阅读说明：本技术 基于基因检测与血栓弹力图结合预测氯吡格雷疗效及应用 (Prediction of clopidogrel curative effect based on combination of gene detection and thromboelastogram and application ) 是由 蓝晓红 周永刚 魏玮 臧菊香 李祥 张烨 于浩 陈颖 唐佳丽 陈曙东 吴冬妮 于 2021-03-09 设计创作，主要内容包括：本发明属于生物技术领域,提供基于基因检测与血栓弹力图结合预测氯吡格雷疗效及其应用,旨在提出在冠心病患者中采用CPY2C19基因多态性与血栓弹力图(Thrombelastograghy,TEG)检测结合预测氯吡格雷抗血小板疗效,可对高危患者、高密度脂蛋白水平降低或者基因测定为中代谢、慢代谢型的冠心病患者进行TEG的二磷酸腺苷(adenosine diphosphate,ADP)抑制率结果确认。确保氯吡格雷的治疗疗效,减少急性心血管不良事件发生,提升临床药师参与个体化药物治疗的服务水平。(The invention belongs to the technical field of biology, provides a method for predicting clopidogrel curative effect based on combination of gene detection and a thromboelastogram and application thereof, and aims to provide a method for predicting clopidogrel antiplatelet curative effect by combining CPY2C19 gene polymorphism and Thromboelastogram (TEG) detection in patients with coronary heart disease, and can confirm the Adenosine Diphosphate (ADP) inhibition rate result of TEG on patients with high risk and high-density lipoprotein level reduction or patients with coronary heart disease with gene determination of medium metabolism and slow metabolism. The therapeutic effect of clopidogrel is ensured, the occurrence of acute cardiovascular adverse events is reduced, and the service level of clinical pharmacists participating in individualized drug therapy is improved.)

1. Predicting the clopidogrel curative effect based on the combination of gene detection and a thromboelastogram, and is characterized by comprising the following steps of:

1) drawing venous blood of a patient taking clopidogrel coronary heart disease, and detecting the CYP2C19 gene polymorphism metabolic type by utilizing a CYP2C19 kit;

2) additionally extracting venous blood of a coronary heart disease patient, and adding an anticoagulant;

3) preparing the blood sample in the step 2) into a thromboelastogram by a thromboelastogram analyzer to detect the clopidogrel ADP inhibition rate;

4) analyzing the inhibition effect of the patient on clopidogrel by combining the CYP2C19 gene polymorphism metabolic type in the step 1) and the ADP inhibition rate obtained in the step 3).

2. The method of claim 1, wherein the CYP2C19 kit detects the genetic polymorphisms CYP2C19 x 1, CYP2C19 x 2, CYP2C19 x 3 and CYP2C19 x 17.

3. The method for predicting clopidogrel therapeutic effect based on gene testing in combination with thromboelastogram of claim 1, wherein the anticoagulant is sodium citrate anticoagulant or heparin sodium anticoagulant.

4. The method for predicting the curative effect and the application of clopidogrel in patients with coronary heart disease based on the combination of gene detection and thromboelastogram as claimed in any one of claims 1 to 3.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to prediction of clopidogrel curative effect and application based on combination of gene detection and a thromboelastogram, in particular to application of a CYP2C19 gene polymorphism result in prediction of clopidogrel antiplatelet curative effect.

Background

CYP2C19 is one of the most important drug metabolizing enzymes in the CYP450 family, a plurality of endogenous substrates and about 2% of clinical drugs are catalyzed and metabolized by the enzymes, and the research finds that CYP2C19 can affect the metabolism of a plurality of drugs such as clopidogrel and the like. The gene polymorphism is an important reason for causing individuals and ethnic groups to show different metabolic capacities to the same drug.

The CYP2C19 gene wild type is CYP2C19 x 1/x 1 type, the more common alleles of Chinese population are CYP2C19 x 2 type, CYP2C19 x 3 type and CYP2C19 x 17 type, wherein CYP2C19 x 2 and CYP2C19 x 3 type can cause the enzyme activity encoded by CYP2C19 gene to be weakened, the capability of metabolizing substrate is weakened, active metabolite cannot be generated, and clopidogrel resistance is caused, and the carrier of the genotype is called weak metabolizer. 99% of the weak metabolizers in the chinese population are x 2 and x 3 alleles. The CYP2C19 gene encodes an increased enzyme activity and the carrier of the gene is called a strong metabolizer. Therefore, in 2010, clopidogrel specifications are required by the FDA in the united states to recommend using this prodrug for detecting polymorphisms in the CYP2C19 gene.

Clopidogrel is one of the most widely used antiplatelet drugs at present, belongs to a platelet membrane surface ADP receptor blocker, is a prodrug, and has the metabolic process influenced by the polymorphism of liver P450 enzyme genes (such as CYP2C19, CYP2C9, CYP3A4/5, 2B6, 1A2 and the like). The studies showed that, among the above-mentioned various gene polymorphisms, the gene polymorphism of CYP2C19 is most strongly correlated with clopidogrel resistance. Some gene mutation individuals cannot metabolize the drug to generate activity, so that the drug is ineffective, and the life of the patient is seriously threatened. Therefore, how to accurately predict the difference of clopidogrel in antiplatelet reaction and realize the individual treatment of patients with coronary heart disease is always the focus of research in the field of clinical pharmacy and an urgent problem to be solved.

The CYP2C19 gene polymorphism has larger distribution difference among different human species, and CYP2C19 x 1, x 2, x 3 and x 17 account for stable and higher proportion among different human populations. The distribution of CYP2C19 x 2 alleles in yellow, white and black races, respectively: 29%, 12% and 15%. There are many mutant alleles of the CYP2C19 enzyme, of which CYP2C19 x 1 is the most common and determines the normal metabolism of the drug; CYP2C19 x 2 and CYP2C19 x 3 are loss-of-function alleles, both can cause the reduction or complete loss of CYP2C19 enzymatic activity, thus produce the influence on the curative effect of the medicament metabolized by it, detect these two sites can cover more than 99% of Chinese population.

The TEG method was invented by Harter, German scholars, 1948 and is currently the most important index for monitoring blood coagulation function clinically in perioperative period. The monitoring method of the blood coagulation function in the extracorporeal circulation operation which takes the blood coagulation function as a main monitoring means is widely used in more than 50 countries. Recently, the effect of the antiplatelet drug evaluated by the TEG technology is also rapidly developed, and the method plays an important role in guiding individualized antiplatelet drug treatment.

At present, the platelet aggregation rate detection generally adopted clinically only roughly estimates the functions of platelets, but has poor accuracy and cannot determine whether the inhibition of the platelets caused by clopidogrel resistance is abnormal or not, but the TEG can dynamically monitor the whole blood coagulation and fibrinolysis processes without special treatment.

The thrombosis of the cardiovascular system is closely related to factors such as damage and falling of vascular endothelium, change of blood flow speed, increase of blood coagulability, placement of a stent in a blood vessel and the like. Thrombosis in the PCI postoperative stent of the coronary heart disease patient is mainly related to poor adherence of the stent, risk factors of coronary heart disease which are not strictly controlled, poor endothelial healing, clopidogrel resistance and the like. Besides clinical environmental factors such as drug interaction and combined diseases, genetic factors are largely (the degree of heredity is 73%) related to clopidogrel resistance. Studies have shown that clopidogrel reactivity is closely related to the genetic determinants of cardiovascular events. The new england journal performed an RCT study on 1535 patients on PCI and the results showed: the incidence rates of the cardiovascular events of the CYP2C19 slow metabolism type and the normal metabolism type are respectively 21.5 percent and 13.3 percent, and the risk of the cardiovascular events of the patients with the slow metabolism type is increased by 60 percent and 3.58 times compared with the risk of the cardiovascular events of the patients with the normal metabolism type. However, the slow metabolic form of CYP2C19 can explain only about 12% of differences between clopidogrel-reactive individuals, and the prediction of the antiplatelet drug effect of the variant on ACS and PCI post-operative patients is limited. Therefore, the pure pharmacogenomics research is difficult to provide sufficient individual medication information for patients treated by clopidogrel, and a new method for predicting clopidogrel antiplatelet reactivity is necessary to be explored and combined with the method for reducing the occurrence of acute cardiovascular events of patients with coronary heart disease.

Disclosure of Invention

The invention aims to provide a method for predicting clopidogrel antiplatelet effect based on combination of gene detection and a thromboelastogram and application thereof, which can be used for determining the ADP inhibition rate result of TEG (glucose oxidase) for high-risk patients and patients with coronary heart disease with high-density lipoprotein level reduction or moderate-slow metabolic type genetic determination. The therapeutic effect of clopidogrel is ensured, the occurrence of acute cardiovascular adverse events is reduced, and the service level of clinical pharmacists participating in individualized drug therapy is improved.

The invention aims to provide a method for predicting clopidogrel curative effect based on combination of gene detection and a thromboelastogram, which comprises the following steps:

1) venous blood of a coronary heart disease patient is extracted, and the CYP2C19 kit is utilized to detect the CYP2C19 gene polymorphism metabolic type;

2) additionally extracting venous blood of a coronary heart disease patient, and adding an anticoagulant;

3) preparing the blood sample in the step 2) into a thromboelastogram by a thromboelastogram analyzer to detect the clopidogrel ADP inhibition rate;

4) analyzing the inhibition effect of the patient on clopidogrel by combining the CYP2C19 gene polymorphism metabolic type in the step 1) and the ADP inhibition rate obtained in the step 3).

Preferably, the CYP2C19 kit detects gene polymorphisms of CYP2C19 x 1, CYP2C19 x 2, CYP2C19 x 3 and CYP2C19 x 17.

Preferably, the anticoagulant is a sodium citrate anticoagulant or a heparin sodium anticoagulant.

The invention also provides the application of the clopidogrel based on the combination of the gene detection and the thromboelastogram for predicting the curative effect of the clopidogrel in a coronary heart disease patient.

Compared with the prior art, the invention has the beneficial effects that:

the method reduces the occurrence of acute adverse cardiovascular events by detecting the CYP2C19 gene polymorphism of different patients and analyzing ADP inhibition rate by combining with a thromboelastogram, and giving treatment scheme decisions to patients with coronary heart disease.

Drawings

FIG. 1 is a CYP2C19 gene polymorphism profile of 2054 patients with coronary heart disease; wherein a is superstrong metabolic type, b is strong metabolic type, c is medium metabolic type, and d is slow metabolic type.

Detailed Description

In order to understand the present invention, the following examples are given to further illustrate the present invention. The technical problem to be solved by the invention is how to predict the curative effect or clopidogrel resistance of the antiplatelet drug clopidogrel for patients with coronary heart disease, and avoid or reduce the occurrence of acute cardiovascular events caused by poor curative effect of the antiplatelet drug.

In order to solve the problems, the invention firstly discloses the combination of the gene polymorphism of CYP2C19 detected by PCR of a coronary heart disease patient taking clopidogrel and ADP inhibition rate detected by a thromboelastogram, and the systematic prediction of the antiplatelet effect of clopidogrel and the clinical application.

Example 1 detection of CYP2C19 Gene polymorphism Metabolic type Using CYP2C19 kit

(1) Reagent preparation

And (3) taking the CYP2C19 kit, the PCR reaction solution, the positive control and the blank control out of the refrigerator, balancing to room temperature, shaking and uniformly mixing after all the components are fully dissolved, and quickly centrifuging for 10 s. According to the reaction number (n) required by the experiment, each reaction solution is respectively subpackaged into n reaction tubes according to the subpackaging amount of 23 ul/hole, the PCR reaction tubes are transferred to a sample preparation area, and the rest reagents are put back to a refrigerator at minus 20 +/-5 ℃ for freezing and light-shielding storage. n-number of samples + blank + weak positive control.

(2) Sample DNA extraction

The reagent is added with absolute ethyl alcohol into the solution rinsing liquid PWB before use, and the volume of the absolute ethyl alcohol is 60 ml. Adding 650ul of cell lysate CL into 250ul of blood, reversing and mixing evenly, centrifuging at 10000rpm for 1min, sucking the supernatant, leaving the cell nucleus sediment, adding 200ul of buffer GS into the cell nucleus sediment, and oscillating until the mixture is thoroughly mixed. 200ul of buffer GB and 20ul of proteinase K were added to the above samples, mixed by inversion thoroughly, left at 56 ℃ for 10min (several times during the mixing), and finally the solution was cleared. After the sample is placed at room temperature for 2-5min, 350ul of buffer BD is added, and the mixture is fully inverted and mixed. The sample is added into an adsorption column (the adsorption column is placed into a collection tube), centrifugation is carried out at 12000rpm for 30 seconds, the solution in the collection tube is poured out, and the adsorption column is placed into the collection tube. 500ul of buffer GDB was added to the adsorption column, centrifuged at 12000rpm for 30S, the solution in the collection tube was decanted, and the adsorption column was placed in the collection tube. Adding 600ul of the rinsing solution PWB into the adsorption column, centrifuging at 12000rpm for 30S, pouring off the solution in the collection tube, putting the adsorption column into the collection tube, centrifuging at 12000rpm for 2min, pouring off the waste liquid, and standing at room temperature for 2 min. Transferring the adsorption column into a 1.5ml centrifuge tube, suspending +70ul elution buffer TB to the middle position of the adsorption membrane for elution, standing at room temperature for 2min, centrifuging at 12000rpm for 2min, and collecting the solution into the centrifuge tube.

(3) Sample application

And (3) respectively adding the genome DNA of the sample to be detected, the weak positive control and the blank control into a reaction tube filled with 3 PCR reaction solutions, namely, respectively detecting each sample to be detected by using the 3 PCR reaction solutions, wherein the addition amount is 2 ul/hole. And covering the PCR reaction tube, and recording the sample adding condition. Transferring the PCR reaction tube to a nucleic acid amplification area for computer detection. If the PCR reaction tube cannot be immediately installed in the machine in the temporary situation, the PCR reaction tube added with the template is recommended to be stored at the temperature of 2-8 ℃ and to be installed in the machine for detection as soon as possible within 24 hours.

(4) PCR amplification

And starting up and carrying out instrument performance self-inspection. And taking the prepared PCR reaction tube in the sample preparation area, placing the PCR reaction tube in a corresponding position of a sample groove of the instrument, and recording the placing sequence. Setting relevant parameters for instrument amplification and starting PCR amplification. After the reaction is finished, appropriate base lines and fluorescence threshold values are defined according to the amplification curve, Ct values of different channels are obtained, and result judgment is carried out. The judgment result is divided into: superstrong metabolic type, strong metabolic type, medium metabolic type, slow metabolic type. Detecting the CYP2C19 metabolic type by using a fluorescence quantitative PCR method, if the metabolic type is a medium-slow metabolic genotype or a high-risk patient at the same time, and the high-density lipoprotein level is reduced, TEG is required to detect the clopidogrel ADP inhibition rate.

Alleles at different sites of CYP2C19 showed different intensity of metabolism of clopidogrel, with 1 being normal functional allele, 2 and 3 being loss-of-function or reduction allele, and 17 being function-enhancing allele. In this example, a total of 2054 venous blood samples of patients with coronary heart disease were collected and analyzed for CYP2C19 gene polymorphism, and the experimental results are shown in Table 1 and FIG. 1.

TABLE 1

Example 2 inhibition of ADP% by thromboelastogram analysis

(1) Liquid preparation before experiment

Taking out the reagent A, the reagent B, the reagent C and the reagent H from the reagent pack respectively₂O; opening the reagent A bottle cap, and taking 40ul H₂Adding the O into a reagent A bottle, covering the reagent A bottle cap, and fully shaking up; opening the reagent bottle B cap, and taking 40ul H₂Adding the O into a reagent B bottle, covering the reagent B bottle cap, and fully shaking up; opening the reagent C bottle cap, and taking 40ul H₂Adding the O into a reagent C bottle, covering the reagent C bottle cap, and fully shaking up.

(2) Packing cup

Four channels of TEG were loaded with a normal cup, a batroxobin cup, an adenosine diphosphate cup, and an arachidonic acid cup in this order.

(3) Activation

Transferring 1ml of sodium citrate anticoagulated whole blood sample into a reagent 1 bottle by using a 1ml pipette, inverting the reagent bottle for 5 times to fully mix the sample uniformly, and standing for 4min to activate blood.

(4) Detection of

And (3) detecting a common cup: after activation, 20ul of reagent 2 was removed to the bottom of the common cup, then blood in a 340ul of reagent 1 bottle was removed to the common cup, the test cup was pushed to the top, and the test bar was pulled into the "test" position.

Batroxobin cup detection: open the reagent A bottle and suck 10ul and add batroxobin cup, add 360ul of heparinized blood sample, aspirate three times, push the test cup to the top, dial the test rod to "test" position.

Detecting adenosine diphosphate in a cup: opening the reagent A bottle to suck 10ul, then opening the reagent B bottle to suck 10ul, adding into an adenosine diphosphate cup, adding 360ul of heparinized blood sample, mixing and sucking three times in the cup, pushing the test cup to the top end, and shifting the test rod to a test position.

And (3) detecting arachidonic acid in a cup: opening the reagent A bottle to suck 10ul, then opening the reagent C bottle to suck 10ul, adding the arachidonic acid cup, adding 360ul of heparinized blood sample, mixing and sucking three times in the cup, pushing the test cup to the top end, and shifting the test rod to a test position.

And (3) ending the blood coagulation process for about 30min, if the fibrinolysis process is observed to be prolonged to 1h, clicking to stop on a software interface after the test is finished, removing the test cup, and discarding according to the laboratory requirements.

Checking the inhibition rate: clicking 'compounding' on software, clicking test result graphs of three channels of a common cup, a batroxobin cup and an adenosine diphosphate cup, carrying out compound display, and checking ADP inhibition rate information after clicking 'finishing'. And clicking a test result graph of three channels of the common cup, the batroxobin cup and the arachidonic acid cup, and performing composite display to check the AA inhibition rate information.

Interpretation of test results: the inhibition rate is less than 50%, which indicates that the medicament cannot have good anticoagulation effect and has certain medicament resistance; the inhibition rate is more than or equal to 50 percent, and the drug is prompted to take effect and has obvious inhibition.

30 coronary heart disease patients are randomly selected, ADP inhibition rate is analyzed according to the thromboelastogram, and clopidogrel antiplatelet treatment scheme is determined by combining CYP2C19 gene polymorphism, and the result is shown in table 2.

TABLE 2

The invention determines the clopidogrel metabolic type of a patient by detecting the CYP2C19 gene polymorphism of the patient with coronary heart disease, determines the administration scheme of clopidogrel by combining the ADP inhibition rate of the patient, avoids delaying the antiplatelet treatment curative effect of the patient with coronary heart disease due to a unified administration scheme, and reduces or avoids the occurrence of acute adverse cardiovascular events.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.