【作者】 陈怡；

【导师】 林茂芳； 俞康；

【作者基本信息】 浙江大学 ， 内科， 2002， 硕士

【摘要】 引言 苯是一种重要的有机溶剂与工业原料。广泛应用于皮革、印刷行业。长期接触苯可引起骨髓损害，导致血细胞减少、再生障碍性贫血，甚至发生白血病。所以寻找安全、有效的细胞保护剂，使苯作业工人免受苯的损害非常重要。目前研究发现苯的毒性作用主要由其代谢产物1，4-氢醌，1，4-苯醌发挥作用。其主要作用机理是促进细胞凋亡，氧自由基的产生增加和DNA损伤引起的。而氨磷汀作为广谱细胞保护剂目前已广泛应用于临床，能选择性地保护正常细胞免受放疗和化疗的毒害作用，而且不降低化疗和放疗的疗效。氨磷汀还是一种多潜能造血刺激剂，保护多系造血祖细胞。它通过稳定正常组织中DNA分子，消除氧自由基，延迟由细胞因子启动的细胞凋亡过程等而发挥保护作用。氨磷汀除了对抗癌药物和放疗有明确的保护作用外，是否能更广泛地应用到对其他毒性物质的保护，是目前研究的一个方向。理论上推测，氨磷汀可以对苯引起的造血系统毒性具有保护作用，但目前国内外尚无此类文献报道。本课题通过研究体外培养条件下，氨磷汀对氢醌诱导的骨髓单个核细胞凋亡的影响，来探讨氨磷汀对苯骨髓毒性的保护作 浙江大学硕士研究生学位论文 用，为氨磷汀有可能作为苯作业工人保护剂提供一个理论依据。 材料和方法 1．取正常志愿者捐献的骨髓，肝素抗凝（50单位／毫升），淋巴细胞分离液 密度离心法分离制备成单个核细胞悬液。台盼蓝染色检测活细胞率，要 求拒染率达95％以上。 2．将细胞悬液置于56OC灭活的10％胎牛血清＋RPMI－1640液的培养体系中， 37℃、5％二氧化碳、饱和湿度条件下培养。 3．氢酿AR由上海凌峰化学试剂有限公司提供，灭菌双蒸水配制成原溶液。 阿米福汀（注射用氨磷汀）由湖南银河生化工程有限公司提供，生理盐 水配制成原溶液。按不同倍数稀释成所需终浓度。 4．细胞凋亡检测方法：OLYMPAS荧光显微镜（型号70DX）HT（凋亡试剂 11）染色观察形态学，凋亡细胞核着色增强、核固缩、染色质浓集沿核 周分布，正常细胞核着色浅而均匀。1．2％琼脂糖凝胶电泳，电压30V， EB染色后出现典型可见DNA降解产物的“梯形”条带。流式细胞术 ANNEXIN V－FITC加碘化丙陡（PI）双染检测细胞凋亡率和坏死率，早期 凋亡细胞为Annexin”／PI，晚期凋亡和继发坏死细胞Annexin”／PI，正常 活细胞为 Annexin／PI。 5．分别加入终浓度为 0，25，50，75，150 W的氢酿，检测作用 2，6，12 小时细胞凋亡，获得氢酮诱导骨髓单个核细胞凋亡的最佳浓度，以此决 定下一步的氢酮浓度。 6．加入根据上一步实验得出的一定浓度氢酮与骨髓单个核细胞培养0，2， 4，6，8，m，12，侣，24小时，检测细胞凋亡，找出细胞凋亡的高峰 时间点，以作为此后实验的细胞凋亡检测时间点。 7．分别加入终浓度为0，2，10，50，100，500帅l氨磷汀力或不加氢醒 浙江大学硕士研究生学位论文 与骨髓单个核细胞培养至上一步实验得到的细胞凋亡高峰时间，比较各 浓度氨磷汀对细胞凋亡率的影响，找出毒性作用小，又能有效抑制氢醒 诱导的细胞凋亡的氨磷汀浓度。 8．分别于氢醒前30分钟加入，同时加入，氢醒后30分钟加入一定浓度的 氨磷汀，检测培养至细胞凋亡高峰时间的细胞凋亡率和坏死率的变化。 9．实验组设置①空白对照组（不加药物处理组）：加入双蒸水60ul和生理 盐水60N。②氨磷汀组：加入一定浓度氨磷汀。③氢醒组：加入一定浓 度氢现④氨磷汀＋氢醒组：同时加入一定浓度氨磷汀和一定浓度氢酿。 检测8，10，12，18，24，48小时各时间点各组凋亡率的变化。 10．统计学处理：用SPSS10．0软件包，均数用了土S表示，多组间比较采用 方差分析，多组间两两比较采用SNK检验，两变量的相关程度用直线相 关分析法分析。 结果 1．荧光染色发现，加用不同浓度氢酮出现细胞核荧光强度增强，核着色加 深，核着色不均匀，染色质浓集，染色质沿核周分布现象。氢醒浓度不同 时，典型的凋亡细胞出现率存在差异。骨髓单个核细胞与不同浓度氢醒培 养6小时都出现DNA“梯形”条带，进一步证实细胞凋亡的存在。 2．流式细胞术检测结果发现，不同浓度氢酿作用后，细胞凋亡率明显高于空 白对?更多还原

【Abstract】 Background Benzene is an important solvent and used broadly in themanufacture of leather and print. Chronic benzene exposure may impair hematopoiesis, and give rise to aplastic anemia and leukemia. Therefore, it is very necessary to search for a safe and effective cytoprotective agent and protect workers of benzene exposure from benzene damage.1,4-hydroquinone and 1,4-benzoquinone are two main metabolites of benzene. Recently research found that these two elements play a critical role in hematopoietic damage. The mechanism is production of free radical of oxygen and impairing DNA, promoting apoptosis of hematopoietic stem cell. Amifostine is a broad-spectrum cytoportective agent, can selectively protect cells from chemotherapeutic and radiative damage, without decrease the effects of chemotherapy and radiotherapy. Amifostine also is a potential hematopoiesis-stimulating agent. It may protect multiple hematopoietic stem cells. It can stabilize DNA molecule, eliminate free radical of oxygen and delay apoptosis by cytokines.It is established that amifostine can protect cell from damage of chemotherapy and radiotherapy. Whether amifostine can protect normal cellfrom other toxic agents is unknown. Recent reports do not involve this aspect. Through in vitro culture, our goals were evaluation of the protective effect of amifostine against hydroquinone-induced apoptosis of bone marrow mononuclear cells. Expect to provide a theoretical basis for protective effect of amifostine against benzene poisoning.Material and method1. Collect of normal volunteer bone marrow in a sterilized tube containing 50U/mL heparin, after separation of mononuclear cells with Ficoll-Hypaque, stained by trypan blue. The rate of trypan blue stain-resistance is more than 95%.2. Use RPMI basic medium supplemented with heat-inactivated 10% FBS, culture under 37癈, saturated humidity and 5%CC>2.3. Hydroquinone AR from shanghai LingFeng chemical agent Ltd, prepare solution with distilled water. Amifostine (for injection) from Hunan sliver liver biochemical engineering Ltd, prepare solution with saline. Diluted needed concentration.4. Methods for examination of apoptosis: HT (apoptosis agent II) stain, observe cell morphology under fluorescent microscope (Olympus 70DX). Apoptotic cell present nucleus dark-colored, nucleus pycnosis, chromatin condensed round the nucleus. Normal cell present nucleus light-colored and uniform. 1.2% agarose gel electrophoresis under 30V voltage, after EB staining, the typical presentation is DNA ladder, due to DNA degradation. Add anti-Annexin V FITC and PI staining, examination apoptotic and necrotic rate by flow cytometer. Early apoptotic cells present Annexin+/PI-, late apoptotic and necrotic cells present Annexin+/PI+ and normal cells present7Annexin-/PI-, respectively.5. Add final concentration of 0, 25, 50, 75 and ISO^M hydroquinone, determine apoptosis after culture for 2, 6 and 12 hours. Acquired the optimal concentration of apoptosis-induced by hydroquinone. Based on this optimal concentration, decide next experimental concentration of hydroquinone.6. According to optimal concentration of hydroquinone, after culture for 0, 2, 4. 6, 8, 10, 12. 18 and 24 hours, determine cell apoptosis, found the peak time of apoptosis, as the optimal culture time.7. Add final concentration of 0, 2, 10, 50, 100 and 50Q\ig/m\ amifostine with or without hydroquinone, determine the peak apoptotic time. Compare apoptotic rate under different concentration of amifostine, found the optimal concentration of amifostine which is lowest toxic effect and most effective apoptosis-inhibited.8. Add amifostine before adding hydroquinone 30 minutes, after adding hydroquinone 30 minutes and at the same time of adding hydroquinone. Determine the apoptotic and necrotic rate at the peak apoptotic time.9. Divide into four groups: ゜lank control group: add distilled water 60ml and saline 60ml; ゛mifostine group: add d更多还原