长波和中波紫外线对HaCaT细胞DNA损伤拉曼光谱对比研究  

Comparative study on Raman spectroscopy of DNA damage of HaCaT cell induced by ultraviolet A and ultraviolet B irradiation

作　　者：刘明[1,2] 潘燕婷[3] 王颖慧[1] 周美娟[1] 黄耀熊[3] 陈建雄[2] 丁振华[1] 

机构地区：[1]南方医科大学,广东广州510515 [2]广东省职业病防治院,广东省职业病防治重点实验室,510310 [3]暨南大学生物医学工程系,510632

出　　处：《中国职业医学》2014年第6期609-614,共6页China Occupational Medicine

基　　金：国家自然科学基金(81172634;81202152);广东省职业病防治重点实验室(2012A061400007);广东省医学科研基金(B2014013)

摘　　要：目的通过分析长波紫外线（UVA）和中波紫外线（UVB）对HaCaT细胞作用后DNA拉曼光谱，从分子水平探索两者对HaCaT细胞DNA损伤作用的差异。方法取对数生长期的HaCaT细胞随机分为对照组、UVA组和UVB组。对照组3个样品，不进行紫外辐照（相当于照射前）；后2组分别于接受UVA和UVB照射（累计照射剂量分别为8．26×101^-2和2．97×101^-2／cm^2）后，培养至照后0．5、1．0、2．0、3．0、4．0和5．0h时收获细胞提取DNA，每个时间点3个样品。采用激光共焦拉曼光谱倒置显微镜系统记录各组HaCaT细胞全基因组DNA的拉曼光谱，对特征谱线进行归属，观察照射前后拉曼光谱特征谱线相对强度（In／Io）变化。结果鸟嘌呤氢键、磷酸基静电斥力、腺嘌呤碱基分子内能和腺嘌呤碱基堆积力特征频率分别是669、1094、1230和1344cm^-1。UVB组鸟嘌呤氢键、磷酸基静电斥力、腺嘌呤碱基分子内能的特征频率In/Io均小于UVA组（P〈0．01）；上述3个指标在紫外辐射类型和照后时间的交互效应上均有统计学意义（P〈0．01），UVB的损伤作用峰值均出现在照后1．0h。UVB组腺嘌呤碱基堆积力特征频率In/Io大于UVA组（P〈0．01）；紫外辐射类型和观察时间的交互效应有统计学意义（P〈0．01），UVB的损伤作用峰值出现在照后3．0h。结论UVB的损伤作用对HaCaT细胞DNA的鸟嘌呤氢键、磷酸基静电斥力、腺嘌呤碱基分子内能和腺嘌呤碱基堆积力的损伤作用比UVA大，UVB的损伤作用峰值出现在照后1．0~3．0h。Objective To explore the effect of DNA damage of HaCaT cells induced by ultra-violet A （UVA） and ultraviolet B （UVB） on molecular level by analyzing the differences of Raman spectroscopy. Methods HaCaT cells cultured in complete medium were divided into control, UVA and UVB groups when they were in logarithmic growth phase. The 3 samples of control group were not exposed to ultraviolet （equivalent to non-irradiation）. Cells of UVA and UVB groups were cultured in complete medium again after exposure to UVA and UVB irradiation with accumulative doses of 8.26 × 10^-2 and 2.97 × 10^ -2 j/cm^2 respectively, and then harvested at the 0.5th, 1.0th, 2.0nd, 3.0rd, 4.0th and 5.0th hour points with 3 samples at each time. Total DNA of HaCaT cell samples was extracted. Laser confocal Raman spectrum inverted microscope was applied to scan DNA and draft Raman spectra based on the reported frequencies. The effects of DNA damage induced by UVA and UVB via relative change of Raman intensity （In/Io） were explored. Results The characteristic frequencies of Guanine hydrogen bond, DNA electrostatic repulsion of phosphate, adenine base molecular energy and adenine base stacking were 669, 1 094, 1 230 and 1 344 cm^-1 respectively. The characteristic frequency In/Io of guanine hydrogen bond, DNA electrostatic repulsion of phosphate and adenine base molecular energy in UVB group were significantly lower than those of UVA group respectively （ P 〈 0. 01 ）, and the interaction effects of the above 3. ind exesexisted between UV radiation type and observation time respectively （ P 〈 0. 01 ） , the damage peaks of UVB were all in 1.0 hour after irradiation. Adenine base stacking bands intensity characteristic frequency In/Io in UVB group was significantly stronger then that of UVA group （ P 〈 0. 01 ）, and interaction effect existed between UV radiation type and observation time （P 〈 0. 01 ） , the damage peak of UVB was in 3.0 hours after irradiation. Conclusion Compared with UVA, UVB was much harmful to

关 键 词：紫外辐射 长波紫外线 中波紫外线 DNA损伤 拉曼光谱 HACAT细胞 

分 类 号：R114[医药卫生—卫生毒理学] R122.4[医药卫生—公共卫生与预防医学]