品对细胞的形态、生长、周期、迁移、凋亡、代谢途径及信号传导等方面的影响,从单一实验中获取大量有关信息,从而确定化合物的生物活性以及潜在毒性。目目前HCA能够实时监测体外肝细胞与毒性机制相关的多个重要标志分子,包括细胞核的形态和数量、线粒体膜电位以及氧化应激状态、凋亡与早期DNA损伤的变化等,是体外评价化合物潜在肝毒性、从机制上预测候选药物安全性的高效手段。该方法能够弥补动物试验灵敏度低、试验周期长,不能量化的缺点,因此,高通量毒性评价技术已成为药品质量风险与安全风险评估的新的技术趋势。本课题将对本实验室已经建立的高内涵肝毒筛选方法进行进一步的考察,并对高内涵肝毒筛选方法在注射液中的应用进行初步探索。本课题研究工作分为三个部分:第一部分,对高内涵肝毒筛选方法的影响因素考察;第二部分,高内涵肝毒筛选方法的方法学考察;第三部分,高内涵肝毒筛选方法在注射液中的应用。第一部分对高内涵肝毒筛选方法的影响因素考察一、细胞初始接种密度对高内涵肝毒筛选方法结果的影响实验结果显示,1×104个· mL-1、1×105个· mL-1、2×105个· mL-1这三种初始接种密度的阳性药噻氯吡啶均在在细胞数目、DNA含量、GSH降低水平、ROS含量及MMP五大指标检测结果均为阳性,阴性药阿司匹林在细胞数目、DNA含量、GSH降低水平、ROS含量及MMP五大指标检测结果均为阴性。这表明阿司匹林对Hep G2肝细胞无毒性作用。对五个指标的检测结果进行综合分析,2×105个·mL-1这一密度的各个指标的结果相对较好。二、不同细胞模型对高内涵肝毒筛选方法结果的影响实验结果显示,药物作用于Hep G2细胞和L-02细胞时,阳性药噻氯吡啶在细胞数目、DNA含量、GSH降低水平、ROS含量及MMP五大指标检测结果均为阳性,这表明阳性药噻氯吡啶对Hep G2肝细胞和L-02细胞具有明显的毒性作用;阴性药阿司匹林在细胞数目、DNA含量、GSH降低水平、ROS含量及MMP五大指标检测结果均为阴性。这表明阿司匹林对HepG2肝细胞和L-02细胞无毒性作用。由于HepG2细胞被认为是适宜的肝脏毒性测试模型,而且其所含的生物转化代谢酶与人正常肝实质细胞具有同源性,分化程度较高,并且保留了较完整且活性稳定的生物转化代谢酶,因此接下来的实验会选择Hep G2细胞为肝毒性测试模型。第二部分高内涵肝毒筛选方法的方法学考察一、高内涵肝毒筛选方法的重复性考察实验结果显示,在对每个药物浓度的三个平行孔间进行了误差分析后,其中细胞数目、DNA含量和GSH降低水平这三个指标的误差相对较小,而ROS含量和MMP这两个指标的误差相对较大。Hep G2肝细胞毒性三次高内涵分析测定的重复性考察结果显示,细胞数目、DNA含量和GSH降低水平的重复性较好,而ROS含量和MMP改变的重复性较差。二、高内涵肝毒筛选方法的重现性考察实验结果显示,细胞数目、DNA含量和GSH降低水平的重复性较好,而ROS含量和MMP改变的重复性较差。通过对高内涵分析方法的重复性及重现性考察,细胞数目、DNA含量和GSH降低水平都有较好的重复性和重现性,而ROS含量和MMP改变这两个指标的重复性和重现性都相对较差,其原因有待进一步研究。第三部分高内涵肝毒筛选方法在注射液中的应用一、多批次氟康唑注射液高内涵分析结果实验结果显示,在本试验体系中,氟康唑注射液90%以上均会出现一定程度的肝毒性,但检测到的出现"毒性预警"的指标及毒性程度存在一定的差异。提示氟康唑注射液临床应用可能会出现不同程度的肝损伤或肝生化异常现象。对74批氟康唑注射液样品结果分析显示,70批的氟康唑注射液谷胱甘肽(GSH)指标提示"毒性预警";43批样品过氧化物(ROS)指标提示"毒性预警";18批样品细胞核DNA指标提示"毒性预警";14批样品细胞数减少(Cell Loss)指标提示"毒性预警";12样品过线粒体膜电位(MMP)指标提示"毒性预警"。提示90%以上氟康唑注射液肝毒性作用机制可能与其干扰肝脏GSH生成有关。二、十种中药注射液高内涵分析结果实验结果显示,除了盐酸川穹嗪注射液外,其他九种中药注射液均存在肝毒性安全风险,其中穿心莲注射液的肝毒性安全风险最大,盐酸川穹嗪的肝毒性安全风险最小。通过对十种中药注射液肝毒性分析各指标结果的总结,四种中药注射液谷胱甘肽(GSH)指标提示"毒性预警";八种中药注射液细胞核DNA指标提示"毒性预警";五种中药注射液细胞数减少(Cell Loss)指标提示"毒性预警"。提示中药注射液引起肝细胞毒性的机制可能为直接细胞毒性作用。
[Abstract]:The so-called drug toxicity is due to the excessive dosage of the drug, the long time of drug use or the harmful response to the high sensitivity of the body to the drug. The liver is the most important organ, including the metabolism and transformation of drugs in the body, which is especially vulnerable to drug damage and is an important target organ for the drug production. Sex evaluation plays an important role in the toxicological evaluation of new drug R & D and the risk assessment of drug safety. However, the experimental animals have a long period of evaluation of the toxicity of the body liver, the large dosage of the tested drugs, the large wood consumption and the experimental results can not fully reflect the specific point of human hepatotoxicity. Therefore, in recent years, the drug safety of the hepatocytes in vitro is in vitro. .HCA (High Content Analysis, HCA) is a highly effective new drug screening technique based on cell phenotype analysis, which is widely used in the study of cytotoxicity of drugs, which is widely used in the study of cytotoxicity of drugs. The effects of form, growth, growth, cycle, migration, apoptosis, metabolic pathways and signal transduction, and to obtain a large number of relevant information from a single experiment to determine the biological activity and potential toxicity of the compound. Currently, HCA can monitor in real time a number of important biomarkers related to the system of hepatocytes and toxic machines in vitro, including the shape of the nucleus. State and quantity, mitochondrial membrane potential, oxidative stress state, apoptosis and changes in early DNA damage are effective methods to evaluate the potential toxicity of compounds in vitro and predict the safety of candidate drugs in mechanism. This method can make up for the low sensitivity of animal test, long cycle time, and cannot be quantified. Therefore, high throughput toxicity assessment can be used. Price technology has become a new technical trend in the assessment of drug quality risk and safety risk. This subject will further investigate the high intension of liver toxin screening methods established in our laboratory and explore the application of high intension screening method in the injection. The research work is divided into three parts: the first part Study on the influencing factors of high intension screening method of hepatotoxicity; the second part, methodological investigation of high intension screening method of hepatotoxicity; the third part, the application of high intension liver toxin screening method in injection. The first part of the study on the influencing factors of high intension hepatotoxicity screening method: the initial density of cell inoculation on high intension hepatotoxicity screening prescription The results of the experimental results showed that 1 * 104 mL-1,1 * 105. ML-1,2 * 105. ML-1, the three initial inoculation density of thichlorpyridine, were in cell number, DNA content, GSH level, ROS content and MMP five major index test results were all positive, negative drug aspirin in cell number, DNA content, GSH to reduce water ROS content and MMP five major indexes were all negative. This showed that aspirin had no toxic effect on Hep G2 hepatocytes. The results of five indexes were analyzed synthetically. The results of each index of 2 x 105. ML-1 were relatively good. Two, the influence of different cell models on the results of high intension hepatotoxicity screening method The results showed that when the drug acted on Hep G2 cells and L-02 cells, the positive drug was positive for the number of cells, the content of DNA, the level of GSH, the content of ROS and the major MMP five indicators, which showed that the positive drug was toxic to Hep G2 hepatocytes and L-02 cells, and the negative drug aspirin was in the number of cells and D. NA content, GSH level, ROS content and MMP five major indicators were negative. This indicates that aspirin has no toxic effect on HepG2 hepatocytes and L-02 cells. Because HepG2 cells are considered a suitable test model for liver toxicity, and the biotransformation enzyme contained in the liver is homologous to human normal liver parenchyma cells, and the differentiation process is similar to those of human normal liver parenchyma cells. High degree, and retained a more complete and active bioconversion enzyme, so the next experiment will select the Hep G2 cell as the liver toxicity test model. Second part of the methodology of high intension hepatotoxicity screening method. After the error analysis of the parallel holes, the number of cells, the content of DNA and the reduction of GSH were relatively small, and the error of the ROS content and the two indexes of MMP were relatively larger than that of the three high intension analysis of.Hep G2 liver cytotoxicity, the number of cells, the content of DNA and the decrease of GSH. The reproducibility of the ROS content and MMP changes was poor. Two, the reproducibility of the high intension hepatotoxicity screening test showed that the number of cells, the content of DNA and the decrease of GSH were better, but the repeatability of the ROS content and the MMP change was poor. The number, DNA content and the decrease of GSH level both have good repeatability and reproducibility, and the repeatability and reproducibility of the two indexes of ROS and MMP are relatively poor. The reason is to be further studied. The third part of the high intension hepatotoxicity screening method in injection, many batches of high connotation analysis of Fluconazole Injection fruit The test results showed that in the test system, the liver toxicity of more than 90% of Fluconazole Injection appeared to a certain extent, but there was a certain difference between the indexes and the toxicity of the detected "toxicity warning". It suggested that the clinical application of Fluconazole Injection may appear the liver injury or liver biochemical abnormalities in different degrees. The 74 batches of fluorine Kang could be found. The results of the samples showed that the 70 batch of Fluconazole Injection glutathione (GSH) indicators suggested "toxicity warning"; the 43 batch of sample peroxide (ROS) indicators suggested "toxicity warning"; the 18 batch of sample nuclei DNA indicators suggested "toxicity warning"; the 14 batch of sample cell number reduction (Cell Loss) indicators suggested "toxicity warning"; 12 samples over mitochondrial membrane The potential (MMP) indicator suggests the "toxicity warning". It suggests that the mechanism of hepatotoxicity over 90% of Fluconazole Injection may be related to the interference of liver GSH. Two, the results of the high connotation analysis of ten kinds of traditional Chinese medicine injection show that there is a safety risk of liver toxicity except for the nine kinds of traditional Chinese medicine injection. The hepatotoxicity safety risk of Xin Lian injection is the largest, and the risk of hepatotoxicity is minimal. Through the summary of the results of the hepatotoxicity analysis of ten kinds of traditional Chinese medicine injection, four kinds of traditional Chinese medicine injection glutathione (GSH) indicator suggests "toxicity early warning"; the nuclear DNA index of eight kinds of traditional Chinese medicine injection suggests "toxicity early warning"; five kinds of traditional Chinese medicine. The decrease in the number of injection cells (Cell Loss) indicates "toxicity warning", suggesting that the mechanism of hepatocyte toxicity induced by Chinese medicine injection may be direct cytotoxicity.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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