CN105861617A - 卷烟烟气有害成分诱导细胞氧化应激ros的测定方法 - Google Patents
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Abstract
本发明涉及卷烟烟气的体外毒理学研究,具体是一种卷烟烟气有害成分诱导细胞氧化应激ROS的测定方法,其特征在于:是利用荧光探针2ˊ,7ˊ‑二氢二氯荧光素二乙酸酯(DCFH‑DA)检测细胞氧化损伤后胞内活性氧ROS水平,包括以下具体步骤:1)配置实验试剂;2)细胞接种培养;3)、DCFH‑DA探针装载;4)、卷烟烟气有害成分染毒;5)、ROS检测。本发明的最大特点是可快速、灵敏地检测卷烟烟气有害成分诱导的细胞氧化损伤程度,对研究卷烟烟气有害成分对于诱导细胞内活性氧ROS的影响有非常重要的意义。
Description
技术领域
本发明涉及到卷烟烟气有害成分的体外毒理学研究,具体来说是一种检测卷烟烟气有害成分诱导细胞氧化损伤后胞内活性氧(ROS)的测定方法。
背景技术
卷烟烟气是一种复杂的气溶胶,烟气中的化学成分多达8000余种,同时包含有1014-1016个自由基,具有氧化性。卷烟烟气中氧化性物质会诱导细胞内活性氧(ROS)的增加,诱导机体产生的ROS会对细胞内蛋白质、脂质和遗传物质(DNA)等造成损伤。因此,研究卷烟烟气有害成分对于诱导细胞内活性氧ROS的影响将具有十分重要的意义。
发明内容
本发明的目的正是基于上述状况而提供的一种检测卷烟烟气有害成分诱导细胞氧化损伤后胞内活性氧(ROS)的测定方法。应用建立的方法可以对卷烟烟气中的有害成分进行体外毒性测试,评价其诱导细胞氧化损伤的程度,进而从化合物水平评价卷烟烟气的毒性影响,可以获得不同有害成分的毒性差异数据。为烟草制品的健康风险评估研究提供方法基础。
本方法依据的发明机理在于:利用荧光探针2ˊ,7ˊ-二氢二氯荧光素二乙酸酯(DCFH-DA)检测细胞内ROS,DCFH-DA作为一种非极性化合物,能够迅速扩散到细胞内,细胞内的酯酶将其水解为非荧光的二氯荧光素(DCFH),从而使探针被装载到细胞内,在ROS存在的条件下,DCFH被氧化生成荧光物质DCF,发出绿色荧光,荧光强度与细胞内活性氧水平成正比。
本发明的目的是通过一下技术方案来实现的:
一种卷烟烟气有害成分诱导细胞氧化应激ROS的测定方法,是利用荧光探针2ˊ,7ˊ-二氢二氯荧光素二乙酸酯(DCFH-DA)检测细胞氧化损伤后胞内活性氧ROS水平,包括以下具体步骤:
1)配置实验试剂:(1)生长培养液,(2)无血清培养液,(3)荧光探针DCFH-DA,(4) 磷酸缓冲盐溶液PBS;
2)细胞接种培养:经扩增培养的人肺腺癌细胞A549或人肝癌细胞HepG2经胰酶消化后,制备细胞悬液1.5×105 个/ml,96孔板每孔加入100 μl细胞悬液,于37 ℃、5% CO2条件下培养24 h;
3)、DCFH-DA探针装载:96孔板中的细胞培养24h后,弃去培养基,无血清培养液洗涤1次,每孔加入100 μl含有10μM DCFH-DA的无血清培养液,在37℃, 5%CO2条件下孵育1 h,弃去上清并用无血清培养液洗涤三次,以充分除去多余探针;
4)、卷烟烟气有害成分染毒:使用无血清培养液将卷烟烟气有害成分纯品母液稀释到不同浓度,设置至少两个非零浓度使细胞存活率在80%以上,染毒2 h;
5)、ROS检测:细胞染毒2 h后,弃去上清,每孔加入PBS洗涤两次,在488 nm激发波长,525 nm发射波长处,用荧光酶标仪测定荧光强度。
在本发明中,各实验试剂的成分及配制方法如下:
(1)生长培养液:RPMI-1640 + 10%胎牛血清 + 2 mM L-谷氨酰胺 + 100 IU/ml青霉素+ 100 μg/ml 链霉素;
(2)无血清培养液:RPMI-1640 + 2 mM L-谷氨酰胺 + 100 IU/ml青霉素 + 100 μg/ml链霉素;
(3)荧光探针DCFH-DA:用二甲基亚砜(DMSO)溶解DCFH-DA成10 mM的母液,使用时用无血清培养基稀释成10 μM的工作液;
(4)磷酸缓冲盐溶液(PBS):0.2 g KCl,0.2 g KH2PO4,8 g NaCl,2.9 g Na2HPO4·12H2O,加双蒸水至1 L,pH 7.2~7.4,高压灭菌。
所述卷烟烟气有害成分具体是指2–氨基–9–氢–吡啶并[2,3–b]吲哚(AαC)。
所述胰酶具体为胰蛋白酶。
本发明的最大特点是可快速、灵敏地检测卷烟烟气有害成分诱导的细胞氧化损伤程度,应用建立的方法对卷烟烟气中的有害成分进行体外毒性测试,评价其诱导细胞氧化损伤的程度,从单一化合物水平评价卷烟烟气体外毒性的化学物质基础,可以获得不同有害成分的毒性数据。有助于更好地解释卷烟烟气的毒性机制,为烟草制品的健康风险评估研究提供方法基础和技术支撑。同时,建立的方法适用性广,可应用于不同细胞的体外试验,测试结果重复性好,稳定性高,特异性强。
具体实施方案
本发明以下结合具体实施例作进一步描述:
实施例1
将A549细胞以1.5×104个/孔的密度接种于96孔板,于37℃、5%CO2条件下培养24h后,弃去培养基,每孔加入100 μl含有10 μM DCFH-DA的无血清培养液,于37℃、5%CO2条件下孵育1 h,弃去上清并用无血清培养液洗涤三次,以去除多余探针。使用无血清培养液稀释卷烟烟气有害成分AαC母液,设置染毒浓度分别为5、10、20和30 μg /ml,对照组均加入0.1%DMSO的无血清培养液,染毒2 h。弃去上清,加入PBS洗涤两次,在488 nm激发波长,525 nm发射波长处,用荧光酶标仪测定荧光强度。检测结果(表1)显示,与对照组细胞相比,烟气有害成分AαC在20-30 μg/ml浓度范围内,A549 细胞内ROS水平显著增加。
表1 烟气有害成分AαC对A549细胞中ROS的影响
AαC (μg/mL) | 荧光强度 |
0 | 19.70±1.15 |
5 | 23.14±3.09 |
10 | 25.50±4.03 |
20 | 30.41±1.27** |
30 | 34.29±3.88** |
** P﹤0.01
实施例2
将HepG2细胞以1.5×104个/孔的密度接种于96孔板,于37℃、5%CO2条件下培养24h后,弃去培养基,每孔加入100 μl含有10 μM DCFH-DA的无血清培养液,于37℃、5%CO2条件下孵育1 h,弃去上清并用无血清培养液洗涤三次,以去除多余探针。使用无血清培养液稀释卷烟烟气有害成分AαC母液,设置染毒浓度分别为5、10、15和20 μg /ml,对照组均加入0.1% DMSO的无血清培养液,染毒2 h。弃去上清,加入PBS洗涤两次,在488 nm激发波长,525nm发射波长处,用荧光酶标仪测定荧光强度。检测结果(表2)显示,与对照组细胞相比,烟气有害成分AαC在10-20 μg/ml浓度范围内,HepG2细胞内ROS水平显著增加。
表2 烟气有害成分AαC对HepG2细胞中ROS的影响
AαC (μg/mL) | 荧光强度 |
0 | 24.88±2.35 |
5 | 30.24±5.89 |
10 | 37.63±3.56* |
15 | 45.41±6.32** |
20 | 49.65±3.93** |
* P﹤0.05,** P﹤0.01。
Claims (4)
1.一种卷烟烟气有害成分诱导细胞氧化应激ROS的测定方法,其特征在于:是利用荧光探针2ˊ,7ˊ-二氢二氯荧光素二乙酸酯(DCFH-DA)检测细胞氧化损伤后胞内活性氧ROS水平,包括以下具体步骤:
1)配置实验试剂:(1)生长培养液,(2)无血清培养液,(3)荧光探针DCFH-DA,(4) 磷酸缓冲盐溶液PBS;
2)细胞接种培养:经扩增培养的人肺腺癌细胞A549或人肝癌细胞HepG2经胰酶消化后,制备细胞悬液1.5×105 个/ml,96孔板每孔加入100 μl细胞悬液,于37 ℃、5% CO2条件下培养24 h;
3)、DCFH-DA探针装载:96孔板中的细胞培养24h后,弃去培养基,无血清培养液洗涤1次,每孔加入100 μl含有10μMDCFH-DA的无血清培养液,在37℃, 5%CO2条件下孵育1 h,弃去上清并用无血清培养液洗涤三次,以充分除去多余探针;
4)、卷烟烟气有害成分染毒:使用无血清培养液将卷烟烟气有害成分纯品母液稀释到不同浓度,设置至少两个非零浓度使细胞存活率在80%以上,染毒2 h;
5)、ROS检测:细胞染毒2 h后,弃去上清,每孔加入PBS洗涤两次,在488 nm激发波长,525 nm发射波长处,用荧光酶标仪测定荧光强度。
2.根据权利要求1所述的测定方法,其特征在于:各实验试剂的成分及配制方法如下:
(1) 生长培养液:RPMI-1640 + 10%胎牛血清 + 2 mM L-谷氨酰胺 + 100 IU/ml青霉素 + 100 μg/ml 链霉素;
(2) 无血清培养液:RPMI-1640 + 2 mM L-谷氨酰胺 + 100 IU/ml青霉素 + 100 μg/ml 链霉素;
(3) 荧光探针DCFH-DA:用二甲基亚砜DMSO溶解DCFH-DA成10 mM的母液,使用时用无血清培养基稀释成10 μM的工作液;
(4) 磷酸缓冲盐溶液PBS:0.2 g KCl,0.2 g KH2PO4,8 g NaCl,2.9 g Na2HPO4·12H2O,加双蒸水至1 L,pH 7.2~7.4,高压灭菌。
3.根据权利要求1所述的测定方法,其特征在于:使用无血清培养液将卷烟烟气有害成分纯品母液分别稀释到5、10、15、20和30 μg /ml的浓度。
4.根据权利要求1所述的测定方法,其特征在于:所述卷烟烟气有害成分具体是指2–氨基–9–氢–吡啶并[2,3–b]吲哚(AαC)。
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