CN119120709A - A vascular inflammation-related protein marker for assisting diagnosis or detection of cerebral aneurysms and its application - Google Patents
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Abstract
本发明公开了一种用于辅助诊断或检测脑动脉瘤的血管炎症相关蛋白标志物，特点是蛋白标志物为OLR1蛋白、IL6蛋白、PTX3蛋白和LPL蛋白的组合或者OLR1蛋白，制备的辅助诊断或检测脑动脉瘤的试剂盒包括OLR1转录组、IL6转录组和PTX3转录组的RT‑qPCR定量扩增引物，优点是以检测OLR1标志物，IL6、PTX3、OLR1、LPL组合标志物以及OLR1、IL6和PTX3组合标志物的转录组/蛋白表达水平为基础的检测试剂盒可以方便快捷地在分子水平上实现对脑动脉瘤的检测，检测效率高，灵敏度高，针对性强。
The invention discloses a vascular inflammation-related protein marker for auxiliary diagnosis or detection of cerebral aneurysm, characterized in that the protein marker is a combination of OLR1 protein, IL6 protein, PTX3 protein and LPL protein or OLR1 protein, and the prepared kit for auxiliary diagnosis or detection of cerebral aneurysm comprises RT-qPCR quantitative amplification primers of OLR1 transcriptome, IL6 transcriptome and PTX3 transcriptome. The advantage is that the detection kit based on detecting the transcriptome/protein expression level of OLR1 marker, IL6, PTX3, OLR1, LPL combination marker and OLR1, IL6 and PTX3 combination marker can conveniently and quickly realize the detection of cerebral aneurysm at the molecular level, and has high detection efficiency, high sensitivity and strong pertinence.
Description
Technical Field
The invention relates to the technical field of auxiliary diagnosis of cerebral aneurysms, in particular to a vascular inflammation related protein marker for auxiliary diagnosis or detection of cerebral aneurysms and application thereof.
Background
Cerebral aneurysms (INTRACRANIAL ANEURYSMS, abbreviated IA) are a potentially fatal asymptomatic disease with high morbidity and mortality, particularly affecting people between 40 and 70 years old. Cerebral aneurysms are characterized by abnormal distension of the cerebral arteries, which may not be found for many years until symptoms are caused by intracranial nerve compression or catastrophic rupture causes subarachnoid hemorrhage. About 30-50% of patients have cerebral aneurysms which are broken and not timely rescuing and dying from the disease, about 30% of patients have cerebral aneurysms which are broken and timely rescuing to hospitals can leave different degrees of disabilities, and few survivors can resume normal life. Therefore, there is an urgent need for a technique that enables early diagnosis and prediction of cerebral aneurysms.
Detection of protein biomarkers plays a vital role in disease prediction, screening, treatment, and long-term outcome assessment. Animal model study data for cerebral aneurysms show that macrophages infiltrate the arterial wall at the beginning of the disease. Inflammatory factors such as tumor necrosis factor (abbreviated TNF- α), interleukin-1β (abbreviated IL-1β) and interleukin-6 (abbreviated IL-6) then cause elastic membrane loss and collagen matrix damage, promoting aneurysm formation by outward remodeling. Persistent inflammation is characterized by infiltration of macrophages, T lymphocytes and B lymphocytes, ultimately leading to tissue fibrosis, leading to rupture of cerebral aneurysms, which is life threatening. Thus, free inflammation-associated proteins in the blood have great potential as early cerebral aneurysm diagnostic biomarkers.
Oxidized low density lipoprotein receptor 1 (abbreviated OLR 1) is a pattern recognition receptor and plays an important role in cardiovascular and cerebrovascular diseases. In cerebrovascular disease, OLR1 can promote oxidative stress to produce a large amount of oxidized low density lipoprotein (abbreviated as ox-LDL). After OLR1 binds to ox-LDL, a series of signaling pathways are activated, further exacerbating oxidative stress, promoting inflammatory responses, leading to vascular endothelial injury and vascular lesions. At present, researches and reports related to OLR1 and cerebral aneurysms are rarely performed at home and abroad.
Disclosure of Invention
The invention aims to solve the technical problem of providing a vascular inflammation related protein marker with high detection efficiency, high sensitivity and strong specificity for auxiliary diagnosis or detection of cerebral aneurysms and application thereof.
The technical scheme adopted for solving the technical problems is that the protein marker is used for assisting in diagnosing or detecting vascular inflammation related to cerebral aneurysms, and the protein marker is OLR1 protein.
Further, the application of the vascular inflammation-related protein marker in preparing a kit for assisting in diagnosing or detecting cerebral aneurysms is utilized, the kit comprises RT-qPCR quantitative amplification primers of an OLR1 transcriptome, the nucleotide sequence of a forward amplification primer of the OLR1 transcriptome is shown as SEQ ID NO.1 to be 5'-TTGCCTGGGATTAGTAGTGACC-3', and the nucleotide sequence of a reverse amplification primer of the OLR1 transcriptome is shown as SEQ ID NO.2 to be 5'-GCTTGCTCTTGTGTTAGGAGGT-3'.
The invention also provides a protein marker related to vascular inflammation for assisting in diagnosis or detection of cerebral aneurysms, wherein the protein marker is a combination of OLR1 protein, IL6 protein, PTX3 protein and LPL protein.
The invention also provides a vascular inflammation related protein marker for assisting diagnosis or detection of cerebral aneurysms, wherein the protein marker is a combination of OLR1 protein, IL6 protein and PTX3 protein.
Further, the application of the vascular inflammation related protein marker in preparing a kit for assisting in diagnosing or detecting cerebral aneurysms is utilized, the kit comprises an OLR1 transcriptome, an IL6 transcriptome and a RT-qPCR quantitative amplification primer of a PTX3 transcriptome, the nucleotide sequence of a forward amplification primer of the OLR1 transcriptome is shown as SEQ ID NO.1 to be 5'-TTGCCTGGGATTAGTAGTGACC-3', the nucleotide sequence of a reverse amplification primer of the OLR1 transcriptome is shown as SEQ ID NO.2 to be 5'-GCTTGCTCTTGTGTTAGGAGGT-3', the nucleotide sequence of a forward amplification primer of the IL6 transcriptome is shown as SEQ ID NO.3 to be 5'-TAGAGTACCTCCAGAACAGATT-3', the nucleotide sequence of a reverse amplification primer of the IL6 transcriptome is shown as SEQ ID NO.4 to be 5'-AATAGTGTCCTAACGCTCATAC-3', the nucleotide sequence of a forward amplification primer of the PTX3 transcriptome is shown as SEQ ID NO.5 to be 5'-GCATAATAGGAACACTTGAGAC-3', and the nucleotide sequence of a reverse amplification primer of the PTX3 transcriptome is shown as SEQ ID NO.6 to be 5'-CTGACAGAGACACAGCATT-3'.
Compared with the prior art, the vascular inflammation related protein marker for assisting in diagnosing cerebral aneurysms and the application thereof have the advantages that IL6, PTX3, OLR1 and LPL and combined markers thereof for assisting in diagnosing cerebral aneurysms are disclosed for the first time, and the expression of IL6, PTX3, OLR1 and LPL transcriptome and protein levels in blood of cerebral aneurysms patients are increased. The mechanism is probably that the proteins related to vascular inflammation are continuously and highly expressed after the vascular inflammation injury, and the proteins are involved in the pathological development process of cerebral aneurysm by increasing the secretion level of inflammatory factors. Therefore, the detection kit based on the detection of the OLR1 marker, the combined markers of IL6, PTX3, OLR1 and LPL and the transcriptome/protein expression level of the combined markers of IL6, PTX3 and OLR1 can conveniently and rapidly realize the detection of cerebral aneurysms on the molecular level, has high detection efficiency and sensitivity and strong pertinence, and is beneficial to early discovery and timely treatment of cerebral aneurysms patients.
Drawings
Fig. 1 is a graph of differential expression of all vascular inflammation-related biomarkers between a cerebral aneurysm group and a control group, wherein a is a volcanic graph of 92 inflammation-related biomarkers, B is a graph of differential expression levels of 11 inflammation-related protein markers, P <0.05, P <0.01, P <0.001;
FIG. 2 is a graph of GO and KEGG enrichment analysis of differentially expressed vasculoinflammatory related proteins, where A is the first 20 GO enrichments against background of all annotated proteins, and B is the first 20 KEGG enrichment pathways against background of all annotated proteins;
Fig. 3 is a thermal plot of correlation between differentially expressed vasculoinflammatory related proteins in patients with cerebral aneurysms, P <0.05, P <0.01, P <0.001;
fig. 4 is a heat map of the correlation of differentially expressed vascular inflammatory proteins with clinical features, wherein a is the correlation between 11 inflammatory proteins in all groups and clinical features, B is the correlation between 11 inflammatory proteins in cerebral aneurysms groups and clinical features, C is the correlation between 11 inflammatory proteins in control groups and clinical features, P <0.05, P <0.01, P <0.001;
Fig. 5 shows the results of ELISA assays for PTX3 protein, IL6 protein, OLR1 protein and LPL protein in plasma of control and cerebral aneurysms, wherein a is PTX3, B is IL6, C is OLR1, D is LPL, P <0.05, P <0.01, P <0.001;
FIG. 6 is a graph of ROC curve analysis of early diagnosis value of cerebral aneurysm by ELISA for verifying PTX3 protein, IL6 protein, OLR1 protein and LPL protein and their combination;
FIG. 7 shows the relative RT-qPCR expression results of PTX3, IL6 and OLR1 transcripts in control and blood leukocytes, wherein A is PTX3, B is IL6, C is OLR1, and P <0.0001;
FIG. 8 is a ROC graph of RT-qPCR verifying expression levels of PTX3, IL6 and OLR1 transcriptomes in blood leukocytes.
Detailed Description
The invention is described in further detail below with reference to the embodiments of the drawings.
In a first embodiment, a protein related to vascular inflammation of cerebral aneurysm is selected.
1. Clinical data of stage volunteers the study was collected from volunteers voluntarily enrolled in a first hospital neurosurgical hospitalization department affiliated with Ningbo university, including 30 cases of cerebral aneurysm patients (53.23.+ -. 7.12, 15 men) confirmed by cerebral angiography, and 29 cases of control group population (53.27.+ -. 6.96, 15 men) clinically confirmed to have no cerebral aneurysm lesions. The case group and the control group personnel are subjected to strict one-to-one matching in terms of gender and age, and all the participants are collected into fasting venous blood samples, the general biochemical indexes such as blood fat and blood sugar and the nucleic acid content in blood are detected, and the results of clinical data such as gender, age, history of tobacco and wine, hypertension and general blood biochemical detection are recorded, wherein the clinical data of the first cerebral aneurysm case for screening differential protein and the clinical data of the control group population are compared as shown in the following table 1.
2. Plasma extraction and biochemical analysis 4 mL peripheral venous blood was collected within 6 hours after admission to the hospital after informed consent of all participants and placed in an anticoagulant tube and then centrifuged at 3000 rpm for 15 minutes at 4 ℃ followed by careful extraction of the upper plasma layer and the middle leukocyte layer. Conventional biochemical indicators including blood glucose, total cholesterol, triglycerides, high density lipoproteins, low density lipoproteins, apolipoprotein a, apolipoprotein B and apolipoprotein E were examined using an automated biochemical analyzer (Olympus AU2700, japan) and the results are shown in table 1.
TABLE 1 comparison of clinical data for the population of the first screening stage cerebral aneurysm case group and the control group
As can be seen from table 1, both apolipoprotein B and glucose concentrations were higher in the cerebral aneurysms group than in the control group (P < 0.05). There were no statistical differences in age, sex profiles, total cholesterol, high density lipoprotein, low density lipoprotein, apolipoprotein a and apolipoprotein E (P > 0.05) in the case and control groups.
3. Ultrasensitive multiplex targeting protein detection assay (abbreviated Olink) analysis of vascular inflammation-related proteins of cerebral aneurysms plasma samples from 30 patients with cerebral aneurysms and 29 patients in the control group were analyzed using Olink beta target 92 CVD II panel (Olink Proteomics AB, sweden) according to manufacturer's guidelines. The technique involves highly specific binding of target proteins to antibody probes labeled with double oligonucleotides, followed by detection and quantification using a microfluidic real-time PCR instrument (Biomark HD, usa), and the final detection readings are displayed as normalized protein expression values that are log2 transformed for various biological analyses.
The present study analyzed the expression levels of 92 vascular inflammation-related proteins in group Olink CVD-II. As shown in fig. 1a, there were 11 differentially expressed inflammation-associated proteins between the cerebral aneurysm group and the control group, 8 proteins up-regulated in the cerebral aneurysm group, including interleukin-6 (IL 6), pentameric protein 3 (PTX 3), hepatitis a virus cell receptor 1 (KIM 1), carcinoembryonic antigen-associated cell adhesion molecule 8 (CEACAM 8), OLR1, interleukin-1 receptor antagonist (IL-1 ra), angiopoietin-1 receptor (TIE 2), and programmed cell death 1 ligand 2 (PD-L2). In contrast, there were 3 proteins down-regulated in the cerebral aneurysm group, including lipoprotein lipase (LPL), fatty acid binding protein 2 (FABP 2) and interleukin-27 (IL-27), and the remaining 81 vascular inflammation-related proteins were partially overlapped and non-differentially expressed, not specifically shown. The scatter plot of fig. 1B further illustrates the fold-change differences in differential protein expression between the case group and the control group. Table 2 shows detailed information of 11 differential proteins between cerebral aneurysm cases and control groups.
Table 2 is detailed information on vascular inflammatory proteins that vary significantly between cerebral aneurysms and controls
Note that Fold Change (FC) between cerebral aneurysms and control was calculated as log2. Calculation of P-values (P < 0.05) using t-test indicated significant differences in protein.
4. Enrichment and correlation analysis of differentially expressed proteins in analysis of the set of Differentially Expressed Proteins (DEPs) between the cerebral aneurysms and the control, we used the R software (Version 4.1.3) to determine significant differentially expressed proteins using the R package "Olink Analyze". We used ggplot packages in R software for data visualization and analysis, generating heat and volcanic maps to visualize the data. In addition, we used the multi-functional data visualization package ggplot in R software for Gene Ontology (GO) enrichment analysis, kyoto gene and genome encyclopedia (KEGG) pathway enrichment analysis. In addition, in order to study the protein expression pattern relationship between the two groups, spearman correlation analysis was performed. We constructed a protein-protein interaction (PPI) network for differentially expressed proteins. Subsequently, we visualize these networks using igraph [1.4.1] and ggraph [2.1.0 ]. And finally, generating an ROC curve by using an ROCR (remote control unit) package, combining two or more indexes by using Logistic regression analysis, displaying Area Under Curve (AUC) values of combined diagnosis in a lower right corner legend, and evaluating the classification performance of the data.
Functional enrichment assays were used to elucidate the potential function of differentially expressed proteins in cerebral aneurysms and control plasma. As shown in fig. 2a, GO enrichment analysis showed that differentially expressed proteins were enriched in leukocyte migration, immune response, triglyceride metabolism, lipoprotein metabolism, acute phase response, regulation of T cell proliferation, etc. As shown in FIG. 2B, KEGG enrichment analysis showed that differentially expressed inflammation-associated protein factors were mainly focused on PPAR signaling, HIF-1 signaling, cytokine-cytokine interactions, PI3K-Akt signaling, and the like. In addition, PPI network analysis of differentially expressed inflammation-associated proteins, as shown in FIG. 3, with the highest score for OLR1, suggests that it plays a critical role in the pathogenesis of IA, while we also found that LPL exhibited negative correlation with IL-1ra, IL6, OLR1 and CEACAM8, FABP2 and TIE2, while positive correlation occurred between other protein factors, and CEACAM8 exhibited the most significant positive correlation with OLR 1. These different factors may be interrelated and play an important role in the course of cerebral aneurysm disease or in poor prognosis.
5. Correlation of different inflammatory factors with clinical characteristics we performed correlation analysis of the 11 differentially expressed proteins screened against all groups, group IA and control groups, respectively. As shown in FIG. 4A, in all participants, apolipoprotein B correlated strongly with IL-1ra (P < 0.001), and TIE2 correlated positively with low density lipoprotein and apolipoprotein B levels (P < 0.01). Notably, glucose concentrations were positively correlated with IL-6 (P < 0.01) and with IL-1ra, OLR1, KIM-1 and PD-L2 (P < 0.05). In contrast, apolipoprotein A correlated positively with IL-27 (P < 0.05), whereas triglycerides correlated positively with IL-1ra (P < 0.05), correlated negatively with IL-27 (P < 0.05). Gender was inversely related to CEACAM8 (P < 0.05). As shown in FIG. 4B, in the IA queue, IL-1ra correlated positively with triglycerides (P < 0.01), apolipoprotein B correlated positively with IL-1ra (P < 0.05), glucose correlated positively with IL-6 (P < 0.05), and apolipoprotein E correlated positively with TIE2 (P < 0.05). In addition, gender was inversely related to IL-1ra (P < 0.05), OLR1 (P < 0.05), CEACAM8 (P < 0.01). Apolipoprotein E is inversely related to FABP2 (P < 0.01), LPL (P < 0.05). High density lipoprotein is inversely related to IL-1ra (P < 0.05). In contrast, as shown in FIG. 4C, in the control group, apolipoprotein A correlated positively with LPL, PD-L2 (P < 0.05), high density lipoprotein correlated positively with IL-27 (P < 0.05), whereas age correlated negatively with TIE2 (P < 0.05), triglyceride correlated negatively with IL-27 (P < 0.05). These findings indicate that there is a complex interdependence between the biomarker examined and the clinical attributes.
In summary, by collecting blood from 59 patients with cerebral aneurysms and a control group, and detecting 92 vascular inflammation-related proteins in the plasma of the patients by using Olink ultrasensitive multiple-targeting protein detection and analysis technology, 11 inflammation-related proteins are found to be differentially expressed between the two groups of patients, namely IL6, PTX3, LPL, KIM1, CEACAM8, OLR1, IL-1ra, FABP2, TIE2, IL-27 and PD-L2.
And secondly, verifying the diagnostic value of the vascular inflammation-related protein screened in the first embodiment on cerebral aneurysms.
To further clarify the accuracy of Olink proteome testing, we further collected samples from the second validation set of clinical cases, control group, for a total of 30 gender-matched IA patients and neurosurgical hospitalized non-IA patients as control groups. We selected the 4 proteins IL6, PTX3, OLR1 and LPL with the most significant differences in protein expression between the case group and the control group and validated the differences in protein expression in patient plasma using an enzyme-linked immunosorbent assay (ELISA).
Table 3 shows a comparison of clinical data for the population of the cerebral aneurysm case group and the control group in the second verification stage
As can be seen from table 3, the total cholesterol (p=0.013) and apolipoprotein E (p=0.043) levels in the plasma of group IA patients were significantly lower than in the control group. As shown in figure 5A, B, C, D, the ELISA results showed that the expression pattern of IL-6, PTX3 and OLR1 in the plasma of IA patients was significantly higher than that of the control group, while the expression pattern of LPL was significantly lower than that of the control group. As shown in FIG. 6, the ROC diagnostic analysis showed that PTX3 had an AUC of 0.90, an optimal sensitivity of 86.20%, a specificity of 90.00%, an area under the curve (AUC) of IL-6 of 0.786, an optimal sensitivity of 73.33%, a specificity of 90.00%, an AUC of OLR1 of 0.754, an optimal sensitivity of 70.00%, a specificity of 76.66%, an AUC of LPL of 0.717, an optimal sensitivity of 76.66% and a specificity of 73.33%. We combined the four predictors and found that the combined AUC was 0.922, the optimal sensitivity was 93.10% and the specificity was 90.00%. These results underscore the potential of IL6, PTX3, OLR1 and LPL as IA diagnostic biomarkers, and also demonstrate that the diagnostic effect can be improved by a combination of multiple markers.
Specific example III, enlarging cases, and controlling group real-time quantitative polymerase chain reaction RT-qPCR verification experiment.
According to the ELISA detection result in the second embodiment, the expression condition of the mRNA level of 3 related inflammatory factors IL-6, PTX3 and OLR1 with AUC >0.75 in the blood white blood cells of patients is further verified by RT-qPCR. Total RNA was isolated from blood leukocyte layer samples using TRIzol reagent. Subsequently, cDNA was synthesized from the RNA template using cDNA reverse transcription kit (TransGen Biotech, china). RT-qPCR was performed on the Roche LightCycler 480 system using SYBR Green SuperMix kit (TransGen Biotech, china). RT-qPCR quantitative analysis was performed using Primer6 software (Premier Biosoft, USA) to create RT-qPCR specific primers. The primer sequences were as follows:
The nucleotide sequence of the forward amplification primer of the OLR1 transcriptome is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse amplification primer of the 5'-TTGCCTGGGATTAGTAGTGACC-3', OLR transcriptome is shown as SEQ ID NO.2, 5'-GCTTGCTCTTGTGTTAGGAGGT-3';
The nucleotide sequence of the forward amplification primer of the IL6 transcriptome is shown as SEQ ID NO.3, and the nucleotide sequence of the reverse amplification primer of the 5'-TAGAGTACCTCCAGAACAGATT-3', IL transcriptome is shown as SEQ ID NO.4, 5'-GCTTGCTCTTGTGTTAGGAGGT-3';
the nucleotide sequence of the forward amplification primer of the PTX3 transcriptome is shown as SEQ ID NO.5, and the nucleotide sequence of the reverse amplification primer of the 5'-GCATAATAGGAACACTTGAGAC-3', PTX transcriptome is shown as SEQ ID NO.6, 5'-CTGACAGAGACACAGCATT-3';
The nucleotide sequence of the reference gene ACTB forward amplification primer is 5'-ATTGCCGACAGGATGCAGA-3' shown in SEQ ID NO.7, the nucleotide sequence of the reference gene ACTB reverse amplification primer is 5'-CAGGAGGAGCAATGATCTTGAT-3' shown in SEQ ID NO.8, and the relative expression amount of mRNA is analyzed by adopting a 2 −△△Ct method.
The results are shown in FIG. 7, and the RT-qPCR results are consistent with ELISA results. Specifically, as shown in fig. 7A, B, C, mRNA expression levels of PTX3 (P < 0.001), IL6 (P < 0.01) and OLR1 (P < 0.001) were significantly increased in blood leukocytes of patients with cerebral aneurysms as compared with the control group. As shown in fig. 8, ROC curve analysis results show that IL6 transcriptome (auc=1.00), PTX3 transcriptome (auc=1.00) and KIM1 transcriptome (auc=1.00) in blood leukocytes of patients with cerebral aneurysms have very high diagnostic value.
The above description is not intended to limit the invention, nor is the invention limited to the examples described above. Variations, modifications, additions, or substitutions will occur to those skilled in the art and are therefore within the spirit and scope of the invention.
Claims (5)
1. A vascular inflammation related protein marker for assisting diagnosis or detection of cerebral aneurysms is characterized in that the protein marker is OLR1 protein.
2. The application of the vascular inflammation-related protein marker in preparing a kit for assisting in diagnosing or detecting cerebral aneurysms, which is characterized in that the kit comprises RT-qPCR quantitative amplification primers of an OLR1 transcriptome, wherein the nucleotide sequence of a forward amplification primer of the OLR1 transcriptome is shown as SEQ ID NO.1 and 5'-TTGCCTGGGATTAGTAGTGACC-3', and the nucleotide sequence of a reverse amplification primer of the OLR1 transcriptome is shown as SEQ ID NO.2 and 5'-GCTTGCTCTTGTGTTAGGAGGT-3'.
3. A vascular inflammation related protein marker for assisting diagnosis or detection of cerebral aneurysms is characterized in that the protein marker is a combination of OLR1 protein, IL6 protein, PTX3 protein and LPL protein.
4. A vascular inflammation related protein marker for assisting diagnosis or detection of cerebral aneurysms is characterized in that the protein marker is a combination of OLR1 protein, IL6 protein and PTX3 protein.
5. The application of the vascular inflammation-related protein marker in preparing a kit for assisting in diagnosing or detecting cerebral aneurysms, which is characterized in that the kit comprises an OLR1 transcriptome, an IL6 transcriptome and a RT-qPCR quantitative amplification primer of a PTX3 transcriptome, wherein the nucleotide sequence of a forward amplification primer of the OLR1 transcriptome is 5'-TTGCCTGGGATTAGTAGTGACC-3' shown in SEQ ID NO.1, the nucleotide sequence of a reverse amplification primer of the OLR1 transcriptome is 5'-GCTTGCTCTTGTGTTAGGAGGT-3' shown in SEQ ID NO.2, the nucleotide sequence of a forward amplification primer of the IL6 transcriptome is 5'-TAGAGTACCTCCAGAACAGATT-3' shown in SEQ ID NO.3, the nucleotide sequence of a reverse amplification primer of the IL6 transcriptome is 5'-AATAGTGTCCTAACGCTCATAC-3' shown in SEQ ID NO.4, the nucleotide sequence of a forward amplification primer of the PTX3 transcriptome is 5'-GCATAATAGGAACACTTGAGAC-3' shown in SEQ ID NO.5, and the nucleotide sequence of a reverse amplification primer of the PTX3 transcriptome is 5'-CTGACAGAGACACAGCATT-3' shown in SEQ ID NO. 5.
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