CN115820841A - Biomarker for combined diagnosis and detection of arteriosclerosis and cerebral infarction and related application thereof - Google Patents

Abstract

The invention belongs to the field of biological medicine, and relates to a biomarker for joint diagnosis and detection of arteriosclerosis and cerebral infarction and related application thereof, wherein the biomarker comprises any one or more of PCBP1 gene, POTEE gene, SIRPG gene and CYP2R1 gene. Compared with the prior art, the four biomarkers provided by the invention are used independently, have better specificity and sensitivity, are particularly suitable for combined use, and have higher detection stability for arteriosclerotic cerebral infarction.

Description

Biomarker for combined diagnosis and detection of arteriosclerosis and cerebral infarction and related application thereof

Technical Field

The invention belongs to the field of biological medicines, and relates to a biomarker for joint diagnosis and detection of arteriosclerosis and cerebral infarction and related application thereof.

Background

The cerebral infarction is also called ischemic stroke, which is called stroke or stroke in traditional Chinese medicine. The disease is caused by blood supply disorder of local brain tissue areas caused by various reasons, and the ischemic and hypoxic lesion necrosis of the brain tissue is caused, so that the clinically corresponding nerve function deficiency expression is generated. The clinical symptoms of patients with cerebral infarction are shown in the aspects of consciousness, cognition, movement, language, sensation and the like, and the phenomena of numbness, weakness, dizziness and the like of limbs can occur in a short time in patients with mild illness; in severe cases, hemiplegia, cerebral hernia and even death occur easily. The cerebral infarction has the characteristics of high morbidity, high mortality and low prevention rate, is one of main diseases seriously harming human health, is also the first cause of disability, and has the mortality rate which is only lower than that of myocardial infarction and cancer and is the third place.

Arteriosclerotic cerebral infarction is also called arteriosclerotic thrombotic cerebral infarction. Is the atherosclerosis and thrombosis of brain, which makes the vessel lumen of cerebral blood vessel narrow or block, resulting in acute cerebral blood supply insufficiency and ischemic necrosis of local brain tissue. The patients can have the symptoms of cerebral focal damage such as hemiplegia, aphasia and the like, and belong to ischemic cerebrovascular diseases. It is common to the elderly. The incidence of diseases is high in patients with high fat diet, diabetes, smoking and the like.

Current diagnoses of cerebral infarction are based on clinical history, physical examination, neuroimaging and laboratory examinations. There are many diagnostic methods in imaging, among which CT examination (electronic computed tomography), MRI (magnetic resonance imaging), DSA (digital subtraction angiography) are the most commonly used imaging methods. However, various examination methods have certain limitations, such as low sensitivity of CT examination to ischemic stroke, inappropriate MRI examination for pregnancy or pacemaker patients, and inability to perform DSA examination for contrast medium-allergic or anesthetic-allergic patients; therefore, a new method for early and accurate diagnosis of cerebral infarction is urgently needed.

In this regard, several cerebral infarction biomarkers have also been studied, and prior art references are as follows:

CN109061139A discloses the application of a serum inflammatory biomarker comprising at least one cytokine of IL-4, IL-5, IL-6, IL-9, MDC, MIP-1 alpha, sLOX-1, IL-33, trx, YKL-40 in preventing and treating acute ischemic cerebral infarction.

CN112305118A discloses L-octanoyl carnitine as a biomarker for the diagnosis of atherosclerotic cerebral infarction.

CN112305119A discloses a biomarker for atherosclerotic cerebral infarction and an application thereof, and specifically discloses that the level of the biomarker PC (P-18.

CN113063718A discloses plasma endothelial cell membrane microparticles (EMVs) as biomarkers for cerebral infarction.

CN113447601B discloses galactosyl ceramide as a biomarker for diagnosing cerebral infarction and leukoencephalopathy.

CN114137226A discloses a cerebral infarction diagnosis marker, which is composed of the following 10 substances: 4-dimethylallyltryptophan (4-dimethylallyltryptophan), taurochenodeoxycholic acid-3-sulfate (Taurochenodeoxycholate-3-sulfate), trihexosylceramide (d 18: 1/18) 0 [ Trihexosylceramide (d 18:1/18: 0).

Although these biomarkers can detect the cerebral infarction to some extent, they are not comprehensive, and it is necessary to further develop more suitable biomarkers with more stable detection results. Furthermore, since arteriosclerosis has a risk of causing cerebral infarction, it is particularly necessary to find a marker that can diagnose and detect arteriosclerosis and cerebral infarction in combination.

Disclosure of Invention

The invention aims to provide a combined diagnosis and detection of arteriosclerosis and cerebral infarction biomarkers and related application, so that the results of arteriosclerosis and cerebral infarction are more stable, and a related biomarker library is enriched.

In a first aspect of the invention, a brain stem biomarker is provided, comprising any one or more of a PCBP1 gene, a POTEE gene, a SIRPG gene, and a CYP2R1 gene.

In a second aspect, the invention provides the use of a biomarker according to the first aspect of the invention in the preparation of a product for diagnosing or detecting arteriosclerotic cerebral infarction.

Preferably, the biomarkers PCBP1 gene, POTEE gene, SIRPG gene and CYP2R1 gene are used in combination, and the kit has the advantages of high sensitivity, high specificity, good stability, rapid detection and the like.

In a third aspect, the present invention provides an arteriosclerotic cerebral infarction diagnostic or detection kit comprising a reagent for detecting the expression level of the biomarker according to the first aspect of the present invention.

Preferably, the reagents for detecting the expression level of the biomarkers include RNA extraction reagents, reverse transcription reagents, and PCR amplification reagents.

Preferably, the PCR amplification primers for detecting the expression level of the biomarker are:

the primer sequence of the PCBP1 is shown as SEQ No. 1-2; the primer sequence of POTEE is shown in SEQ No. 3-4; the primer sequence of the SIRPG is shown as SEQ No. 5-6; the primer sequence of CYP2R1 is shown in SEQ No. 7-8.

Compared with the traditional diagnostic method only using imaging, the diagnostic kit provided by the invention has the application value of early diagnosis, is beneficial to early detection of preventive treatment of diseases, reduces the medical cost of disease development, reduces the probability of occurrence of severe cerebral infarction and improves the quality of life of patients.

Compared with the prior art, the four biomarkers provided by the invention are used independently, have better specificity and sensitivity, are particularly suitable for combined use, and have higher detection stability.

Drawings

FIG. 1 shows the results of the q-pcr gene detection of four biomarkers of the invention in different samples.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

Screening of biomarkers

Samples of 25 atherosclerotic patients, 25 cerebral infarction patients and 25 healthy persons were collected with the following information in table 1:

TABLE 1 sample information

1. Sample platelet isolation and RNA extraction

The blood is sampled and platelets are isolated from the blood sample within 24 hours after collection to minimize loss of platelet RNA quality and quantity. To separate platelets, platelet Rich Plasma (PRP) was separated in a centrifugation step at 1000 rpm. The PRP was extracted and transferred to a 1.5ml Eppendorf tube, and then platelets were precipitated by a centrifugation step at 3000rpm for 20 minutes. The supernatant was then removed and platelets were observed as white precipitates. Platelets were washed with Phosphate Buffered Saline (PBS) solution and then removed after a 15 minute 3000rpm centrifugation step, followed by flash centrifugation and removal of residual PBS solution. Precipitated platelets were treated with TRIzol reagent (Invitrogen) to purify total RNA.

2. Library construction and mRNA sequencing

The sample is detected to be qualified (OD 260/OD280 value is 1.8-2.0), and the RNA is enriched by magnetic beads with Oligo (dT). Fragmentation buffer was then added to randomly break the mRNA. First strand cDNA was synthesized using a hexabase random primer using mRNA as a template, and then second strand cDNA was synthesized by adding buffer, dNTPs and DNA polymerase I, followed by purification of double-stranded cDNA using AMPure XP beads. And (3) carrying out end repair on the purified double-stranded cDNA, adding A tail and connecting a sequencing joint, then carrying out fragment size selection by using AMPure XP beads, and finally carrying out PCR enrichment to obtain a final cDNA library. And after the library is qualified, sequencing different libraries by Illumina Novaseq 6000 according to effective concentration and target offline data volume after posing.

3. Differential gene analysis

Using DESeq2 to respectively compare the expression difference of mRNA of an atherosclerosis patient group with an cerebral infarction group (group C), a healthy person with the cerebral infarction group (group B) and the healthy person with the atherosclerosis group (group A), and preprocessing all differential expression genes (| Log2FC | > 2 and q-value ≦ 0.05), wherein 1716 differential genes are screened out by the healthy person and the atherosclerosis group, 618 differential genes are screened out by the healthy person and the cerebral infarction group, and 197 genes are screened out by the atherosclerosis and cerebral infarction group. To identify valuable genes, we further selected the intersection gene of three sets of differential genes, comprising: the average values of gene expression levels of MT-ND4L, MTND2P28, MT-TT, TAGLN2P1, MTCO1P12, PCBP1, ACTBP2, POTEE, SIRPG, EIF1P3 and CYP2R1 in different samples are shown in Table 2.

TABLE 2 mean values of gene expression amounts of intersection genes in different samples

Symbol	Gene ID	Average value of healthy people	Mean value of atherosclerosis	Mean cerebral infarction value
					MT-ND4L	4539	11926.60	101095.33	8099.67
MTND2P28	100652939	934.40	16244.67	420.11
					MT-TT	4576	77.60	2246.00	39.44
TAGLN2P1	4576	1461.60	34.67	986.89
					MTCO1P12	100418887	151.60	1173.67	68.00
PCBP1	5093	98.80	710.67	265.44
					ACTBP2	62	630.80	7.33	322.00
POTEE	445582	354.80	4.33	208.56
					SIRPG	55423	39.60	0.00	164.78
EIF1P3	730144	88.60	0.67	57.11
					CYP2R1	120227	43.60	0.00	51.11

PCBP1, POTEE, SIRPG and CYP2R1 are preselected by expression quantity values to be early screening target genes of the cerebral infarction caused by atherosclerosis.

Example 2

Gene verification

Target gene sequences were obtained by sequencing, qpcr primers for 4 selected biomarker genes were designed using Primer Premier 6 software, and the Primer sequences are shown in table 3.

TABLE 3 qpcr primers for the 4 biomarker genes

25 healthy human samples, 20 atherosclerosis samples and 16 cerebral infarction (early stage) samples are collected, q-pcr gene copy number detection is carried out by using designed primers, the experimental flow is as follows, and the results are shown in figure 1.

After the sample is subjected to platelet separation and RNA extraction quality inspection, reverse transcription is carried out, and a transcription system is as follows (reverse transcription reagents are all collected from Saimer fly):

50uM OligodT(20)primer：1ul；

10mM dNTP mix：1ul；

RNA：2ul；

Rnasy-free H2O：9ul。

the reaction procedure is as follows: 5min at 65 ℃ and 1min;

5X SSIV buffer：4ul；

100mM DTT：1ul；

RRI：1ul；

SuperScript IV：1ul；

the reaction procedure is as follows: 10min at 50 ℃ and 10min at 80 ℃.

The transcribed product (cDNA) can be subjected to q-PCR in the following reaction system:

2x SYBR GREEN Mix：5ul；

Primer-F(10uM)：0.2ul；

Primer-R(10uM)：0.2ul；

RNeasy-free H2O：3.6ul；

cDNA：1ul；

the reaction procedure is as follows:

the final experimental results: PCBP1, POTEE, SIRPG and CYP2R1 have statistical difference (p is less than or equal to 0.05) in the relative expression quantity in health, atherosclerosis and cerebral infarction. Wherein the relative expression level of the marker PCBP1 in health, atherosclerosis and cerebral infarction is very obvious difference (P is less than or equal to 0.01).

TABLE 4 relative expression and significance of 4 biomarker genes in different samples

Note: capital ABC represents extremely significant (P is less than or equal to 0.01); the lower case letters abc indicate significant differences (p ≦ 0.05)

Cutoff value

The cutoff value is a judgment standard, for data with two classes of variables, the continuous independent variable cutoff value is determined by ROC analysis, a point (maximum value) with the maximum Youden index (sensitivity + specificity-1) is selected as an optimal cutoff value point, the point is a boundary value for judging health, atherosclerosis and cerebral infarction, and the range among all values in the relative expression quantity of the genes is determined so as to distinguish disease stages. And respectively calculating to obtain cutoff values corresponding to the maximum values of the joden indexes of the four genes of PCBP1, POTEE, SIRPG and CYP2R1 in three periods of health, atherosclerosis and cerebral infarction.

TABLE 5 specificity, sensitivity, jordan index and cut-off values of the 4 biomarker genes

When the relative expression quantity of the gene is lower than the corresponding cut-off value, the progress of the patient in three stages of health, atherosclerosis and cerebral infarction is evaluated according to the sensitivity and specificity corresponding to the maximum value of the john's index of the gene. When PCBP1 is used as a diagnostic gene, the health stage can be judged when the relative expression level of the gene is less than 1.235, the cerebral infarction stage can be judged when the gene expression level is more than 1.235 and less than 3.300, and the atherosclerosis stage can be judged when the expression level is more than 3.300; POTEE is used as a diagnosis gene, the health stage can be judged when the relative expression quantity of the gene is more than 0.870 and less than 1.380, the atherosclerosis stage can be judged when the relative expression quantity of the gene is less than 0.870, and the cerebral infarction stage can be judged when the relative expression quantity of the gene is more than 1.380; taking SIRPG as a diagnostic gene, judging the state as a healthy stage when the relative expression quantity of the gene is more than 0.965, judging the state as an atherosclerosis stage when the relative expression quantity of the gene is less than 0.965 and more than 0.760, and judging the state as a cerebral infarction stage when the relative expression quantity of the gene is less than 0.760; CYP2R1 is taken as a diagnosis gene, the health stage can be judged when the relative expression quantity of the gene is more than 0.585, the atherosclerosis stage can be judged when the relative expression quantity of the gene is less than 0.585 and more than 0.105, and the cerebral infarction stage can be judged when the relative expression quantity of the gene is less than 0.105.

TABLE 6 cut-off values for the different stages in which the 4 biomarker genes are located

The verification shows that the 4 genes PCBP1, POTEE, SIRPG and CYP2R1 can be independently used as biomarkers for diagnosis of three stages of health, atherosclerosis and cerebral infarction.

Claims (6)

1. A biomarker for combined diagnosis and detection of arteriosclerosis and cerebral infarction, which is characterized in that the biomarker comprises any one or more of PCBP1 gene, POTEE gene, SIRPG gene and CYP2R1 gene.

2. Use of a biomarker according to claim 1 in the manufacture of a product for diagnosing or detecting cerebral infarction.

3. The use of claim 2, wherein the biomarker comprises a combination of a PCBP1 gene, a POTEE gene, a SIRPG gene, a CYP2R1 gene.

4. An arteriosclerotic cerebral infarction diagnostic or detection kit comprising a reagent for detecting the expression level of the biomarker according to claim 1.

5. The arteriosclerotic diagnostic or test kit of claim 4 wherein the reagents for detecting the expression level of said biomarkers comprise RNA extraction reagents, reverse transcription reagents and PCR amplification reagents.

6. The arteriosclerotic diagnosis or detection kit according to claim 5, wherein the PCR amplification primers for detecting the expression level of the cerebral infarction biomarker are:

the primer sequence of the PCBP1 is shown as SEQ No. 1-2;

the primer sequence of the POTEE is shown in SEQ No. 3-4;

the primer sequence of the SIRPG is shown as SEQ No. 5-6;

the primer sequence of CYP2R1 is shown in SEQ No. 7-8.

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