A SNP marker associated with the antiplatelet drug clopidogrel in the treatment of ischemic stroke and its application
A clopidogrel and marker technology, applied in the fields of genetic engineering and cerebrovascular medicine, can solve the problems of different drug responsiveness and so on
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Embodiment 1
[0053] Example 1 Selection and Collection of Research Samples and Arrangement of Sample Data
[0054] 1. Sample selection
[0055] (1) Aged 40-80 years old, gender is not limited;
[0056] (2) Conform to the diagnostic criteria revised by the Fourth National Cerebrovascular Disease Conference and confirmed by CT or MRI;
[0057] (3) Oral clopidogrel at 75mg / day (including the first loading dose of 300mg) for more than 5 days (including 5 days).
[0058] 2. The inventor selected a total of 313 samples that met the criteria for a single Sequenom MassARRAY genotyping experimental sample, and systematically collected the demographic data and clinical data of these samples.
Embodiment 2
[0059] Example 2 Detection of Platelet Aggregation Rate of Sample——Determination of Clopidogrel Resistance
[0060] 1. The platelet aggregation test was carried out by light turbidimetry. Take 2.7 ml of EDTA anticoagulated whole blood (1:9), centrifuge at 800rpm for 5min to extract platelet-rich plasma, then centrifuge at 3000rpm for 15min to extract platelet-poor plasma, the platelet count is 10-20×10 9 / L, use PPP to adjust PRP to 200~300×10 9 / L, PPP was used as blank control, preheated at 37°C for 3 minutes, and 10 μM ADP was used as inducer, and the platelet aggregation rate of patients was measured by light turbidimetry.
[0061] 2. Determination of the platelet aggregation rate in patients with ischemic stroke before taking clopidogrel (day0) and after taking clopidogrel (day5). If the platelet inhibition rate is less than 10% or the blood aggregation value is >50% after taking the drug, it is considered that clopidogrel resistance occurs; according to this standard, ...
Embodiment 3
[0063] Example 3 Genotyping and scanning of SNP markers in peripheral blood DNA
[0064] Among the above-mentioned 313 eligible ischemic stroke patients (including 106 cases in the resistance group and 207 cases in the non-resistance group), relevant results were obtained by Sequenom MassARRAY typing. The specific steps are:
[0065] 1. Extraction of genomic DNA from peripheral blood by phenol-chloroform method
[0066] (1) Take 100 μl of EDTA anticoagulated whole blood, add 200 μl of sterile double distilled water and mix well;
[0067] (2) Add 200 μl of 6 M sodium iodide and mix well;
[0068] (3) Add 400 μl of chloroform / isoamyl alcohol (24:1, v / v), mix repeatedly, let stand for 10 minutes; centrifuge at 12000 rpm for 10 minutes, draw the supernatant and put it into another centrifuge tube;
[0069] (4) Add 300 μl of isopropanol to the supernatant, mix well, let stand for 3 minutes, centrifuge at 12000 rpm for 10 minutes, and discard the supernatant;
[0070] (5) Add 50...
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