ID-LC-MS/MS方法对高糖血症SD大鼠肾组织DNA中8-oxo-dGsn的检测  

Determination of the Oxidative Damage Production of DNA( 8- oxo- dGsn) in SD Rats Kidney of Hyperglycemia by ID- LC- MS /MS

作　　者：罗顺斌[1,2] 夏梦明[1,2] 邱相君[3] 孙未[4] 王哲[4] 胡国新[1,2] 蔡剑平[1] 

机构地区：[1]温州医科大学药学院,325035 [2]温州医科大学药学院药理教研室,325035 [3]河南省河南科技大学医学院,471003 [4]温州医科大学附属第二医院,325027

出　　处：《医学研究杂志》2014年第4期32-37,共6页Journal of Medical Research

基　　金：国家自然科学基金资助项目(81171028;81341023)

摘　　要：目的用同位素稀释高效液相-串联质谱法(Isotope-Diluted LC-MS/MS,ID-LC-MS/MS)和免疫组化法检测高糖血症SD大鼠肾组织DNA氧化产物8-羟基脱氧鸟苷(8-oxo-dGsn)的含量,评价高糖血症对大鼠肾组织DNA氧化损伤的影响。方法健康雄性SD(Sprague-Dawley rats)大鼠28只,随机分为4组:3月糖尿病模型组(3DR组)及其对照组(3CR组),6月糖尿病模型组(6DR组)及其对照组(6CR组)。糖尿病模型组接受腹腔单次链脲佐菌素(streptozotosin,STZ)每公斤70mg注射,对照组接受腹腔生理盐水注射。糖尿病模型组造模成功3个月及6个月后,分别抽提大鼠肾组织DNA,用ID-LC-MS/MS方法检测其DNA氧化产物8-oxo-dGsn的含量;用免疫组化法观察肾组织中DNA氧化损伤部位及程度。结果糖尿病模型组造模成功3个月时肾组织DNA中8-oxo-dGsn高于对照组高,造模成功6个月时肾组织DNA中8-oxodGsn含量比正常对照组增加显著(P<0.01);免疫组化结果显示3个月模型组DNA氧化损伤水平高于对照组,6个月时模型组DNA氧化损伤更为严重。结论高糖血症3个月可造成大鼠肾组织DNA的氧化损伤,高糖血症6个月时大鼠肾组织中DNA氧化损伤程度呈显著增加,高糖血症是肾组织中DNA氧化损伤的重要原因。Objective To evaluate the oxidative damage production of DNA （8-oxo-dGsn） in SD rats kidney induced by i.p.injection of STZ through ID-LC-MS/MS and Immunohistochemistry method.Methods Rats aged 2 months were randomly assigned to three months （start from injection） of control group,three months of diabetic group,six months of control group,six months of diabetic group （3CR,3DR,6CR,6DR,respectively） receiving an intraperitoneal （i.p.） saline injection （n =8） or a single i.p.injection of STZ （70mg/kg body weight; n =20）.The content and degree of the 8-oxo-dGsn are determinated by ID-LC-MS/MS and Immunohistochemistry,respectively.Results The levels of 8-oxo-dGsn in the diabetes group were generally higher than those in the control group,and there was a significant difference between 6DR and 6CR （P ＜ 0.01）.The results of the immunohistochemistry of 8-oxo-dG-sn showed that a relative apparent oxidative damage of DNA had been observed in 3DR,while it was more serious in 6DR than the others （3CR,3DR,6CR）.Conclusion The oxidative damage to DNA in the kidney occurred in hyperglycemia state for 3 month,while it became more serious for 6 month.So hyperglycemia may be a major factor which cause oxidative damage to DNA in kidney.

关 键 词：同位素稀释高效液相-串联质谱法(ID-LC-MS MS) 高糖血症 DNA氧化损伤 8-oxo-dGsn 

分 类 号：R318[医药卫生—生物医学工程]