Use of a reagent for detecting CCL4 expression in the preparation of a kit for differentiating glioma from central nervous system inflammatory diseases

Abstract

The present invention relates to the technical field of biomarkers, in particular to the application of a reagent for detecting the expression of CCL4 in the preparation of a kit for differentiating glioma and central nervous system inflammatory diseases. The present invention discovers that CCL4 can be used as a potential biomarker for differentiating glioma and central nervous system inflammatory diseases through serum, which can avoid the secondary damage to patients caused by invasive pathological brain tissue biopsy. The present invention finds that the serum expression of 10 factors including CCL4 is highly expressed in central nervous system inflammatory diseases compared with glioma, but there is no significant effect in cerebrospinal fluid. In addition, among these 10 factors in the central nervous system inflammatory disease group, only CCL4 is highly correlated between serum and cerebrospinal fluid. Therefore, CCL4 can be used as a biomarker for differentiating glioma and central nervous system inflammatory diseases through serum.

Description

Technical Field

[0001] The present invention relates to the technical field of biomarkers, and particularly to the application of a reagent for detecting CCL4 expression in the preparation of a kit for differentiating glioma and central nervous system inflammatory diseases. Background Art

[0002] Glioma is one of the most lethal tumors, causing a high incidence and mortality worldwide. The mortality rate of adult brain tumors is high. Compared with brain tumors, the mortality rate of central nervous system inflammatory diseases is relatively low.

[0003] Computed tomography (CT) or magnetic resonance imaging (MRI) is commonly used to detect brain tumors or central nervous system inflammatory diseases. However, it is difficult to obtain a correct diagnosis solely relying on medical images ( Figure 15 ), which may lead to incorrect medications or treatments. To avoid medical misdiagnosis, such patients need to undergo brain biopsy or surgery to obtain brain lesion tissues for pathological diagnosis. Brain biopsy or surgery is a process of obtaining typical brain lesion areas for subsequent pathological diagnosis and analysis, and is the gold standard for diagnosing brain tumors or central nervous system inflammatory diseases. However, brain biopsy or surgical examination also brings secondary injuries, such as bleeding, neurological deficits, cerebrospinal fluid leakage, cerebral edema, etc. Therefore, there is an urgent need for non-invasive methods for differentiating and diagnosing glioma and central nervous system inflammatory diseases. Summary of the Invention

[0004] To solve the above problems, the present invention provides the application of a reagent for detecting chemokine 4 (CCL4) expression in the preparation of a kit for differentiating glioma and central nervous system inflammatory diseases. The present invention discovers that CCL4 can be used as a biomarker for differentiating glioma and central nervous system inflammatory diseases through serum, which can avoid the secondary injuries brought to patients by invasive pathological brain tissue biopsy.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the application of a reagent for detecting chemokine 4 expression in the preparation of a kit for differentiating glioma and central nervous system inflammatory diseases.

[0007] Preferably, the central nervous system inflammatory diseases include central nervous system inflammatory demyelinating diseases and autoimmune encephalitis.

[0008] Preferably, the central nervous system inflammatory demyelinating diseases include myelitis, intracranial inflammatory demyelinating diseases, and demyelinating lesions.

[0009] The present invention provides a kit for differentiating glioma and central nervous system inflammatory diseases, comprising a reagent for detecting chemokine 4 expression.

[0010] Preferably, the reagent includes a reagent for detecting chemokine 4 by flow cytometry or a chemokine 4 antibody chip.

[0011] Preferably, the reagent for detecting chemokine 4 by flow cytometry includes fluorescent microspheres conjugated with a chemokine 4 antibody, a biotin-labeled chemokine 4 antibody, a fluorescein solution, a sample diluent, a concentrated washing solution, and a chemokine 4 standard.

[0012] Preferably, the fluorescein solution includes a streptavidin-phycoerythrin solution.

[0013] Preferably, the sample diluent includes a diluent containing BSA.

[0014] Preferably, the concentrated washing solution includes a 20-fold concentrated PBS buffer.

[0015] Beneficial effects:

[0016] The present invention provides the use of a reagent for detecting the expression of CCL4 in the preparation of a kit for differentiating glioma from central nervous system inflammatory diseases. The present invention has found that CCL4 can be used as a potential biomarker for differentiating glioma from central nervous system inflammatory diseases through serum, which can avoid the secondary damage caused by invasive pathological brain tissue biopsy to patients. The present invention collected 4 glioma samples (pathological diagnosis, serum, and cerebrospinal fluid) and 5 samples of patients with central nervous system inflammatory diseases (serum and cerebrospinal fluid). By detecting the expression levels of 40 human immune cytokines in the serum and cerebrospinal fluid samples of 9 samples, the present invention has found that the serum expression of 10 factors, namely IL-4, IFN-α, IFN-γ, IL-6, TNF-α, CCL4, P-Cadherin, TRAIL, CCL11, and VEGF, is higher in central nervous system inflammatory diseases than in glioma, but there is no significant effect in cerebrospinal fluid. The present invention also analyzed the correlations between serum and cerebrospinal fluid of these 10 factors in the glioma group and between serum and cerebrospinal fluid in the central nervous system inflammatory disease group, and found that only CCL4 is highly correlated in the serum and cerebrospinal fluid of the central nervous system inflammatory disease group (r = 0.92, 95% CI = 0.20 - 0.99, p = 0.027). Therefore, CCL4 can be used as a biomarker for differentiating glioma from central nervous system inflammatory diseases through serum. Description of the drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0018] Figures 1 to 3 Pathological results of patient A with glioma;

[0019] Figure 4 andFigure 5 The MRI results of patient A with glioma

[0020] Figures 6 to 8 The pathological results of patient B with glioma

[0021] Figures 9 to 11 The MRI results of patient B with glioma

[0022] Figure 12 The differential expression results of 10 inflammatory factors in serum and cerebrospinal fluid

[0023] Figure 13 and Figure 14 The differential expression results of 30 inflammatory factors in serum and cerebrospinal fluid

[0024] Figure 15 The clinical imaging results of patients with undetermined glioma or intracranial demyelinating changes; among them, A is T1, B is T2, C is Flair, and D is enhancement

[0025] Figure 16 The results of the difference interval of the mean expression abundance of CCL4 in the sera of different patients and the mean of CCL4 clinical specimens; among them, A is the result of the expression abundance of CCL4 in the sera of 19 patients with glioma and 22 patients with central nervous system inflammatory diseases, which is statistically significant, p < 0.001; B is the result of the difference interval of the mean of CCL4 clinical specimens Detailed implementation mode

[0026] The present invention provides the use of a reagent for detecting the expression of chemokine 4 in the preparation of a kit for differentiating glioma and central nervous system inflammatory diseases. In the present invention, the central nervous system inflammatory diseases preferably include central nervous system inflammatory demyelinating diseases and autoimmune encephalitis; the central nervous system inflammatory demyelinating diseases preferably include myelitis, intracranial inflammatory demyelinating diseases, and demyelinating lesions.

[0027] In the embodiments of the present invention, serum and paired cerebrospinal fluid samples were collected from 4 patients with glioma and 5 patients with central nervous system inflammatory diseases (the imaging manifestations of these 5 patients were intracranial space-occupying lesions and spinal cord space-occupying lesions). 40 human immune cytokines were detected in these samples, and it was found that the expression levels of serum IL-4, IFN-α, IFN-γ, IL-6, TNF-α, CCL4, P-Cadherin, TRAIL, CCL11, and VEGF in the central nervous system inflammatory disease group were higher than those in the glioma group (p < 0.05). The present invention then performed receiver operating characteristic (ROC) analysis on the immune molecules in the serum and cerebrospinal fluid of these groups. Serum factors such as IL-4, IFN-α, IFN-γ, IL-6, TNF-α, CCL4, CCL11, and VEGF had certain sensitivity and specificity for differentiating central nervous system inflammatory diseases and gliomas (p < 0.05). The present invention further examined the consistency and stability of the abundances of these factors in serum and cerebrospinal fluid, and analyzed the correlations between serum and cerebrospinal fluid in the glioma or central nervous system inflammatory disease groups. There was a significant correlation between serum and cerebrospinal fluid CCL4 in the central nervous system inflammatory disease group (r = 0.92, 95% CI = 0.20 - 0.99, p = 0.027). From the 40 immune factors tested, CCL4 could distinguish central nervous system inflammatory diseases and gliomas, and CCL4 could be used as a biomarker for differentiating gliomas and central nervous system inflammatory diseases through serum.

[0028] After detection, the average expression level of serum CCL4 in the glioma group patients was 916.8 pg / ml, and the average expression level of serum CCL4 in the central nervous system inflammatory disease patients was 1279.0 pg / ml. When differentiating gliomas and central nervous system inflammatory diseases, the reference criteria preferably include:

[0029] 1) If the expression level of CCL4 in the serum to be detected > 1279.0 pg / ml, then the patient is most likely a patient with central nervous system inflammatory diseases;

[0030] 2) If the expression level of CCL4 in the serum to be detected is 916.8 - 1279.0 pg / ml, then a clinical comprehensive analysis of the patient's central nervous system inflammatory diseases is carried out for diagnosis, including: imaging diagnosis and co-diagnosis with other glioma markers;

[0031] 3) If the expression level of CCL4 in the serum to be detected < 916.8 pg / ml, then the patient is most likely a glioma.

[0032] The present invention provides a kit for differentiating central nervous system inflammatory diseases, including reagents for detecting the expression of CCL4.

[0033] In the present invention, the reagent includes a reagent for detecting CCL4 by flow cytometry or a CCL4 antibody chip; the reagent for detecting CCL4 by flow cytometry preferably includes fluorescent microspheres conjugated with CCL4 antibody, biotin-labeled CCL4 antibody, fluorescein solution, sample diluent, concentrated washing solution, and CCL4 standard; the fluorescein solution preferably includes streptavidin-phycoerythrin solution; the sample diluent preferably includes a diluent containing BSA; the concentrated washing solution preferably includes 20-fold concentrated PBS buffer. The reagent for detecting CCL4 expression used in the examples of the present invention is preferably purchased from Beijing Qiangxin Borui Biotechnology Co., Ltd.

[0034] To further illustrate the present invention, the application of the reagent for detecting CCL4 expression provided by the present invention in the preparation of a kit for differentiating central nervous system inflammatory diseases will be described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1

[0036] Inclusion criteria and detection of patients

[0037] A total of 9 patients were enrolled in the present invention, including 4 patients with glioma and 5 patients with central nervous system inflammatory diseases. Serum and cerebrospinal fluid of the above patients were collected after admission and stored in liquid nitrogen in the specimen bank. The 4 patients with glioma were diagnosed by two independent clinical pathologists. All 5 patients with central nervous system inflammatory diseases had evidence of improvement in imaging (magnetic resonance images of the head or spinal cord at admission and at discharge for reexamination).

[0038] After the diagnosis of the patients was confirmed, the serum and cerebrospinal fluid were released from liquid nitrogen in the specimen room, thawed overnight at 4°C, and then sent for detection. Among the 4 patients diagnosed with glioma (partial patient cases and MRI results are shown in Figures 1 to 11 ). Among the 5 patients with central nervous system inflammatory diseases, they were diagnosed as myelitis (2 cases), intracranial inflammatory demyelinating disease (2 cases), and demyelinating lesion (1 case) respectively.

[0039] Using the ABplex Human Cytokine 40 Detection Kit (purchased from Qiangxin Borui Biotechnology Co., Ltd., Beijing, China, product number: RK04368), the expression levels of 40 immune factors in the cerebrospinal fluid and serum of the above 9 patients were detected. The 40 immune factors were: IL-4, IFN-α, IFN-r, IL-6, IL-2, IL-8, IL-10, TNF-a, IFN-β, PDGF-AA, CCL4, PD-L1, P-Cadherin, CCL7, LEPTIN, CCL20, CCL27, G-CSF, CCL3, CCL5, LTF, TRAIL, CCL11, CXCL10, CFD, GRANZYME, β-NGF, EGF, TGF-β1, CCL2, PDGF-BB, NEFL, VEGF, CD152, GM-CSF, CD14, SCF, S100A8, Siglec-5, SST2. According to the kit instructions and combined with the reagents in the kit, the detection steps were as follows: Prepare the standards and reagents, add 50 μL of the standard or add the test sample to each well, add 50 μL of the encoded microspheres to each well, add 50 μL of the detection working antibody and incubate at 37 °C for 0.5 hours, wash once, add 50 μL of SA-PE and incubate, incubate at 37 °C in the dark for 15 minutes, and detect after adding 70 μL of the wash buffer.

[0040] Statistical analysis

[0041] GraphPad Prism version 9.0.0 was used for data analysis and chart production. To compare the concentration differences between central nervous system inflammatory diseases and gliomas, two-way ANOVA variance analysis and multiple comparisons of serum and cerebrospinal fluid of the same patient were used in the present invention, and the results are shown in Figures 12 to 14 , where Brain tumor is glioma, Braininflammation is central nervous system inflammatory disease, serum is serum, and CSF is cerebrospinal fluid. In addition, the present invention also performed receiver operating characteristic (ROC) curve analysis to determine whether the measured potential immune factors could distinguish central nervous system inflammatory diseases and gliomas. This analysis provided the area under the curve (AUC) value and information on specificity, sensitivity, and p-value, and the results are shown in Table 1. Calculation was performed through ROC analysis. Finally, the present invention used the Pearson correlation coefficient to evaluate the correlation of immune factors between serum and cerebrospinal fluid, and the results are shown in Table 2. p < 0.05 was defined as having statistical significance.

[0042] Conclusion

[0043] The present invention found that the expressions of IL-4, IFN-α, IFN-γ, IL-6, TNF-α, CCL4, P-Cadherin, TRAIL, CCL11, and VEGF in the serum between the glioma group and the central nervous system inflammatory disease group were higher than those in the cerebrospinal fluid (IL-4, p = 0.023; IFN-α, p = 0.027; IFN-γ, p = 0.049; IL-6, p = 0.028; TNF-α, p = 0.024; CCL4, p = 0.038; P-Cadherin, p = 0.035; TRAIL, p = 0.022; CCL11, p = 0.013; VEGF, p = 0.012)( Figure 13 and Figure 14 ). The expressions of these 10 factors in the serum of the central nervous system inflammatory disease group were higher than those in the serum of glioma. However, there was no significant difference in the cerebrospinal fluid between the central nervous system inflammatory disease group and the glioma group( Figure 12 ). The expression levels of IL-4, IFN-α, IFN-γ, IL-6, TNF-α, CCL4, CCL11, and VEGF had high sensitivity and specificity between the central nervous system inflammatory disease group and the glioma group (p < 0.05), as shown in Table 1.

[0044] Table 1 Results of ROC analysis of immune molecules in cerebrospinal fluid and serum

[0045]

[0046]

[0047] The present invention also analyzed the correlation between the serum and cerebrospinal fluid of the central nervous system inflammatory disease group and the glioma group. It was found that the expression of CCL4 in the serum of the central nervous system inflammatory disease group was significantly higher than that of the glioma group, and there was a significant correlation between the serum and cerebrospinal fluid of the central nervous system inflammatory disease group (r = 0.92, 95% CI = 0.20 - 0.99, p = 0.027). However, other factors had no obvious statistical significance in different groups, as shown in Table 2.

[0048] Table 2 Correlation analysis of immune molecules in cerebrospinal fluid and serum

[0049]

[0050]

[0051] Example 2

[0052] To further verify the titer of CCL4, 19 glioma patients (imaging + pathological pictures) who had been diagnosed in Tiantan Hospital were collected, and sera were collected from the 19 glioma patients. 22 encephalitis patients (imaging + clinical laboratory tests) who had been diagnosed were collected in Tiantan Hospital, and sera were collected from the 22 encephalitis patients. Through ELISA detection, the test results are shown in Figure 16 .

[0053] As can be seen from Figure 16 , CCL4 in encephalitis sera > glioma sera, which is statistically significant. CCL4 > 1279.0 pg / ml is more likely to be a central nervous system inflammatory disease, and if CCL4 < 916.8 pg / ml, it is more likely to be glioma.

[0054] In summary, CCL4 can be used as a biomarker to distinguish glioma from central nervous system inflammatory diseases through sera. Detecting the expression level of CCL4 in sera can identify or assist in the diagnosis of glioma and central nervous system inflammatory diseases, and can avoid the secondary damage caused by invasive pathological brain tissue biopsy to patients.

[0055] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting the expression of chemokine 4 in the preparation of a kit for differentiating glioma from central nervous system inflammatory diseases.

2. The application according to claim 1, characterized in that, The central nervous system inflammatory diseases include central nervous system inflammatory demyelinating diseases and autoimmune encephalitis.

3. The application according to claim 1, characterized in that, The central nervous system inflammatory demyelinating diseases include myelitis, intracranial inflammatory demyelinating diseases, and demyelinating lesions.

4. The application according to claim 1, characterized in that, The kit for differentiating glioma from central nervous system inflammatory diseases includes a reagent for detecting the expression of chemokine 4.

5. The application according to claim 4, wherein The reagent includes a reagent for detecting chemokine 4 by flow cytometry fluorescence method or a chemokine 4 antibody chip.

6. The application according to claim 5, wherein The reagent for detecting chemokine 4 by flow cytometry fluorescence method includes fluorescent microspheres conjugated with chemokine 4 antibody, biotin-labeled chemokine 4 antibody, fluorescein solution, sample diluent, concentrated washing solution, and chemokine 4 standard.

7. The application according to claim 6, characterized in that, The fluorescein solution includes streptavidin phycoerythrin solution.

8. The application according to claim 6, wherein The sample diluent includes a diluent containing BSA.

9. The application according to claim 6, wherein The concentrated washing solution includes 20-fold concentrated PBS buffer.

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