摘要目的 探讨不同EPHX1基因型慢阻肺患者外周血淋巴细胞中mRNA和蛋白的表达水平及外周血抗氧化能力指标的差异.方法 采用病例对照研究的方法,收集昆明医科大学第一附属医院2016年10月至2018年2月门诊和既往住院的220例有吸烟史的稳定期慢阻肺患者(慢阻肺组)及230名健康吸烟者(对照组)外周血,按照BigDye Terminator v1.1 DNA测序试剂盒操作说明进行基因检测.按不同的EPHX1 exon3和exon4的基因多态性可以将EPHX1分为正常活性、慢活性、极慢活性和快活性四种基因活性型.慢阻肺患者按EPHX1基因型及基因活性分为慢活性组(慢及极慢基因活性)和快活性组(正常及快基因活性).采用qRT?PCR以及western blot检测各组外周血淋巴细胞EPHX1 mRNA和蛋白表达水平,并用相应试剂盒分别测定血清各项抗氧化能力指标.结果(1)对照组的2-△△Ct为1.000,慢阻肺组的2-△△Ct为1.052±0.023,两组EPHX1基因mRNA表达水平差异无统计学意义(t=1.992,P=0.865).慢活性组EPHX1基因mRNA表达水平(1.053±0.023)与快活性组(1.048±0.021)比较,差异无统计学意义(t=1.133,P=0.260).(2)Western blot检测EPHX1蛋白表达水平结果表明,慢阻肺组与对照组EPHX1/GAPDH灰度比值(分别为0.613±0.089和0.602±0.075)差异无统计学意义(t=0.805,P=0.422);慢活性组EPHX1蛋白表达量与快活性组(分别为0.606±0.088和0.622± 0.092)相比,差异无统计学意义(t=-0.786 P=0.434).(3)对照组及慢阻肺组各项抗氧化能力指标差异有统计学意义(均P<0.05).慢活性组和快活性组慢阻肺患者的各项抗氧化能力指标差异有统计学意义(均P<0.05).结论 不同EPHX1基因型慢阻肺患者抗氧化能力的不同,可能与EPHX1基因多态性影响了微粒体环氧化水解酶的酶活性有关,而与EPHX1 mRNA和蛋白表达水平无关.

abstractsObjective To explore the difference of mRNA, protein expression levels and the indexes of peripheral blood antioxidant capacity in peripheral blood lymphocytes of different EPHX1 genotypes in chronic obstructive pulmonary disease(COPD). Methods A case?control study was conducted to collect peripheral blood samples of 220 stable chronic COPD patients with smoking history and 230 healthy smokers (control group) from October 2016 to February 2018 in the First Affiliated Hospital of Kunming Medical University, and the genetic testing was carried out according to the operation instructions of BigDye Terminator v1.1 DNA Sequencing Kit. Based on their EPHX1 exon 3 and exon 4 polymorphism status, the EPHX1 was classified into 4 groups, i. e., normal activity, slow activity, extremely slow activity and fast activity. Then COPD patients were allocated to either a slow activity group (slow and very slow activity) or a fast activity group (normal and fast activity) according to EPHX1 genotype and gene activity. The expression of EPHX1 mRNA and protein in peripheral blood lymphocytes were detected by qRT?PCR and Western blot, and indexes of serum antioxidant capacity was detected by corresponding kits. Results (1)The 2-ΔΔCt of the control group was 1.000, and the 2-ΔΔCt of the COPD group was 1.052±0.023. There was no significant difference in the level of EPHX1 mRNA expression between the two groups (t=1.992 P=0.865). The level of EPHX1 mRNA expression in the slow activity group was not different significantly compared to that in the fast?active group (1.053 ± 0.023 vs 1.048 ± 0.021, t=1.133, P=0.260). (2)The level of EPHX1 protein expression by Western blot analysis showed that the EHPX1/GAPDH gray ratio was not different significantly between the COPD group and the control group (0.613 ± 0.089 vs 0.602 ± 0.075, t=0.805, P=0.422). The level of EPHX1 protein expression in the slow activity group was not significantly different compared to that in the fast activity group (0.606±0.088 vs 0.622±0.092, t=-0.786 P=0.434). (3)There were significant differences in indexes of antioxidant capacity between the control group and the COPD group (P<0.05). There were significant differences in indexes of antioxidant capacity between the slow activity group and the fast activity group of COPD patients (P<0.05). Conclusions The different antioxidant capacity of COPD patients with different EPHX1 genotypes may be related to the polymorphism of EPHX1 gene affecting the activity of microsomal epoxidase, but not to the level of EPHX1 mRNA and protein expression.