火药烟雾致支气管上皮细胞损伤体外研究平台建立及应用
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摘要
目的：1、设计制作烟雾发生装置及烟雾与细胞反应装置,组建烟雾吸入性损伤细胞分析平台。2、使用烟雾吸入性损伤细胞分析平台,检测民用黑火药烟雾对培养人支气管上皮细胞(BEAS-2B)的损伤作用,研究其损伤机制。
方法：1、制作烟雾发生装置：使用无线电磁加热器引燃民用黑火药,制作实验烟雾。通过计算火药燃烧率检查火药质量。通过检测实验烟雾对细胞的损伤作用,检查实验烟雾质量。2、制作烟雾与细胞反应装置：装置内温度恒定为37。C,湿度恒定≥90%。3、细胞与烟雾反应影响因素：(1)细胞密度：(2)细胞与烟雾接触方式；(3)烟雾量大小；(4)实验后检测时间：(5)细胞培养液体积；(6)发烟材料燃烧速度：4、使用剂量为0.5g、1g、2g、4g火药制作实验烟雾,与细胞接触10min后,观察其对细胞损伤作用大小；使用相同剂量为2g火药制作实验烟雾,分别与细胞接触5min、10min、15min、20min,观察细胞损伤情况。5、观察烟雾损伤细胞内ROS含量,使用DHE染色,荧光显微镜下观察拍照,IPP软件进行数据处理分析。6、观察烟雾损伤细胞核内DNA损伤情况,使用单细胞凝胶电泳(又称彗星试验)检测火药烟雾造成的细胞核内DNA断裂情况。荧光显微镜下观察拍照,使用casp软件进行数据处理分析。7、用ELISA方法检测烟雾损伤细胞分泌IL-8的情况。
结果：1、火药质量检测变异系数<5%。烟雾质量检测变异系数<10%。2、火药与细胞反应的影响因素,适宜火药检测指标为(1)细胞密度,12孔板,每孔细胞量为4×104个。(2)细胞与烟雾接触方式,使用滤网。(3)烟雾总体积>10L。(4)细胞培养液体积为1ml。(5)检测时间为24h。(6)火药5s内燃尽。3、实验烟雾火药剂量增加及与细胞接触时间的延长,对细胞的损伤加重。4、荧光染色检测单个细胞内ROS含量,随着细胞活性的降低而升高。5、单细胞凝胶电泳检测细胞核内DNA断裂,随着细胞活性降低而加重。6、细胞分泌的IL-8量随着单个细胞损伤的加重而升高。
结论：设计制作的发烟装置能够稳定安全制作实验烟雾,烟雾与细胞反应装置能够实现烟雾致细胞损伤中烟雾浓度、接触时间与损伤效应呈正相关。火药烟雾对支气管上皮细胞有损伤作用,损伤机制与ROS含量增加、细胞核内DNA断裂及IL-8分泌增多相关。
Objective:1, a smoke-generating device and an apparatus used in smoke treating cell were designed, and the cell research platform of smoke inhalation injury were established basing on the two previous.2, cultured human bronchial epithelial cell injured by civilian black powder smoke were studied through the cell research platform.
Methods:1, in the smoke generating device, a wireless electromagnetic heater was used to ignite black powder and produce smoke. The quality of black powder was checked by black powder burn rate. The quality of experimental smoke was indirectly checked through the cell damage.2, the apparatus used in the study of smoke treating cell, was set to a constant temperature 37℃and the humidity was greater than constantly.3, factors which influence smoke treating cell were:cell density, the exposure mode, volume of smoke, testing time, volume of cell culture medium, combustion velocity.4, one test smoke was produced by a dose of 0.5g, 1g,2g,4g black powder burning and test time was set up to 10 minute. The other test smoke was produced by a dose of 2g black powder burning and test time was set up to 5min, 1Omin,15min and 20min. Then status of cells was evaluated to decide their injury by test smoke. 5, intracellular ROS levels were detected by DHE, and were analyzed by fluorescent microscope and IPP software.6, DNA damage in cell nuclei was detected by SCGE, and was analyzed by fluorescent microscope camera and casp software.7, IL-8 secreted by cells was detected through ELISA.
Results:1, coefficient variation of different batches of black powder was less than 5%. Coefficient variation of different batches of smoke was less than 10%.2, factors that influence smoking-treating cells:(1) cell density in each hole of 12-well plate was 4×104. (2) the filter were used in the exposure mode. (3) the total smoke volume was greater than 10L. (4) cell culture medium volume was 1ml in each hole of 12-well plate. (5) cell activity was detected after 24h. (6) combustion time was less than 5 seconds.3, cell injury were greater with dose of black powder smoke increased and test time Extended.4, ROS levels were higher with cell injury greater in single cell.5, nuclear DNA breakage was more severe with cell injury greater.6, the amount of IL-8 secreted by cell was increased with cell injury greater in single cell.
Conclusion:The experimental devices produced test smoke stably and safely. The apparatus of smoke treating cell fulfilled that cell injury was positively correlated with smoke concentration and exposure time. Bronchial epithelial cell was injured by black powder smoke, and the mechanism was relevant with increased ROS levels, nuclear DNA breakage and the increased secretion of IL-8.
方法：1、制作烟雾发生装置：使用无线电磁加热器引燃民用黑火药,制作实验烟雾。通过计算火药燃烧率检查火药质量。通过检测实验烟雾对细胞的损伤作用,检查实验烟雾质量。2、制作烟雾与细胞反应装置：装置内温度恒定为37。C,湿度恒定≥90%。3、细胞与烟雾反应影响因素：(1)细胞密度：(2)细胞与烟雾接触方式；(3)烟雾量大小；(4)实验后检测时间：(5)细胞培养液体积；(6)发烟材料燃烧速度：4、使用剂量为0.5g、1g、2g、4g火药制作实验烟雾,与细胞接触10min后,观察其对细胞损伤作用大小；使用相同剂量为2g火药制作实验烟雾,分别与细胞接触5min、10min、15min、20min,观察细胞损伤情况。5、观察烟雾损伤细胞内ROS含量,使用DHE染色,荧光显微镜下观察拍照,IPP软件进行数据处理分析。6、观察烟雾损伤细胞核内DNA损伤情况,使用单细胞凝胶电泳(又称彗星试验)检测火药烟雾造成的细胞核内DNA断裂情况。荧光显微镜下观察拍照,使用casp软件进行数据处理分析。7、用ELISA方法检测烟雾损伤细胞分泌IL-8的情况。
结果：1、火药质量检测变异系数<5%。烟雾质量检测变异系数<10%。2、火药与细胞反应的影响因素,适宜火药检测指标为(1)细胞密度,12孔板,每孔细胞量为4×104个。(2)细胞与烟雾接触方式,使用滤网。(3)烟雾总体积>10L。(4)细胞培养液体积为1ml。(5)检测时间为24h。(6)火药5s内燃尽。3、实验烟雾火药剂量增加及与细胞接触时间的延长,对细胞的损伤加重。4、荧光染色检测单个细胞内ROS含量,随着细胞活性的降低而升高。5、单细胞凝胶电泳检测细胞核内DNA断裂,随着细胞活性降低而加重。6、细胞分泌的IL-8量随着单个细胞损伤的加重而升高。
结论：设计制作的发烟装置能够稳定安全制作实验烟雾,烟雾与细胞反应装置能够实现烟雾致细胞损伤中烟雾浓度、接触时间与损伤效应呈正相关。火药烟雾对支气管上皮细胞有损伤作用,损伤机制与ROS含量增加、细胞核内DNA断裂及IL-8分泌增多相关。
Objective:1, a smoke-generating device and an apparatus used in smoke treating cell were designed, and the cell research platform of smoke inhalation injury were established basing on the two previous.2, cultured human bronchial epithelial cell injured by civilian black powder smoke were studied through the cell research platform.
Methods:1, in the smoke generating device, a wireless electromagnetic heater was used to ignite black powder and produce smoke. The quality of black powder was checked by black powder burn rate. The quality of experimental smoke was indirectly checked through the cell damage.2, the apparatus used in the study of smoke treating cell, was set to a constant temperature 37℃and the humidity was greater than constantly.3, factors which influence smoke treating cell were:cell density, the exposure mode, volume of smoke, testing time, volume of cell culture medium, combustion velocity.4, one test smoke was produced by a dose of 0.5g, 1g,2g,4g black powder burning and test time was set up to 10 minute. The other test smoke was produced by a dose of 2g black powder burning and test time was set up to 5min, 1Omin,15min and 20min. Then status of cells was evaluated to decide their injury by test smoke. 5, intracellular ROS levels were detected by DHE, and were analyzed by fluorescent microscope and IPP software.6, DNA damage in cell nuclei was detected by SCGE, and was analyzed by fluorescent microscope camera and casp software.7, IL-8 secreted by cells was detected through ELISA.
Results:1, coefficient variation of different batches of black powder was less than 5%. Coefficient variation of different batches of smoke was less than 10%.2, factors that influence smoking-treating cells:(1) cell density in each hole of 12-well plate was 4×104. (2) the filter were used in the exposure mode. (3) the total smoke volume was greater than 10L. (4) cell culture medium volume was 1ml in each hole of 12-well plate. (5) cell activity was detected after 24h. (6) combustion time was less than 5 seconds.3, cell injury were greater with dose of black powder smoke increased and test time Extended.4, ROS levels were higher with cell injury greater in single cell.5, nuclear DNA breakage was more severe with cell injury greater.6, the amount of IL-8 secreted by cell was increased with cell injury greater in single cell.
Conclusion:The experimental devices produced test smoke stably and safely. The apparatus of smoke treating cell fulfilled that cell injury was positively correlated with smoke concentration and exposure time. Bronchial epithelial cell was injured by black powder smoke, and the mechanism was relevant with increased ROS levels, nuclear DNA breakage and the increased secretion of IL-8.
引文
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[3]Rasmussen RE. In vitro systems for exposure of lung cell to NO2 and 03. J Toxicol Environ Health.1984;13(2-3):397-411.
[4]Potera C. More human, more humane:a new approach for testing airborne pollutants. Environ Health Perspect.2007;115(3):A148-151. PMCID: 1849917.
[5]Komori K, Murai K, Miyajima S, et al. Development of an in vitro batch-type closed gas exposure device with an alveolar epithelial cell line, A549, for toxicity evaluations of gaseous compounds. Analytical Sciences. 2008;24(8):957-962.
[6]Wallaert B, Fahy 0, Tsicopoulos A, et al. Experimental systems for mechanistic studies of toxicant induced lung inflammation. Toxicology Letters.2000;112-113:157-163.
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