1. A platelet treatment guidance method based on drug gene polymorphism and thromboelastogram is characterized by comprising the following steps:

s1, drug gene detection: detecting gene polymorphic sites related to aspirin and clopidogrel by using PCR and Sanger sequencing technologies;

s2, whole blood genome DNA sequencing: taking 2ml of venous blood in an EDTA anticoagulant tube, extracting DNA by using a whole blood genome DNA extraction kit, carrying out PCR amplification on the extracted DNA, and finally sequencing the amplified product by using a sequencer;

s3, carrying out metabolic type classification: carrying out metabolic type classification according to genotypes, and classifying the metabolic types into strong metabolic types and weak metabolic types;

s4, TEG detection: elbow vein blood was analyzed using a TEG5000 type coagulation analyzer, and the platelet inhibition rate (AA%) induced by the AA pathway and the platelet inhibition rate (ADP%) induced by the ADP receptor pathway were measured within 2 h;

s5, result analysis: AA inhibition < 50%, defined as AR; ADP inhibition < 30%, defined as CR.

2. The method for guiding platelet therapy based on drug gene polymorphism and thromboelastogram as claimed in claim 1, wherein in S1, aspirin related gene detects COX-1 (A > G), ITGB3 (T > C) gene, wherein G, C is aspirin resistance related allele; the clopidogrel related gene detects CYP2C19 gene x 2 (681G/A), x 3 (636G/A), and x 17 (-806C/T).

3. The method for guiding platelet therapy based on drug gene polymorphism and thromboelastogram of claim 2, wherein in S2, the DNA is subjected to enzyme digestion reaction before DNA sequencing, and the conditions of the enzyme digestion reaction are as follows: denaturation: 94 ℃, 5min, 94 ℃, 30 s; annealing: 56 ℃ for 20 s; extension: 72 ℃ for 20 s;

for a total of 35 cycles, and a final extension at 72 ℃ for 5 min.

4. The method for guiding platelet therapy based on pharmacogenomic polymorphism and thromboelastogram according to claim 3, wherein the carrier of loss-of-function allele is a patient with at least 1 loss-of-function allele: 1/' 2, ' 1/' 3, ' 2/' 2, ' 2/' 3, ' 3/' 3, ' 2/' 17, or ' 3/' 17; non-carriers of loss-of-function alleles are patients without loss-of-function alleles: 1/' 1, ' 1/' 17, or ' 17/' 17.

5. The method for guiding platelet therapy based on drug gene polymorphism and thromboelastogram according to claim 4, wherein said S4 is classified into strong metabolic types according to CYP2C19 x 2, x 3 and x 17 genotypes: wild type homozygous subgroups CYP2C19 x 1/'1 fast metabolizing form, CYP2C19 x 1/' 17 ultrafast metabolizing form;

weak metabolic type: wild type and mutant gene heterozygous set: intermediate metabotropic, mutant gene homozygote or heterozygous subgroup of CYP2C19 x 1/'2 and x 1/' 3: slow metabolic forms of CYP2C19 x 2/' 2, 2/' 3 and 3/' 3.

6. The method for guiding platelet therapy based on drug gene polymorphism and thromboelastogram according to claim 1 or 5, wherein the coagulation analysis reagent comprises kaolin, an activator, arachidonic acid and adenosine diphosphate.

7. The method for guiding platelet therapy based on drug gene polymorphism and thromboelastogram of claim 6, wherein all data are processed by SPSS17.0 statistical software package.

8. The method for guiding platelet therapy based on drug gene polymorphism and thromboelastogram according to claim 7, wherein the measurement data conforms to the normal distribution adopted mean ± standard deviation () Expressing that the normal distribution is not met by adopting a median, comparing the mean between two groups by adopting an independent sample t test, expressing the number of cases and percentage of the counting data by adopting a chi-type comparison²And (6) checking.

9. The method of claim 8, wherein all the tests are bilateral tests, and the difference is statistically significant when P < 0.05.

Background

Patients with light ischemic stroke have a high short-term risk of subsequent stroke, and are highly likely to be disabled, especially in the first 90 days after stroke. The CHANCE study found that aspirin administered within 24h after a mild ischemic stroke or Transient Ischemic Attack (TIA) in combination with clopidogrel treatment for 21d reduced the risk of stroke recurrence by 32% compared to aspirin alone, and did not increase the bleeding complications. Secondary analysis of the POINT study also demonstrated that the benefit of reducing ischemic events with dual-antibody treatment was greatest at the first 21d after onset, while the benefit of continued dual-antibody treatment after 21d would be offset by increased bleeding risk. Therefore, the current domestic and foreign guidelines recommend that aspirin and clopidogrel double antiplatelet therapy be started as early as possible and maintained for 21 days for patients with non-cardiogenic light ischemic stroke who have not received alteplase intravenous thrombolysis. However, in the literature, 3% to 85% of patients who take aspirin and 4% to 30% of patients who take clopidogrel are reported to have the risk of cerebral stroke recurrence even if the patients regularly take the drugs for a long time, and the clinical prognosis is not improved, which may be related to the reduction of the reactivity of the patients to antiplatelet drugs. This phenomenon of decreased responsiveness of patients to antiplatelet drugs is known as "resistance" by scholars. Aspirin Resistance (AR), Clopidogrel Resistance (CR) are important causes for the prevention and treatment of partial ischemic stroke failure and will greatly increase the risk of recurrence of ischemic stroke. The mechanism of occurrence of AR and CR is relatively complex and not yet completely clear, and previous researches show that related gene polymorphism related to the metabolism and action process of antiplatelet drugs can be an important factor. At present, no individualized and standardized antiplatelet therapy system according to the state of Chinese people exists in China. The platelet inhibition effect varies from person to person, and the realization of individualized antiplatelet therapy is a problem which needs to be solved by research on antithrombotic therapy.

Therefore, we have proposed a platelet therapy guidance method based on drug gene polymorphism and thromboelastogram to solve the above problems.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a platelet treatment guidance method based on drug gene polymorphism and thromboelastogram.

The platelet treatment guidance method based on the drug gene polymorphism and the thromboelastogram comprises the following steps:

s1, drug gene detection: detecting gene polymorphic sites related to aspirin and clopidogrel by using PCR and Sanger sequencing technologies;

s2, whole blood genome DNA sequencing: taking 2ml of venous blood in an EDTA anticoagulant tube, extracting DNA by using a whole blood genome DNA extraction kit, carrying out PCR amplification on the extracted DNA, and finally sequencing the amplified product by using a sequencer;

s3, carrying out metabolic type classification: carrying out metabolic type classification according to genotypes, and classifying the metabolic types into strong metabolic types and weak metabolic types;

s4, TEG detection: elbow vein blood was analyzed using a TEG5000 type coagulation analyzer, and the platelet inhibition rate (AA%) induced by the AA pathway and the platelet inhibition rate (ADP%) induced by the ADP receptor pathway were measured within 2 h;

s5, result analysis: AA inhibition < 50%, defined as AR; ADP inhibition < 30%, defined as CR.

Preferably, in S1, the aspirin related gene detects COX-1 (A > G) and ITGB3 (T > C) genes, wherein G, C is aspirin resistance related allele; the clopidogrel related gene detects CYP2C19 gene x 2 (681G/A), x 3 (636G/A), and x 17 (-806C/T).

Preferably, in S2, the DNA is subjected to an enzyme digestion reaction before DNA sequencing, and the enzyme digestion reaction conditions are as follows: denaturation: 94 ℃, 5min, 94 ℃, 30 s; annealing: 56 ℃ for 20 s; extension: 72 ℃ for 20 s;

for a total of 35 cycles, and a final extension at 72 ℃ for 5 min.

Preferably, the loss-of-function allele carrier is a patient having at least 1 loss-of-function allele: 1/' 2, ' 1/' 3, ' 2/' 2, ' 2/' 3, ' 3/' 3, ' 2/' 17, or ' 3/' 17; non-carriers of loss-of-function alleles are patients without loss-of-function alleles: 1/' 1, ' 1/' 17, or ' 17/' 17.

Preferably, in S4, the metabotropic classification is performed according to CYP2C19 x 2, x 3 and x 17 genotypes, and is classified into strong metabotropic type: wild type homozygous subgroups CYP2C19 x 1/'1 fast metabolizing form, CYP2C19 x 1/' 17 ultrafast metabolizing form;

preferably, the weak metabotropic form: wild type and mutant gene heterozygous set: intermediate metabotropic, mutant gene homozygote or heterozygous subgroup of CYP2C19 x 1/'2 and x 1/' 3: slow metabolic forms of CYP2C19 x 2/' 2, 2/' 3 and 3/' 3.

Preferably, the coagulation assay reagent includes kaolin, an activator, arachidonic acid, and adenosine diphosphate.

Preferably, all data is processed using the SPSS17.0 statistical software package.

Preferably, the metrology data conforms to a normal distribution using the mean ± standard deviation of () Expressing that the average between two groups is compared by using independent sample t testThe counting data are expressed by number and percentage, and the comparison among groups adopts chi²And (6) checking.

Preferably, all tests are two-sided tests, with differences of P < 0.05 being statistically significant.

The invention has the beneficial effects that: the risk of new stroke within 90d can be effectively reduced without increasing the bleeding risk according to the drug gene polymorphism and TEG (TEG-mediated platelet resistance) for guiding the platelet treatment of light ischemic stroke. The gene detection and TEG can find patients insensitive to the treatment of the antiplatelet drugs in time, can provide a new basis for the selection of the antiplatelet drugs, so that the patients with ischemic stroke can select the antiplatelet drugs more accurately and reasonably, thereby formulating an individualized antiplatelet treatment scheme, effectively reducing the risk of stroke recurrence, having important guiding significance for the secondary prevention of ischemic stroke, and being worthy of clinical popularization and application.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples.

In the embodiment, clinical data of 250 acute light ischemic stroke patients receiving treatment in the affiliated Wujian hospital of Jiangsu university from 1 month in 2018 to 04 months in 2020 are selected and included into the standard, (1) the age is more than or equal to 18 years; (2) the first non-cardiogenic acute mild ischemic stroke is defined as sudden focal neurological dysfunction caused by vascular reasons, and the stroke scale score (NIHSS) of the American national institute of health is less than or equal to 3 points; (3) cranial CT or MRI excluded bleeding or confirmed new cerebral infarction; (4) the disease onset time is less than or equal to 48 hours, and antiplatelet drug treatment can be started within 48 hours after symptoms appear. Patients will be excluded from study participation if one of the following criteria is met: (1) bleeding or other conditions, such as vascular malformations, trauma, tumors, abscesses, neurodegenerative diseases, etc.; (2) acute ischemic cerebrovascular disease caused by non-atherosclerosis such as arteritis and hereditary acute ischemic cerebrovascular disease; (3) no evidence of acute infarction on CT or MRI; (4) has definite anticoagulation treatment indication (cardiac embolism such as atrial fibrillation or artificial heart valve); (5) clopidogrel or aspirin contraindications such as bleeding tendency, active peptic ulcer and recent active bleeding are presented; (6) patients with clinically unadaptable severe psychotic symptoms; (7) systemic infectious diseases, autoimmune diseases, severe heart, liver, kidney diseases; (8) there is history of intracranial hemorrhage, and there is history of alimentary tract hemorrhage or major operation in alimentary tract before 3 months; (9) administering a proton pump inhibitor; (10) the blood disease is combined, the platelet count is more than 450 multiplied by 10/L or less than 100 multiplied by 10/L or the hemoglobin is less than 80 g/L; (11) non-steroidal anti-inflammatory drugs or anti-platelet drugs except aspirin and clopidogrel are taken for a long time; (12) revascularization plans are possible within 3 months after screening; (13) there is a severe co-existence of non-cardiovascular diseases with a life expectancy of less than 3 months. All patients enrolled in the study were eligible for atherosclerotic acute ischemic stroke diagnosis, were outside the venous thrombolysis window, received no thrombolysis treatment, and were treated with clopidogrel and aspirin for antithrombotic treatment. The study was approved by the ethical committee of the institutional advancement hospital affiliated to Jiangsu university, and the patients participating in the study had complete clinical data, and all patients or guardians signed informed consent.

Random grouping and treatment: patients with acute mild ischemic stroke were diagnosed and randomly assigned to the test group or the control group at a ratio of 1:1 within 48h of the onset of symptoms. The test group selects anti-platelet treatment according to the detection result of the polymorphism of the gene 1d and the ADP and AA inhibition rate of TEG detection of the patient 8 d. The main objective was to assess the impact of both treatment regimens on the recurrence rate of stroke in 90d of patients with acute mild ischemic stroke. The selected drugs were aspirin (trade name: Aspirin B, Bayer pharmaceuticals, Inc.) and clopidogrel (trade name: Boravine, Xenofoamantant, Inc.).

Drug gene detection, namely detecting gene polymorphic sites related to aspirin and clopidogrel by PCR and Sanger sequencing technologies, wherein a PC instrument and a sequencer are purchased from Thermo Fisher Scientific company in the United states. After random grouping, 2ml of venous blood of a patient in a selected test group is immediately pumped into an EDTA anticoagulant tube, DNA is extracted according to the specification of a whole blood genome DNA extraction kit, PCR amplification is carried out on the extracted DNA according to the specification of a related gene detection kit (Shanghai Baiao Biotechnology Co., Ltd.), and finally sequencing is carried out on an amplification product by using a sequencer. The enzyme digestion reaction conditions are that denaturation: 94 ℃, 5min, 94 ℃, 30 s; annealing: 56 ℃ for 20 s; extension: 72 ℃ for 20 s. For a total of 35 cycles, and a final extension at 72 ℃ for 5 min. The amplification product was subjected to gel electrophoresis in 1.5% agarose, and the electrophoretic band was scanned using a gel imaging system and analyzed. Detecting COX-1 (A > G) and ITGB3 (T > C) genes by using aspirin related genes, wherein G, C is aspirin resistance related allele; detection of clopidogrel-related genes CYP2C19 gene x 2 (681G/a), x 3 (636G/a), x 17 (-806C/T), and loss of function allele carriers were patients with at least 1 loss of function allele (i.e.. x 2 or x 3): 1/' 2, ' 1/' 3, ' 2/' 2, ' 2/' 3, ' 3/' 3, ' 2/' 17, or ' 3/' 17; non-carriers of loss-of-function alleles are patients without loss-of-function alleles: 1/' 1, ' 1/' 17, or ' 17/' 17. Primers were generated by Primer 3 software (table 2). Metabotropic classification was performed according to CYP2C19 x 2, x 3 and x 17 genotypes, classified as strong metabotropic: wild type homozygous subset (CYP 2C19 x 1/'1, fast metabolizing) + (CYP 2C19 x 1/' 17 ultrafast metabolizing); weak metabolic type: wild type and heterozygous subset of mutant genes (CYP 2C19 x 1/'2 and 1 x 3, intermediary metabotropic) + mutant gene homozygotes or heterozygous subset (CYP 2C19 x 2/' 2, 2/'3 and 3/' 3, slow metabotropic) [10 ].

TEG detection Using TEG5000 type blood coagulation analyzer, reagents including Kaolin (containing 1% Kadin solution), activator, Arachidonic Acid (AA) and Adenosine Diphosphate (ADP), all of which are products of Haemoscope corporation of America, were used. All patients sampled 6mL of elbow venous blood on fasting from 8d morning and placed in heparin tube and citrate tube, respectively, and tested the AA pathway-induced platelet inhibition rate (AA%) and ADP receptor pathway-induced platelet inhibition rate (ADP%) within 2 h.

And (4) judging a result: AA inhibition < 50%, defined as AR; ADP inhibition < 30%, defined as CR.

Clinical data collection and follow-up

Baseline data were collected for all patients enrolled, including age, gender, Body Mass Index (BMI), time to onset, NIHSS score, stroke risk factors (e.g., hypertension, diabetes, hypercholesterolemia, hyperhomocysteinemia, smoking, drinking history), lesion distribution, etiology typing. All patients were followed by phone or outpatient visits after discharge to see if they had new strokes (ischemic and hemorrhagic) within 90d, as well as complex cardiovascular events (including non-cardiogenic TIA, new strokes, angina, myocardial infarction, vascular death) and hemorrhagic events (including eye, nasal, gum, skin, intracranial, digestive and urinary system hemorrhage). Vascular death is defined as death due to stroke (ischemic or hemorrhagic), systemic hemorrhage, myocardial infarction, congestive heart failure, pulmonary embolism, sudden death, arrhythmia. Hemorrhagic stroke is defined as acute extravasation of blood into the parenchyma of the brain or subarachnoid space with associated neurological symptoms. Recurrent stroke is considered disabling if the modified rankin (mrs) score is 2 or more.

And (4) a statistical method, wherein all data are processed by adopting an SPSS17.0 statistical software package. The measurement data conform to the normal distribution by the mean + -standard deviation) The mean values between the two groups were compared using independent sample t test, indicating that the mean values did not fit the normal distribution. The counting data are expressed by number and percentage, and the comparison among groups adopts chi² And (6) checking. All tests were two-sided, with differences of P < 0.05 being statistically significant.

Results

Comparison of clinical baseline data between two groups of patients

A total of 250 acute light ischemic stroke patients were included in the study from month 1 in 2017 to month 4 in 2020, and randomly assigned to the test group and the control group at a ratio of 1: 1. The median age in total was 66 years, 39.2% of the patients were females, and the mean BMI was 24.4. + -. 3.0 Kg/m 2. The median time from the occurrence of stroke to randomized cohort was 20 h. In total 94.4% of patients had a history of hypertension, 43.2% of patients had a history of diabetes, 48.0% of patients had a history of smoking, 32.8% of patients had a history of drinking, 22.4% of patients had hyperhomocysteinemia, and 41.2% of patients had hypercholesterolemia. The two groups of patients have no statistical difference (P is more than 0.05) in the aspects of sex, age, Body Mass Index (BMI), NIHSS score at the time of admission, time from onset to random admission, hypertension, diabetes, hyperlipidemia, homocysteaminemia, smoking, drinking, focus parts, etiology typing and the like, and have comparability.

Test group drug gene detection and TEG detection results

According to COX-1 and ITGB3 gene detection, 69 of COX-1-1676A > G sites are wild AA type, 56 are variants accounting for 44.8%, wherein 42 are AG type heterozygous variants, and 14 are GG type homozygous variants. No mutant allele was found in the-842A > G site. In the 1565T > C locus of ITGB3, 111 cases are wild TT type, 14 cases are variant, account for 11.2%, 12 cases are TC type heterozygous variant, and 2 cases are CC type homozygous variant. Only 5 cases carried mutant alleles of the COX-1 and ITGB3 genes, and 65 cases carried AR-related alleles, accounting for 52.0%. Through CYP2C19 gene detection, 54 cases of clopidogrel strong metabolism type account for 43.20%, wherein 52 cases of CYP2C19 x 1/x 1 and 2 cases of CYP2C19 x 1/x 17 are included; 71 cases of clopidogrel weak metabolism type account for 56.80%, wherein 44 cases of CYP2C19 x 1/x 2, 6 cases of CYP2C19 x 1/x 3, 13 cases of CYP2C19 x 2/x 2, 2 cases of CYP2C19 x 3/3 and 6 cases of CYP2C19 x 2/3. 33 cases (26.4%) all carried related alleles of aspirin resistance and clopidogrel weak metabolism. The TEG detection result shows that the AA inhibition rate is less than 50 percent, 3 cases (2.4 percent) exist, and the mutant allele of PTGS1 and ITGB3 genes is not carried; ADP inhibition rate < 30%, 12 cases (9.6%), 1 case CYP2C19 x 2/' 3; no cases were found with simultaneous AA inhibition < 50% and ADP inhibition < 30%.

Within the follow-up visit of 90d, 4 patients (3.2%) in the test group had stroke, 12 patients (9.6%) in the control group had stroke, and the difference between the two groups was statistically significant (P < 0.05). In the test group, 2 patients (1.6%) had disabling stroke, in the control group 7 patients (5.6%) had no statistical difference (P > 0.05). The experimental group and the control group have no hemorrhagic stroke. In the test group 5 (4.0%) developed complex vascular events and in the control group 14 (11.2%), the difference was statistically significant (P < 0.05). In the test group 1 (0.8%) of the patients developed TIA and in the control group 2 (1.6%), the difference between the two groups was not statistically significant (P > 0.05). Neither group experienced cardiovascular events as well as vascular death or other cause death events.

TEG is a method for dynamically reflecting blood coagulation, platelet aggregation and fibrinolysis functions in a graphical manner by detecting changes in the viscoelastic force of thrombi. In recent years, TEG is increasingly applied to detect AR and CR. The reaction condition of an organism after taking different antiplatelet drugs can be detected through TEG, wherein the platelet aggregation inhibition rate detection induced by ADP can reflect the reactivity of the organism to clopidogrel, and the platelet aggregation inhibition rate induced by AA can reflect the reactivity of the organism to aspirin. In China, research reports that after double-antibody treatment is given to patients with non-cardiogenic ischemic stroke in the acute stage, the clinical prognosis of the patients can be improved by selecting sensitive anti-platelet aggregation drugs according to TEG results without increasing the bleeding risk. Therefore, the research guides the selection of the antiplatelet drugs and the maintenance dosage by jointly detecting the related gene polymorphism of aspirin and clopidogrel and detecting the platelet inhibition rate by TEG, and defines the application value of the gene polymorphism and TEG in guiding the individualized antiplatelet treatment of patients with light ischemic stroke.

The risk of new stroke within 90d can be effectively reduced without increasing the bleeding risk according to the drug gene polymorphism and TEG (TEG-mediated platelet resistance) for guiding the platelet treatment of light ischemic stroke. The gene detection and TEG can find patients insensitive to the treatment of the antiplatelet drugs in time, can provide a new basis for the selection of the antiplatelet drugs, so that the patients with ischemic stroke can select the antiplatelet drugs more accurately and reasonably, thereby formulating an individualized antiplatelet treatment scheme, effectively reducing the risk of stroke recurrence, having important guiding significance for the secondary prevention of ischemic stroke, and being worthy of clinical popularization and application.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.