香烟烟雾与氡染毒致大鼠肺损伤的蛋白质组学研究
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摘要
目的:从蛋白质组学角度研究香烟烟雾和氡吸入染毒引起的大鼠肺组织蛋白质表达谱的改变,寻找香烟烟雾和氡吸入引起肺损伤的分子标志物,为吸烟和氡暴露健康危害的评价提供实验资料。
方法:(1)采用动式烟雾染毒装置对Wistar大鼠进行香烟烟雾染毒,装置内烟雾浓度控制在(20±5)%,氧气浓度控制在21%,每天染毒2次,每次30min,分别染毒1、2、4个月作为低、中、高剂量组,每组5只动物。(2)采用HD-3型多功能移动式氡室对Wistar大鼠动态吸入氡染毒,通过计算机控制装置内氡浓度,使其剂量率恒定保持在1×105Bq/m3,每天染毒16h。氡及其子体累积染毒剂量分别达100、200、400工作水平月(working level month,WLM)作为低、中、高剂量组,对照组室内氡本底浓度80Bq/m3,每组5只动物。(3)采用上述方法对Wistar大鼠进行香烟烟雾和氡联合交替染毒,使香烟烟雾和氡染毒分别达2个月和200WLM作为联合染毒组。(4)观察染毒模型大鼠肺组织的病理学改变和测定氡染毒大鼠模型体内氡子体210Po的分布。(5)建立双向电泳(2-DE)分离大鼠肺组织总蛋白的技术,提高方法的灵敏度、分离度和重现性。(6)染毒后分别提取大鼠肺组织的总蛋白,采用2-DE分离,考马斯亮蓝染色,差异表达点进行MALDI-TOF或MALDI-TOF-TOF质谱分析,NCBInr数据库搜索鉴定分析结果。(7)免疫组化方法分别验证质谱鉴定出的差异表达蛋白晚期糖基化终产物受体(RAGE)和硫氧还蛋白(Trx)。以GAPDH为内参,运用Western blot法分别验证RAGE、Trx和S100A6蛋白在不同染毒组大鼠肺组织中的表达,以Bandscan4.3软件进行灰度分析并进行统计学检验。(8)测定氡染毒大鼠肺组织中细胞色素C氧化酶(CcO)的活性。
结果:(1)建立了香烟烟雾、氡单独和联合染毒Wistar大鼠模型。HE和Masson染色结果提示各染毒组大鼠肺组织出现了支气管炎症、炎症细胞浸润、肺气肿、胶原纤维化等病变,病变程度随香烟烟雾、氡染毒剂量的加大而加重。染氡组大鼠体内组织器官中氡子体210Po的含量随氡暴露剂量加大而增加。(2)建立了大鼠肺组织总蛋白的2-DE分离技术。银染法中,pH3~10,11cm的胶条分离到约450个蛋白质点,pH3~10,24cm的胶条分离到约625个蛋白质点,pH4~7,24cm的胶条分离到约1248个蛋白质点。确定肺组织蛋白2-DE实验采用pH4~7,24cm的胶条进行分离,并获得较好的分离效果和重复性。(3)各染毒组大鼠肺组织总蛋白经2-DE分离、MS分析,香烟烟雾染毒组共鉴定出18种差异表达蛋白,包括10个上调和8个下调的蛋白;氡染毒组共鉴定出15种差异表达蛋白,包括9个上调和6个下调的蛋白;香烟烟雾与氡联合染毒组共鉴定出20种差异表达蛋白,包括12个上调和8个下调的蛋白。差异蛋白多数与机体代谢、细胞存活与增殖、氧化还原、信号传导、细胞骨架等功能有关。(4)免疫组化结果表明RAGE、Trx和S100A6在不同染毒各剂量组大鼠肺组织中表达随染毒剂量增加而升高;Western blot分析进一步验证了免疫组化结果;氡染毒大鼠肺组织中CcO的活性随染氡剂量的增加而降低,验证结果均与2-DE检测吻合。
结论:(1)通过病理组织学和大鼠体内氡子体210Po的测定,成功建立了香烟烟雾与氡单独和联合吸入染毒的大鼠模型。在优化和发展传统双向凝胶电泳技术、比较蛋白质组方法的基础上,建立了蛋白质组提取-双向凝胶电泳分离-半自动胶内酶解-质谱鉴定的高通量技术分析平台。(2)获得了香烟烟雾与氡染毒大鼠肺组织的蛋白质表达谱及两者单独和联合染毒相关的差异表达蛋白质,初步鉴定出的差异蛋白质数为:香烟烟雾染毒18个,氡染毒组15个,联合染毒组20个。这些蛋白分别与代谢、细胞存活与增殖、氧化还原、信号传导、细胞骨架等功能相关,提示香烟烟雾与氡染毒致肺损伤是多种机制共同作用的结果。(3)应用免疫组化和Western blot等方法对香烟烟雾与氡染毒大鼠肺组织中差异表达蛋白Trx、RAGE、S100A6和CcO的表达水平进行了验证,结果与蛋白质组学检测一致,提示这些蛋白可作为香烟烟雾和氡致肺损伤的蛋白分子标志物。
Objective: Proteomic analysis of the rat lung tissues was conducted upon exposure to cigarette smoke and radon to provide experimental data for evaluating health risks and exploring potential molecular markers.
Methods: (1) Wistar rats were exposed to cigarette smoke in a gas inhalation chamber with the smoke concentration controlled at the range of (20±5)% and oxygen concentration at 21%, twice a day, 30min each at low, middle and high dose for 1, 2 and 4 months with 5 per group. (2) Wistar rats were exposed to radon with a multifunction ecological radon room at a constant level of 1×105Bq/m3. The low, middle and high dose groups were exposed up to a cumulative dose of 100, 200 and 400WLM (working level months) respectively. The control rats were housed in a room with a background concentration of radon at 80 Bq/m3. (3) Wistar rats were exposed to radon for up to cumulative dose of 200 WLM and cigarette smoke for 2 months in the same conditions as (1) and (2) as the joint group. (4) Pathological changes in lung tissue of the rats were observed and the contents of the radon daughter - 210Po in rats were determined. (5) Two-dimensional electrophoresis (2-DE) was used to separate proteins from rats. (6) The differential expression proteins were identified by MALDI-TOF MS or MALDI-TOF-TOF MS. The results were searched in NCBInr database. (7) The differential expression proteins of RAGE, Trx and S100A6 were verified by immunohistochemistry and western blot.
Results: (1) Pathological changes in the lung tissues were observed with findings of inflammatory cell penetrated into mucous membrane, pulmonary edema, hyperaemia in the alveolus interval, fibrosis and so on. The contents of 210Po in rats exposed to radon increased with the exposure dose. (2) 2-DE technique resulted in 450 proteins at condition of pH3~10, 11cm IPG strip, 625 proteins at pH3~10, 24cm IPG strip and 1248 proteins at pH4~7, 24cm IPG strip which was chosen for later experiment. (3) 18 differential expression proteins were found in the lung of rats exposed to cigarette smoke, with 10 up and 8 down-regulated expression proteins. 15 differential expression proteins were identified in the lung of rats exposed to radon, with 9 up and 6 down-regulated expression proteins. 20 differential expression proteins were identified in the lung of rats exposed to cigarette smoke and radon, with 12 up and 8 down-regulated expression proteins. These proteins were functioned in energy metabolism, proliferation, antioxidant, signal transduction, cytoskeleton, and so on. (4) The proteins of RAGE, Trx and S100A6 were confirmed by immunohistochemistry and western blot, which were consistent with the 2-DE results.
Conclusions: (1) The animal models of cigarette smoke and radon exposure were set up and a 2-DE proteomic approach was established. (2) Differential expression proteins were identified with functions involved in energy metabolism, proliferation, antioxidant, signal transduction and cytoskeleton, indicating a multistep process in response to cigarette smoke and radon exposure. (3) Proteins of RAGE, Trx and S100A6 could be applied as potential molecular markers in evaluating adverse effects induced by cigarette smoke and radon exposure.
方法:(1)采用动式烟雾染毒装置对Wistar大鼠进行香烟烟雾染毒,装置内烟雾浓度控制在(20±5)%,氧气浓度控制在21%,每天染毒2次,每次30min,分别染毒1、2、4个月作为低、中、高剂量组,每组5只动物。(2)采用HD-3型多功能移动式氡室对Wistar大鼠动态吸入氡染毒,通过计算机控制装置内氡浓度,使其剂量率恒定保持在1×105Bq/m3,每天染毒16h。氡及其子体累积染毒剂量分别达100、200、400工作水平月(working level month,WLM)作为低、中、高剂量组,对照组室内氡本底浓度80Bq/m3,每组5只动物。(3)采用上述方法对Wistar大鼠进行香烟烟雾和氡联合交替染毒,使香烟烟雾和氡染毒分别达2个月和200WLM作为联合染毒组。(4)观察染毒模型大鼠肺组织的病理学改变和测定氡染毒大鼠模型体内氡子体210Po的分布。(5)建立双向电泳(2-DE)分离大鼠肺组织总蛋白的技术,提高方法的灵敏度、分离度和重现性。(6)染毒后分别提取大鼠肺组织的总蛋白,采用2-DE分离,考马斯亮蓝染色,差异表达点进行MALDI-TOF或MALDI-TOF-TOF质谱分析,NCBInr数据库搜索鉴定分析结果。(7)免疫组化方法分别验证质谱鉴定出的差异表达蛋白晚期糖基化终产物受体(RAGE)和硫氧还蛋白(Trx)。以GAPDH为内参,运用Western blot法分别验证RAGE、Trx和S100A6蛋白在不同染毒组大鼠肺组织中的表达,以Bandscan4.3软件进行灰度分析并进行统计学检验。(8)测定氡染毒大鼠肺组织中细胞色素C氧化酶(CcO)的活性。
结果:(1)建立了香烟烟雾、氡单独和联合染毒Wistar大鼠模型。HE和Masson染色结果提示各染毒组大鼠肺组织出现了支气管炎症、炎症细胞浸润、肺气肿、胶原纤维化等病变,病变程度随香烟烟雾、氡染毒剂量的加大而加重。染氡组大鼠体内组织器官中氡子体210Po的含量随氡暴露剂量加大而增加。(2)建立了大鼠肺组织总蛋白的2-DE分离技术。银染法中,pH3~10,11cm的胶条分离到约450个蛋白质点,pH3~10,24cm的胶条分离到约625个蛋白质点,pH4~7,24cm的胶条分离到约1248个蛋白质点。确定肺组织蛋白2-DE实验采用pH4~7,24cm的胶条进行分离,并获得较好的分离效果和重复性。(3)各染毒组大鼠肺组织总蛋白经2-DE分离、MS分析,香烟烟雾染毒组共鉴定出18种差异表达蛋白,包括10个上调和8个下调的蛋白;氡染毒组共鉴定出15种差异表达蛋白,包括9个上调和6个下调的蛋白;香烟烟雾与氡联合染毒组共鉴定出20种差异表达蛋白,包括12个上调和8个下调的蛋白。差异蛋白多数与机体代谢、细胞存活与增殖、氧化还原、信号传导、细胞骨架等功能有关。(4)免疫组化结果表明RAGE、Trx和S100A6在不同染毒各剂量组大鼠肺组织中表达随染毒剂量增加而升高;Western blot分析进一步验证了免疫组化结果;氡染毒大鼠肺组织中CcO的活性随染氡剂量的增加而降低,验证结果均与2-DE检测吻合。
结论:(1)通过病理组织学和大鼠体内氡子体210Po的测定,成功建立了香烟烟雾与氡单独和联合吸入染毒的大鼠模型。在优化和发展传统双向凝胶电泳技术、比较蛋白质组方法的基础上,建立了蛋白质组提取-双向凝胶电泳分离-半自动胶内酶解-质谱鉴定的高通量技术分析平台。(2)获得了香烟烟雾与氡染毒大鼠肺组织的蛋白质表达谱及两者单独和联合染毒相关的差异表达蛋白质,初步鉴定出的差异蛋白质数为:香烟烟雾染毒18个,氡染毒组15个,联合染毒组20个。这些蛋白分别与代谢、细胞存活与增殖、氧化还原、信号传导、细胞骨架等功能相关,提示香烟烟雾与氡染毒致肺损伤是多种机制共同作用的结果。(3)应用免疫组化和Western blot等方法对香烟烟雾与氡染毒大鼠肺组织中差异表达蛋白Trx、RAGE、S100A6和CcO的表达水平进行了验证,结果与蛋白质组学检测一致,提示这些蛋白可作为香烟烟雾和氡致肺损伤的蛋白分子标志物。
Objective: Proteomic analysis of the rat lung tissues was conducted upon exposure to cigarette smoke and radon to provide experimental data for evaluating health risks and exploring potential molecular markers.
Methods: (1) Wistar rats were exposed to cigarette smoke in a gas inhalation chamber with the smoke concentration controlled at the range of (20±5)% and oxygen concentration at 21%, twice a day, 30min each at low, middle and high dose for 1, 2 and 4 months with 5 per group. (2) Wistar rats were exposed to radon with a multifunction ecological radon room at a constant level of 1×105Bq/m3. The low, middle and high dose groups were exposed up to a cumulative dose of 100, 200 and 400WLM (working level months) respectively. The control rats were housed in a room with a background concentration of radon at 80 Bq/m3. (3) Wistar rats were exposed to radon for up to cumulative dose of 200 WLM and cigarette smoke for 2 months in the same conditions as (1) and (2) as the joint group. (4) Pathological changes in lung tissue of the rats were observed and the contents of the radon daughter - 210Po in rats were determined. (5) Two-dimensional electrophoresis (2-DE) was used to separate proteins from rats. (6) The differential expression proteins were identified by MALDI-TOF MS or MALDI-TOF-TOF MS. The results were searched in NCBInr database. (7) The differential expression proteins of RAGE, Trx and S100A6 were verified by immunohistochemistry and western blot.
Results: (1) Pathological changes in the lung tissues were observed with findings of inflammatory cell penetrated into mucous membrane, pulmonary edema, hyperaemia in the alveolus interval, fibrosis and so on. The contents of 210Po in rats exposed to radon increased with the exposure dose. (2) 2-DE technique resulted in 450 proteins at condition of pH3~10, 11cm IPG strip, 625 proteins at pH3~10, 24cm IPG strip and 1248 proteins at pH4~7, 24cm IPG strip which was chosen for later experiment. (3) 18 differential expression proteins were found in the lung of rats exposed to cigarette smoke, with 10 up and 8 down-regulated expression proteins. 15 differential expression proteins were identified in the lung of rats exposed to radon, with 9 up and 6 down-regulated expression proteins. 20 differential expression proteins were identified in the lung of rats exposed to cigarette smoke and radon, with 12 up and 8 down-regulated expression proteins. These proteins were functioned in energy metabolism, proliferation, antioxidant, signal transduction, cytoskeleton, and so on. (4) The proteins of RAGE, Trx and S100A6 were confirmed by immunohistochemistry and western blot, which were consistent with the 2-DE results.
Conclusions: (1) The animal models of cigarette smoke and radon exposure were set up and a 2-DE proteomic approach was established. (2) Differential expression proteins were identified with functions involved in energy metabolism, proliferation, antioxidant, signal transduction and cytoskeleton, indicating a multistep process in response to cigarette smoke and radon exposure. (3) Proteins of RAGE, Trx and S100A6 could be applied as potential molecular markers in evaluating adverse effects induced by cigarette smoke and radon exposure.
引文
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