缺血性脑卒中患者外周血单个核细胞DNA氧化损伤及修复

DNA damage and repair of mononuclear cells in the peripheral blood of patients with ischemic stroke

目的检测缺血性脑卒中患者急性期和恢复期外周血单个核细胞的DNA氧化损伤及修复情况,与健康者进行比较,以判断缺血性脑卒中患者DNA氧化损伤水平的变化以及DNA氧化损伤水平与病程的关系。方法急性缺血性脑卒中患者36例,于发病3 d内抽取空腹静脉血10 mL进行8-羟基-2’-脱氧鸟嘌呤核苷(8-OhdG)、神经特异性烯醇化酶(NSE)及单个核细胞的彗星率及彗尾DNA百分比的检测,于病程18～21 d抽血2 mL再检测单个核细胞的彗星率及彗尾DNA百分比。同时选取医院门诊体检人群中与研究对象年龄、性别相匹配的健康者20例作为对照。10 mL空腹静脉血中,5 mL分离外周血单个核细胞,提取DNA,将DNA酶解,高效液相色谱—电化学检测方法检测8-OhdG；3 mL双抗体夹心放射免疫法检测血清中NSE；2 mL分离外周血单个核细胞,应用碱性单细胞凝胶电泳的方法检测单个核细胞的彗星率及彗尾DNA百分比,同时对缺血性脑卒中患者的神经功能缺损评分进行比较。结果缺血性脑卒中患者急性期外周血单个核细胞的8-OhdG和NSE含量均较对照组明显升高(P＜0．01),急性期患者外周血单个核细胞彗星率为90．13%±2．22%,彗尾DNA百分比为60．71%±4．18%。较恢复期患者的细胞彗星率60．89%±2．98%、彗尾DNA百分比39．20%±3．61%明显增高(P＜0．01)；恢复期患者神经功能缺损评分较急性期患者明显下降(P＜0．01)。对照组血液中单个核细胞彗星率为6．4%±1．61%,彗尾DNA百分比3．37%±0．63%,较缺血性脑卒中患者明显减少(P＜0．01)。结论缺血性脑卒中患者急性期和恢复期外周血单个核细胞存在DNA氧化损伤。

Objective To investigate DNA damage and repair of mononuclear cells in the peripheral blood of patients with ischemic stroke and to evaluate the relationship between the status of DNA damage and disease course. Methods Bloods from 36 patients were collceted to detect 8-OhdC, NSE, comet cell and tail DNA before treatment. 20 normal healthy counterparts were enrolled as control. （1） DNA of mononuclear cells in the peripherical blood was isolated to detect 8-OhdG by high performance liquid chromatography with electrochemical detection; （2） Serum was isolated to detect NSE by ELISA; （3） Bloods from patients were collceted to detect comet cell and tail DNA by single cell gel electrophoresis assay, and neurological score was assessed. Results （1） A significantly higher concentration of 8-OhdG in lymphocytes was detected in acute ischemic stroke patients when compared with control; （2） The status of neuronal specific enolase in the peripheral blood of patients with ischemic stroke was much higher than that of control; （3） The perccntages of comet cells（90.13%±2.22%） and tail DNA（60.71%±4.18%） in acute patients with ischemic stroke were significantly higher than those in convalescence patients（60.89%±2.98% and 39.2%±3.61%, respectively）. Meanwhile, neurological score in acute patients was significantly higher than that in convalescence patients. The percentages of comet cells（6.4%±1.61%） and tail DNA（3.37%±0.63%） in normal control were significantly lower than those in patients with ischcmic stroke. Conclusion DNA damage exists in mononuclear cells of the peripherical blood of acute and convalescence patients with ischemic stroke.