本文選題：方法學 + 高內(nèi)涵分析技術(shù)��； 參考：《北京中醫(yī)藥大學》2017年碩士論文

【摘要】：所謂藥物毒性反應(yīng)是由于用藥劑量過大、用藥時間過長或機體對藥物敏感性過高時產(chǎn)生的危害性反應(yīng)。肝臟是外源物質(zhì)包括藥物在體內(nèi)代謝和轉(zhuǎn)化的最重要器官,特別容易遭受藥物損傷,是藥物產(chǎn)生毒性的重要靶器官。因此,藥物肝毒性的評價在新藥研發(fā)及藥物安全風險評估的毒理學評價中占重要地位。然而,實驗動物在體肝毒性評價周期長,受試藥品用量大,耗費的研究成木較大,實驗結(jié)果也不能完全反應(yīng)人體肝毒性的特點。因此,近年來體外肝細胞在藥物安全性評價中逐漸成為通用的肝細胞毒性評價的體外試驗工具,并廣泛應(yīng)用于藥物細胞毒性的研究。HCA(High Content Analysis,HCA)是一種基于細胞表型分析的高效新藥篩選技術(shù),能夠在保持細胞結(jié)構(gòu)和功能完整的前提下,同時檢測待測樣品對細胞的形態(tài)、生長、周期、遷移、凋亡、代謝途徑及信號傳導(dǎo)等方面的影響,從單一實驗中獲取大量有關(guān)信息,從而確定化合物的生物活性以及潛在毒性。目目前HCA能夠?qū)崟r監(jiān)測體外肝細胞與毒性機制相關(guān)的多個重要標志分子,包括細胞核的形態(tài)和數(shù)量、線粒體膜電位以及氧化應(yīng)激狀態(tài)、凋亡與早期DNA損傷的變化等,是體外評價化合物潛在肝毒性、從機制上預(yù)測候選藥物安全性的高效手段。該方法能夠彌補動物試驗靈敏度低、試驗周期長,不能量化的缺點,因此,高通量毒性評價技術(shù)已成為藥品質(zhì)量風險與安全風險評估的新的技術(shù)趨勢。本課題將對本實驗室已經(jīng)建立的高內(nèi)涵肝毒篩選方法進行進一步的考察,并對高內(nèi)涵肝毒篩選方法在注射液中的應(yīng)用進行初步探索。本課題研究工作分為三個部分:第一部分,對高內(nèi)涵肝毒篩選方法的影響因素考察;第二部分,高內(nèi)涵肝毒篩選方法的方法學考察;第三部分,高內(nèi)涵肝毒篩選方法在注射液中的應(yīng)用。第一部分對高內(nèi)涵肝毒篩選方法的影響因素考察一、細胞初始接種密度對高內(nèi)涵肝毒篩選方法結(jié)果的影響實驗結(jié)果顯示,1×104個· mL-1、1×105個· mL-1、2×105個· mL-1這三種初始接種密度的陽性藥噻氯吡啶均在在細胞數(shù)目、DNA含量、GSH降低水平、ROS含量及MMP五大指標檢測結(jié)果均為陽性,陰性藥阿司匹林在細胞數(shù)目、DNA含量、GSH降低水平、ROS含量及MMP五大指標檢測結(jié)果均為陰性。這表明阿司匹林對Hep G2肝細胞無毒性作用。對五個指標的檢測結(jié)果進行綜合分析,2×105個·mL-1這一密度的各個指標的結(jié)果相對較好。二、不同細胞模型對高內(nèi)涵肝毒篩選方法結(jié)果的影響實驗結(jié)果顯示,藥物作用于Hep G2細胞和L-02細胞時,陽性藥噻氯吡啶在細胞數(shù)目、DNA含量、GSH降低水平、ROS含量及MMP五大指標檢測結(jié)果均為陽性,這表明陽性藥噻氯吡啶對Hep G2肝細胞和L-02細胞具有明顯的毒性作用;陰性藥阿司匹林在細胞數(shù)目、DNA含量、GSH降低水平、ROS含量及MMP五大指標檢測結(jié)果均為陰性。這表明阿司匹林對HepG2肝細胞和L-02細胞無毒性作用。由于HepG2細胞被認為是適宜的肝臟毒性測試模型,而且其所含的生物轉(zhuǎn)化代謝酶與人正常肝實質(zhì)細胞具有同源性,分化程度較高,并且保留了較完整且活性穩(wěn)定的生物轉(zhuǎn)化代謝酶,因此接下來的實驗會選擇Hep G2細胞為肝毒性測試模型。第二部分高內(nèi)涵肝毒篩選方法的方法學考察一、高內(nèi)涵肝毒篩選方法的重復(fù)性考察實驗結(jié)果顯示,在對每個藥物濃度的三個平行孔間進行了誤差分析后,其中細胞數(shù)目、DNA含量和GSH降低水平這三個指標的誤差相對較小,而ROS含量和MMP這兩個指標的誤差相對較大。Hep G2肝細胞毒性三次高內(nèi)涵分析測定的重復(fù)性考察結(jié)果顯示,細胞數(shù)目、DNA含量和GSH降低水平的重復(fù)性較好,而ROS含量和MMP改變的重復(fù)性較差。二、高內(nèi)涵肝毒篩選方法的重現(xiàn)性考察實驗結(jié)果顯示,細胞數(shù)目、DNA含量和GSH降低水平的重復(fù)性較好,而ROS含量和MMP改變的重復(fù)性較差。通過對高內(nèi)涵分析方法的重復(fù)性及重現(xiàn)性考察,細胞數(shù)目、DNA含量和GSH降低水平都有較好的重復(fù)性和重現(xiàn)性,而ROS含量和MMP改變這兩個指標的重復(fù)性和重現(xiàn)性都相對較差,其原因有待進一步研究。第三部分高內(nèi)涵肝毒篩選方法在注射液中的應(yīng)用一、多批次氟康唑注射液高內(nèi)涵分析結(jié)果實驗結(jié)果顯示,在本試驗體系中,氟康唑注射液90%以上均會出現(xiàn)一定程度的肝毒性,但檢測到的出現(xiàn)"毒性預(yù)警"的指標及毒性程度存在一定的差異。提示氟康唑注射液臨床應(yīng)用可能會出現(xiàn)不同程度的肝損傷或肝生化異�，F(xiàn)象。對74批氟康唑注射液樣品結(jié)果分析顯示,70批的氟康唑注射液谷胱甘肽(GSH)指標提示"毒性預(yù)警";43批樣品過氧化物(ROS)指標提示"毒性預(yù)警";18批樣品細胞核DNA指標提示"毒性預(yù)警";14批樣品細胞數(shù)減少(Cell Loss)指標提示"毒性預(yù)警";12樣品過線粒體膜電位(MMP)指標提示"毒性預(yù)警"。提示90%以上氟康唑注射液肝毒性作用機制可能與其干擾肝臟GSH生成有關(guān)。二、十種中藥注射液高內(nèi)涵分析結(jié)果實驗結(jié)果顯示,除了鹽酸川穹嗪注射液外,其他九種中藥注射液均存在肝毒性安全風險,其中穿心蓮注射液的肝毒性安全風險最大,鹽酸川穹嗪的肝毒性安全風險最小。通過對十種中藥注射液肝毒性分析各指標結(jié)果的總結(jié),四種中藥注射液谷胱甘肽(GSH)指標提示"毒性預(yù)警";八種中藥注射液細胞核DNA指標提示"毒性預(yù)警";五種中藥注射液細胞數(shù)減少(Cell Loss)指標提示"毒性預(yù)警"。提示中藥注射液引起肝細胞毒性的機制可能為直接細胞毒性作用。
[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.
【學位授予單位】：北京中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285.5

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