[0020] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.
[0021] The application principle of the present invention will be described in detail below with reference to the accompanying drawings.
[0022] like figure 1 As shown, the test method based on the EPHX1 genotype COPD response to NAC according to the embodiment of the present invention comprises the following steps:
[0023] S101: DNA is extracted by phenol/chloroform method, followed by polymerase chain reaction PCR amplification;
[0024] S102: Polyacrylamide electrophoresis, laser scanning automatic DNA sequencing, mEPH gene activity is divided into four types according to different gene polymorphisms of EPHX1, exon3 and exon4;
[0025] S103: COPD patients were divided into slow and very slow activity groups and fast and normal activity groups according to the activity of microsomal epoxide hydrolase EPHX1 gene. Both groups were treated with NAC 600 mg twice a day for one year. , 24, 36, 48 weekend follow-up, FVC and FEV were measured on the follow-up day 1 , completed SGRQ and 6MWT at 24 and 48 weeks, and recorded the number of acute exacerbations of COPD in one year;
[0026] S104: Quanto software was used to calculate the sample size, and SPSS 20.0 statistical software was used for statistical analysis of the data.
[0027] The application principle of the present invention will be further described below in conjunction with experiments.
[0028] 1 Materials and methods
[0029] 1.1 Research objects
[0030] The selected cases were all 219 stable COPD patients with a smoking history who were outpatients and inpatients in the Department of Intensive Respiratory Medicine, The First Affiliated Hospital of Kunming Medical University from June 2012 to December 2013. 2011 GOLD guidelines), 223 age-sex-matched healthy smokers from the Physical Examination Center of the First Affiliated Hospital of Kunming Medical University were the control group. Inclusion criteria: (1) FEV 1 /FVC<70%; (2) FEV after inhalation of 400μg salbutamol for 30min 1 % of predicted rise <15% or FEV 1 Increase in absolute value <0.2L; (3) FEV after bronchodilator use 1 <80% predicted value; (4) age ≥ 60 years old. (5) Course of disease ≥ 5 years. Exclusion criteria: (1) bronchial asthma; (2) excluding other chronic bronchopulmonary diseases and allergic diseases; (3) having a history of upper respiratory tract infection within 6 weeks before selection or having upper respiratory tract infection during the 2-week screening period; (4) Patients with a history of pulmonary surgery, malignant tumors, or severe insufficiency of other organs; (5) patients with severe hypoxemia who cannot cooperate with the test.
[0031] All the tested COPD patients did not change the original treatment regimen (including inhaled LABA and hormones). Approved by the hospital ethics committee, all selected patients filled out the informed consent form.
[0032] 1.2 EPHX1 gene activity typing
[0033] DNA was extracted from the peripheral blood of 219 COPD patients and 223 healthy smokers by the phenol/chloroform method, followed by polymerase chain reaction (PCR) amplification: mEPH exon3 PCR amplification upstream primer (5'-GAAACTGCCTTGCCACTC-3') and Downstream primer (5'-CCTGCCTAGCTCTAAAGATG-3'); mEPH exon4 PCR amplification upstream primer (5'-ACATCCACTTCATCCACGT-3') and downstream primer (5'-ATGCCTCTGAGAAGCCAT-3'). PCR reaction conditions: PCR reaction system 20 μl, genomic DNA 100 ng primers, 200 mmol/L dNTP, 1 U Taq DNA polymerase; PCR amplification conditions: pre-denaturation at 95 °C for 5 min, extension at 72 °C for 5 min, denaturation at 94 °C for 1 min, annealing at 63 °C for 1 min , extended at 72 °C for 1 min, a total of 35 cycles. (3) Polyacrylamide electrophoresis, laser scanning automatic DNA sequencing. According to Smith and Harrison [18]According to different EPHX1, exon3 and exon4 gene polymorphisms, mEPH gene activity can be divided into the following four types: normal activity: both exon3 and exon4 genotypes are homozygous wild type, or both exon3 and exon4 genotypes are heterozygous. Zygotype; fast activity: exon3 (homozygous wild type), exon4 at least one mutation (heterozygous or homozygous mutant); slow activity: exon3 has only one slow allele (heterozygous); very slow Alleles: Both exon3 are slow alleles (homozygous mutants).
[0034] 1.3 Experimental Design
[0035] COPD patients were divided into Group 1 (slow and very slow activity) and Group 2 (fast and normal activity) according to the gene activity of microsomal epoxide hydrolase (EPHX1). Bang) orally for one year. Before the start of the test, the pulmonary function parameters were measured at baseline, and the St. George's Respiratory Disease Questionnaire (SGRQ) was used to investigate the health status of the patients and the 6-minute walk test (6MWT): after a 15-min rest, the patients were allowed to walk as fast as they could on the ground, and the 6MWT was recorded. Follow-up at the weekends of 12, 24, 36, 48, respectively, FVC and FEV were measured on the follow-up day 1 , completed SGRQ and 6MWT at 24 and 48 weeks. The number of exacerbations of COPD was recorded during the trial, and antibiotics and/or oral corticosteroids were required, or hospitalization was required. Adverse events were recorded.
[0036] 1.4 Pulmonary function test
