Application of CCL8 cell factor in kit for early screening esophageal cancer, kit and prediction method

Abstract

The invention relates to the technical field of biological detection, in particular to application of a CCL8 cell factor in a kit for early screening of esophageal cancer, the kit and a prediction method, and the corresponding kit and the prediction method are included. By detecting the content of the CCL8, the correlation between the CCL8 and the morbidity condition of a patient with esophageal squamous carcinoma is disclosed, and a new biomarker is provided for early screening of esophageal carcinoma. Secondly, the CCL8 can recruit immunosuppressive CCR1 + neutrophils, which reveals the immunoregulation mechanism possibly involved in the development process of esophageal cancer, deepens the understanding of the pathogenesis of esophageal cancer, and provides a theoretical basis for the intervention strategy development of CCL8. By detecting the content of CCL8 in serum, early warning of a patient can be realized, and the early diagnosis rate of esophageal cancer is expected to be improved, so that the prognosis of the patient is improved.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to the application of CCL8 cytokine in a kit for early screening of esophageal cancer, a kit and a prediction method. Background Art

[0002] As the sixth leading cause of death in the world, esophageal cancer is one of the diseases that seriously threaten human health in today's society. The large proportion of esophageal cancer patients in my country is an important reason why eastern Asia has become the region with the highest incidence of esophageal cancer in the world. More than half of the nearly 600,000 newly diagnosed esophageal cancer patients worldwide each year occur in my country. The incidence and mortality of esophageal cancer in my country are significantly higher than those in other countries and regions in the world. In contrast to the histological type of adenocarcinoma, which accounts for more than 80% of the cases in the West, more than 97% of the histological types of esophageal cancer in my country are esophageal squamous cell carcinoma (ESCC), accounting for only 1.5% of the cases. In addition, there are huge differences in epidemic characteristics, risk factors for the onset of disease, and gene mutation maps with Western countries. It is a digestive tract malignancy with Chinese characteristics. Therefore, it is necessary to take esophageal cancer patients in China as the research subjects, explore the mechanism of its pathogenesis and progression factors, and provide reliable means for the precise treatment of esophageal cancer patients in China.

[0003] Due to the limited efficacy of traditional treatments for esophageal cancer, mutant genes are constantly being studied as ideal targets for targeted therapy. Although targeted mutant gene therapy has achieved good therapeutic effects in the treatment of solid tumors such as lung cancer and breast cancer, unlike other solid tumors such as lung cancer, except for TP53, the mutation rates of other genes in esophageal cancer are relatively low, which hinders the development and application of targeted therapy in esophageal cancer patients.

[0004] In recent years, as the role of the immune system in the occurrence and development of tumors has been gradually clarified, emerging immunotherapies have shown great potential in clinical applications. The tumor microenvironment in solid tumors can not only affect the patient's prognosis, but the cytokines contained in it are also closely related to the occurrence and development of tumors. As for the application of related immunotherapies in the early screening and treatment of esophageal cancer, screening and identifying key molecules that regulate the immune microenvironment of esophageal cancer can lay a good theoretical and application foundation for preventing the occurrence of esophageal cancer in my country and improving the clinical efficacy of esophageal cancer.

[0005] In existing studies, chemokine (CC motif) ligand 8 (CCL8), also known as monocyte chemoattractant protein 2 (MCP-2), is a member of the chemokine CC family and can be secreted by fibroblasts, monocytes-macrophages, epithelial cells, vascular endothelial cells and smooth muscle cells. Its structural feature is that four cysteine ​​residues in the amino acid sequence form two adjacent disulfide bonds; it plays an important role in the body's inflammatory response and immune response. In addition, CCL8 in the tumor microenvironment can also affect the occurrence and development of tumors. Therefore, CCL8 is considered to be a target for the development of drugs for related human diseases. Although there are studies that have verified the interaction between chemokines and esophageal cancer cells, that is, combining esophageal cancer cell lines and using an in vitro cell co-culture system to find that CCL8 secreted by macrophages promotes the progression of esophageal cancer through NF-kB, these studies focus on the effect of CCL8 on the invasion and migration ability of esophageal cancer cells. There is no clear report on how CCL8 can be used in the prevention and screening of esophageal cancer and how it leads to the occurrence of esophageal cancer. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an application of CCL8 cytokine in a kit for early screening of esophageal cancer, a kit and a prediction method. Since the chemokine CCL8 plays a key role in regulating the immune status of the local tissue microenvironment, this target can be used for early screening of esophageal squamous cell carcinoma patients in the population, laying the foundation for the early prevention and treatment of esophageal cancer, and has important application value for prolonging the patient's quality of life and survival period.

[0007] The present invention is achieved through the following technical scheme: providing the use of cytokine CCL8 as a marker in preparing a kit for early screening of esophageal squamous cell carcinoma.

[0008] Through the above technical scheme, the present invention detects the level of cytokine CCL8 in individual serum to predict the cancer status of an individual. Among them, the name of CCL8 is chemokine CC ligand 8, its molecular weight is 11246Da, and its 3D structure is as follows Fig.12 As shown. Cytokine CCL8 promotes the occurrence and progression of esophageal cancer by specifically binding to its receptor and activating different types of immune cells; that is, cytokine CCL8 plays a key role in regulating the immune status of the local tissue microenvironment. This target can be used for early screening of esophageal squamous cell carcinoma patients in the population, laying the foundation for early prevention and treatment of esophageal cancer, and has important application value for prolonging the patient's quality of life and survival.

[0009] Furthermore, cytokine CCL8 showed high expression levels in the serum of esophageal squamous cell carcinoma patients over the age of 55. Cytokine CCL8 is a chemokine, which not only plays an important role in inflammatory response and immune response by recruiting immune cells, but also plays an indispensable role in the tumor microenvironment.

[0010] Furthermore, the esophageal cancer is esophageal squamous cell carcinoma. The mechanism of action of cytokine CCL8 in esophageal cancer (especially esophageal squamous cell carcinoma) has some unique characteristics compared with other tumors, which makes it more applicable in the prevention and early screening of esophageal squamous cell carcinoma.

[0011] The incidence of esophageal squamous cell carcinoma (ESCC) is high in Asia and sub-Saharan Africa, accounting for approximately 90% of esophageal cancer cases. Therefore, research targeting CCL8 may be more relevant and applicable in these regions. CCL8 is an inflammatory cytokine that can stimulate tumor growth and progression, including esophageal cancer. In the tumor microenvironment of esophageal cancer, CCL8 interacts with immune cells such as neutrophils or tumor-associated macrophages (TAMs) to promote tumor migration and invasion. In addition, CCL8 may be associated with the effect of neoadjuvant immunotherapy for esophageal cancer. For example, studies have shown that CXCL13 (a chemokine like CCL8) enhances the effect of immunotherapy in adjuvant therapy, which may indicate that CCL8 also has a similar role in immunotherapy.

[0012] The present invention also provides a kit for early screening of esophageal squamous cell carcinoma, wherein the kit is an ELISA detection kit for detecting the content of CCL8 cytokine.

[0013] Compared with existing technologies, the CCL8 kit has significant technical advantages in esophageal cancer screening and research. First, the CCL8 kit uses specific antibodies to quantitatively detect CCL8 protein, which can provide highly sensitive and specific results. For example, the CCL8 ELISA kit (ab203366) provided by Abcam can quantitatively measure mouse MCP2 (CCL8) with a sensitivity of 1pg / ml, which helps to detect low-level CCL8 expression in esophageal cancer at an early stage. Secondly, the use of the CCL8 kit is relatively simple, and some kits use single-step sandwich ELISA technology, which can significantly reduce the experimental steps and detection time, improve experimental efficiency, and reduce operational errors. At the same time, the sample applicability of the CCL8 kit is wide, and it can be used for the detection of various sample types such as cell culture supernatant, serum, and plasma to meet different clinical needs; in addition, the CCL8 kit has high stability, can accurately reflect the actual concentration of CCL8 in the sample, and its components usually have a long shelf life and can remain stable under appropriate storage conditions; finally, the CCL8 kit supports large-scale sample testing, which can reduce experimental costs and reduce dependence on professionals. In summary, the CCL8 kit has significant technical advantages in esophageal cancer screening and research, can provide highly sensitive and specific test results, and is easy to operate, cost-effective and efficient. These features make the CCL8 kit a powerful tool for early diagnosis and research of esophageal cancer.

[0014] Finally, a method for detecting the risk of esophageal squamous cell carcinoma in an individual is provided, based on the above kit, comprising the following steps:

[0015] a) collecting a serum sample from the individual to be tested, and using the ELISA detection kit to detect the content of CCL8 in the sample;

[0016] b) comparing the CCL8 content detected in step a) with a preset threshold value for esophageal squamous cell carcinoma;

[0017] c) If the CCL8 content detected in step b) is higher than a preset threshold value for esophageal squamous cell carcinoma, the individual is determined to have a risk of esophageal squamous cell carcinoma.

[0018] Furthermore, in step a), the serum sample is subjected to esophageal tissue CCR1 + Detection of neutrophil ratio.

[0019] Through the above technical solution, the inventors conducted a further screening of immune-related cytokines based on the previous study of differentially expressed cytokines in the serum of 6-8-week-old and 18-month-old mice, combined with mouse esophageal tissue specimens. Preliminary verification results showed that the screened chemokine CCL8 recruited CCR1 +Neutrophils change the local microenvironment, thus leading to the occurrence and development of esophageal cancer.

[0020] Beneficial effects:

[0021] The present invention is based on the fact that chemokine CCL8 plays a key role in regulating the immune status of the local tissue microenvironment. This target is used for early screening of patients with esophageal squamous cell carcinoma in the population, laying the foundation for the early prevention and treatment of esophageal cancer, and has important application value for prolonging the patient's quality of life and survival period. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the result of the analysis of esophageal squamous cell carcinoma patient information in the TCGA database. Figure A shows the age distribution of esophageal squamous cell carcinoma patients in the TCGA database, and Figure B shows the 3-year survival rate of esophageal squamous cell carcinoma patients aged 55 years and above and 55 years and below;

[0023] Figure 2 This is the analysis result of sequencing data of esophageal squamous cell carcinoma patients aged 55 years and above and 55 years and below in the TCGA database. Figure A shows the differential gene analysis results of esophageal squamous cell carcinoma patients aged 55 years and above and 55 years and below; Figure B shows the KEGG pathway enrichment analysis results of differentially upregulated genes in esophageal squamous cell carcinoma patients aged 55 years and above;

[0024] Figure 3 These are the mass spectrometry results of the five cytokines with the highest levels in the serum of wild-type mice aged 6-8 weeks and 18 months and above;

[0025] Figure 4 The transcript levels of five molecules obtained by β-galactosidase detection and mass spectrometry screening in the esophagus of mice aged 6-8 weeks and 18 months and older;

[0026] Figure 5 To score and detect the expression level of CCL8 in the esophagus of mice aged 6-8 weeks and 18 months and older by immunohistochemistry;

[0027] Figure 6 The ELISA method was used to detect the CCL8 content in different groups. Figure A shows the level of CCL8 in the serum of wild-type mice aged 6-8 weeks and 18 months and above, Figure B shows the level of CCL8 in the 24-hour culture supernatant of esophageal tissue of mice aged 6-8 weeks and 18 months and above; Figure C shows the level of CCL8 in the serum of patients with esophageal squamous cell carcinoma aged 55 years and below and over 55 years;

[0028] Figure 7 To show the tumor volume growth curve of subcutaneous tumors in mice aged 6-8 weeks and 18 months and older using the mEC25 cell line;

[0029] Figure 8This is an analysis of the recruitment of neutrophils in cancer patients. Figure A shows the results of ssGSEA immune infiltration analysis of esophageal squamous cell carcinoma patients aged 55 and above and 55 and below in the TCGA database; Figure B shows the immune infiltration score of neutrophils in tumor tissues of esophageal squamous cell carcinoma patients aged 55 and above and 55 and below in the TCGA database; Figure C shows the proportion of neutrophils in the total immune cell population in the blood routine test results of esophageal squamous cell carcinoma patients aged 55 and above and 55 and below collected from the First Affiliated Hospital of Zhengzhou University;

[0030] Fig. 9 After subcutaneous tumors were carried out in mice aged 6-8 weeks and 18 months and above using the mEC25 cell line, the infiltration level of neutrophils in the tumor tissue was detected by immunohistochemistry combined with the neutrophil marker Ly6G;

[0031] Fig.10 The results of the migration experiment of neutrophils by CCL8. Neutrophils from the bone marrow of mice aged 18 months and above were collected by density gradient centrifugation and placed in transwell plates containing different concentrations of CCL8. The number of cells that migrated to the lower chamber was counted after 2 hours, and the transcription level of the main receptor of CCL8 in the neutrophils that migrated to the lower chamber was detected by qRT-PCR, and statistical analysis was performed; Figure A shows the statistical results of the chemotaxis index of the neutrophil migration experiment of mice aged 18 months and above, and Figure B shows the expression levels of CCL8 receptors CCR1, CCR2, CCR3, CCR5, and CCR8 on neutrophils that migrated to the lower chamber detected by qRT-PCR;

[0032] Fig.11 Figure 1 shows the effect of CCL8 on neutrophil function. Figure A shows CCR1 isolated from wild-type mice aged 6-8 weeks and 18 months and older. + After neutrophils were stimulated with different concentrations of CCL8 in vitro, the expression of immunosuppressive molecule TGFβ1 by qRT-PCR; Figure B is a western blot detection of CCR1 isolated from mice aged 18 months and above + The expression of TGFβ1 in neutrophils after stimulation with different concentrations of CCL8 in vitro.

[0033] Fig.12 Schematic diagram of the 3D structure of CCL8. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods in the following examples without specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The reagents and raw materials used in the embodiments and comparative examples of the present invention can be obtained through commercial channels unless otherwise specified.

[0036]

[0037]

[0038]

[0039]

[0040]

[0041] Example 1

[0042] Clinical information and bulk RNA sequencing data of esophageal squamous cell carcinoma patients were obtained from http: / / www.cbioportal.org / study?id=esca_tcga#clinical and https: / / portal.gdc.cancer.gov / projects / TCGA-ESCA for analysis. Figure 1 The pie chart in the figure analyzes and shows the population distribution of esophageal squamous cell carcinoma patients at different ages of onset in the TCGA database. Each color in the pie chart represents a different age group and the percentage of that age group in the total population. Among them, pink (50-59 years old) accounts for the largest proportion, which is 43.01%. This means that among the analyzed esophageal squamous cell carcinoma patient population, nearly half of them are in this age group; light green (60-69 years old) accounts for 24.73%, which is the second largest group; light purple (40-49 years old) accounts for 17.20%, which is the third largest group. The proportion of the remaining age groups is less than 10%.

[0043] From this result, we can see that the two age groups of 50-59 years old and 60-69 years old account for the majority, which indicates that the population structure of this group of esophageal squamous cell carcinoma patients is aging, mainly patients aged 50 years and above ( Figure 1 A). The patients were divided into a group of patients aged 55 years and above and a group of patients aged 55 years and below according to their age of onset. Survival analysis was performed under R4.2.1 version. The results showed that the 3-year survival rate of patients aged 55 years and above was worse than that of patients aged 55 years and below ( Figure 1B). Combined with the bulk RNA sequencing data from the above website, the DEseq2 method was used to perform differential gene analysis on esophageal squamous cell carcinoma patients aged 55 years and above and esophageal squamous cell carcinoma patients aged 55 years and below in the TCGA database, and the ggplot2 package (version 3.5.1) was used to visualize the results in the form of a volcano plot, where genes with |log2Foldchange|≥1 and adjustedP-value<0.05 were considered differentially expressed genes. Each point in the figure represents a gene, where red points represent genes that are significantly upregulated in the group of esophageal squamous cell carcinoma patients aged 55 years and above, and blue points represent genes that are significantly downregulated in the group of esophageal squamous cell carcinoma patients aged 55 years and above. ( Figure 2 A). The above differentially expressed genes that were significantly upregulated in the group of esophageal squamous cell carcinoma patients over 55 years old were extracted and KEGG pathway enrichment analysis was performed. It was found that the expression of genes related to the chemokine signaling pathway was upregulated in esophageal squamous cell carcinoma patients over 55 years old ( Figure 2 B). Considering that serum is an important medium for chemokines to exert their effects, the researchers conducted follow-up research based on bioinformatics analysis and combined samples from C57BL / 6 female mice aged 6-8 weeks and 18 months and above. The specific process is briefly described as follows:

[0044] Screening of differentially expressed proteins in serum

[0045] The inventors conducted mass spectrometry on the serum of C57BL / 6 female mice aged 6-8 weeks and 18 months and above. The results showed that the secretory proteins enriched in the serum of C57BL / 6 female mice aged 6-8 weeks and 18 months and above were different and had differences. The five secretory proteins with the highest expression levels (CCL8, MMP2, MPO, S100A9, M-CSF) were further screened out, among which CCL8 was a typical chemokine and had a higher expression level in the peripheral blood serum of mice aged 18 months and above ( Figure 3 ).

[0046] Example 2

[0047] In Example 1, since the detection of cytokines in the serum of mice aged 6-8 weeks and 18 months and above is a preliminary screening using mass spectrometry detection technology, the screened cytokines need to be further verified and analyzed before their expression can be further confirmed. This example briefly introduces the relevant verification process as follows:

[0048] Detection of differentially expressed important cytokines

[0049] Esophageal tissue specimens of C57BL / 6 female mice aged 6-8 weeks and 18 months and above were collected. Each group of esophageal tissues was divided into two parts, one of which was made into frozen sections. After the sections were rewarmed and washed with PBS, the tissue sections were fixed with β-galactosidase staining fixative. The fixed tissue sections were washed 3 times with β-galactosidase washing buffer at room temperature for 15 minutes each time. The tissue was placed in β-galactosidase staining solution and incubated in 5mL or 10mL of polystyrene to prevent evaporation. The incubation temperature was 30°C. After incubation overnight, the working solution was discarded, the sections were sealed with neutral gum, and the samples were observed and photographed under an optical microscope. It was found that the esophageal tissue of C57BL / 6 mice aged 18 months and above highly expressed β-galactosidase, while the esophageal tissue of C57BL / 6 mice aged 6-8 weeks hardly expressed β-galactosidase ( Figure 4 A).

[0050] Another esophageal tissue was ground using a tissue grinder, and then 1 mL of RNA isoPlus was added to extract RNA. 0.5 mL of chloroform was added to the suspension, and the suspension was mixed by inverting at least ten times. During this period, the centrifuge tube cover was tightly closed. After confirming that the solution had no phase separation, it was placed on ice for 5 minutes, and then centrifuged at 4°C, 12000 rpm / min for 15 minutes. The centrifuge tube was carefully removed from the centrifuge, and the upper colorless transparent liquid was aspirated into a new 1.5 mL enzyme-free EP tube with a 100 μL pipette. An equal volume of isopropanol was added to the collected liquid, the centrifuge tube cover was tightly closed, and the suspension was mixed by inverting at least ten times. After sufficient mixing, it was placed on ice for 10 minutes. After 10 minutes, it was centrifuged at 12000 rpm for 10 minutes, and the supernatant was carefully discarded. 1 mL of pre-cooled 75% ethanol was slowly added along the tube wall, and the supernatant was discarded after centrifugation at 4°C, 12000 rpm / min for 5 minutes. The EP tube was inverted on absorbent paper to remove the ethanol. After the tube was dried, 10-50 μL DEPC water was added according to the amount of precipitation to resuspend the precipitation, and the RNA concentration of each sample was detected by NanoDrop2000. Subsequently, reverse transcription and qRT-PCR were performed to verify the difference in the transcription level of cytokines screened by spectrum detection. The reaction system was prepared using 200 μL EP tubes. First, 1 μL Enzyme Mix and 4 μL 5× All-in-one qRT Super Mix were added to all 200 μL EP tubes. The appropriate volume of RNA of each sample was calculated and added according to the RNA concentration. The volume of the reaction system was supplemented to 20 μL with DEPC water, and the bubbles were removed by mixing with a handheld centrifuge. The reaction program was set on the instrument, 50°C, 15 min, and then 85°C, 5 s, for reverse transcription reaction. The product obtained was the cDNA corresponding to the RNA. Search the transcription level genome sequence numbers corresponding to CCL 8, MMP2, MPO, S100A9, and M-CSF on the NCBI website, and enter the sequence numbers on the Primerblast website for primer design. Design principles: The primer length is preferably between 15-30bp; the GC base content is between 30%-80%, and the GC content difference between the upstream and downstream primers should not be too large; the Tm value is between 58-60°C. After obtaining the primers, place the TaqProUniversalSYBRqPCRMasterMix, primers, and cDNA solution on ice, and operate on ice throughout the process. Then centrifuge the primers at 4°C, 12000rpm / min for 1min, add the corresponding amount of DEPC water according to the mark on the tube wall, dilute the primers to a solution with a concentration of 1μM, and dilute the cDNA solution 5 times with DEPC water.Add 10L Taq Pro Universal SYBR qPCR Master Mix, 4μL upstream primer, 4μ downstream primer, and 2μ diluted cDNA solution to each fluorescent quantitative PCR 8 tube, and configure a qPCR reaction system with a total volume of 20ul. At the same time, a set of GAP DH primers needs to be added to each plate of q-PCR reaction as a reference. Use a handheld centrifuge to remove bubbles, and set the reaction program on the q-PCR instrument as follows: Step 1, pre-denaturation, 95℃, 30 seconds, one cycle; Step 2, cycle reaction, first 95℃, 10 seconds, then 60℃, 30 seconds, cycle 40 times and run the program. After statistical analysis, it was found that the transcription level of CCL8 in the esophagus of mice aged 18 months and above was higher than that of mice aged 6-8 weeks (. Figure 4 B).

[0051] Immunohistochemistry was then used to further evaluate the expression of cytokines. The specific operation is as follows:

[0052] Take the esophageal tissue of C57BL / 6 female mice aged 6-8 weeks and 18 months and above, wash the tissue samples and put them in the fixative, and use the alcohol gradient dehydration: 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol I, anhydrous ethanol II, soak for 1 min respectively. After dehydration, use xylene for transparent treatment, soak in xylene twice, 1 min each time. Put the transparent tissue sample into the melted paraffin, let the paraffin fully penetrate into the tissue sample, put the wax-impregnated tissue sample into the mold, pour the melted paraffin, and then let the paraffin cool and solidify. Fix the embedded tissue block on the sample clamp of the slicer, and adjust the slice thickness of the slicer to 5um. Use a brush to gently remove the cut tissue slices from the knife, unfold them in warm water (40℃), and then bake them at 60℃ for 2h. Soak the slices in xylene I, xylene II and xylene III for 5min respectively to dissolve and remove the paraffin on the slices. Then, the sections were placed in anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol and 70% ethanol, soaked once each for 5 minutes each time; then washed and soaked in purified water for 5 minutes. The sections were soaked in EDTA antigen repair working solution and antigen repair was performed for 20 minutes at a slightly boiling state. The sections were treated with 3% hydrogen peroxide aqueous solution at room temperature for 10 minutes to inactivate endogenous peroxidase. The sections were blocked by incubating the sections at room temperature for 1 hour with 5% bovine serum albumin, and then the sections were incubated overnight with CCL8 antibodies diluted to an appropriate concentration, and then the matching secondary antibodies of appropriate concentrations were added to the tissue sections that had been incubated with the primary antibody and washed, incubated at room temperature for 60 minutes, and then DAB coloring solution was used for coloring after washing, and then the nuclei were counterstained with hematoxylin dye. After completing the above staining steps, dehydration was performed, xylene was used for transparent treatment, and then the sections were sealed with neutral gum and observed under an optical microscope. It was found that compared with mice aged 6-8 weeks, CCL8 was highly expressed in the esophagus of mice aged 18 months and above ( Figure 5 );

[0053] Collect fresh anticoagulated whole blood from patients with esophageal squamous cell carcinoma, wild-type mice aged 6-8 weeks and 18 months and above in 1.5mL EP tubes, centrifuge at 4℃, 1800rpm / min for 5min, and aspirate the supernatant with a pipette and transfer it to a new 1.5mL EP tube. Take the esophagus of mice killed by cervical dislocation, wash the esophageal tissue with PBS buffer, wash off the adhering blood, and then cut the esophageal tissue with ophthalmic scissors. Add the chopped esophageal tissue to a 12-well cell culture plate, soak it in RPMI1640 culture medium, place it in a 37℃ carbon dioxide constant temperature incubator for 24h, collect all the contents in the cell culture plate in a 1.5mL EP tube, centrifuge at 4℃, 1800rpm / min for 5min, and aspirate the supernatant with a pipette into a new 1.5mL EP tube. The above specimens were tested with the corresponding ELISA kits for detecting CCL8. The specific steps are as follows:

[0054] 1) Place the ELISA kit at room temperature for 20 minutes;

[0055] 2) Dilute the washing buffer to the appropriate concentration on ice, and after gradient dilution of the standard sample according to the instructions, design the standard sample wells and sample wells. Add 50uL of different concentrations of standard sample to the standard sample wells according to the concentration gradient, and add 50uL of sample serum or esophageal supernatant to the sample wells.

[0056] 3) Except for the blank control, 100uL of the corresponding CCL8 detection antibody labeled with horseradish peroxidase was added to each well, and then all wells were sealed with a sealing film and incubated at room temperature for 2h.

[0057] 4) After incubation, discard the liquid in the wells and turn the wells upside down on absorbent paper to pat dry. Then add 300uL of washing buffer to each well. After standing for 1 minute, discard the washing buffer and turn the wells upside down on absorbent paper to pat dry. Repeat this washing three times. Seal the plate with a sealing film each time to avoid contamination.

[0058] 5) After the washing process is completed, add 100uL of the mixture of substrate A and substrate B of the ELISA kit to each well, seal the plate with a sealing film, and incubate at room temperature in the dark for 20 minutes. Stop the incubation when dark blue holes appear on the plate. After the incubation is completed, add 50uL of the reaction stop solution to each well to stop the reaction. Within 15 minutes after the reaction is stopped, use an ELISA reader to measure the OD value of each well at a wavelength of 450nm and read the value.

[0059] ELISA detection showed that the levels of CCL8 in the serum and esophageal tissue culture supernatant of mice aged 18 months and above were higher than those in the 6-8 week group of mice ( Figure 6 A and 6B), and the CCL8 content in patients over 55 years old was significantly higher than that in patients 55 years old and below ( Figure 6 C).

[0060] Example 3

[0061] The present application relates to the in vitro culture of tumor cells, wherein the mouse esophageal cancer cell line mEC25 cells are cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Since mEC25 cells have the characteristic of adherent growth, mEC25 cells are cultured in cell culture flasks of specifications T75 and T175, and are passaged when the cell density exceeds 75%. Female C57BL / 6J mice aged 6-8 weeks and 18 months and above are raised in an SPF animal room for one week, and tumors are loaded after confirming that the mice are in a stable state and have no abnormalities. Shave the hair on the right side of the mouse with a mouse shaver, and use a 1mL syringe to subcutaneously inject 1x10 6 mEC25 cells were added to the mice bearing tumors, and the changes in tumor volume were detected every 7 days. It was found that the tumor volume of mice aged 18 months and above increased faster than that of mice aged 6-8 weeks ( Figure 7 ).

[0062] Example 4

[0063] Combined with the bulk mRNA sequencing data of esophageal squamous cell carcinoma patients in the TCGA database, the ssGSEA method was used to score and analyze the immune cell infiltration in esophageal squamous cell carcinoma patients aged 55 years and above and those aged 55 years and below. Figure 8 A) found that neutrophil infiltration was higher in patients with esophageal squamous cell carcinoma aged 55 years or older ( Figure 8 B). The blood routine test results of patients with esophageal squamous cell carcinoma aged 55 years and above and those aged 55 years and below were collected from the First Affiliated Hospital of Zhengzhou University. The results showed that the proportion of neutrophils in the total immune cell population in the peripheral blood of patients with esophageal squamous cell carcinoma aged 55 years and above was significantly higher than that of patients with esophageal squamous cell carcinoma aged 55 years and below. Figure 8 C); mEC25 cell line was used to subcutaneously load tumors in mice aged 6-8 weeks and 18 months and above. After obtaining tumor tissues for wax embedding and obtaining sections, the expression level of neutrophil marker Ly6G in the tumor tissue was detected by immunohistochemistry. It was found that the infiltration level of neutrophils in the tumor tissue of mice aged 18 months and above was significantly higher than that of mice aged 6-8 weeks ( Fig. 9 ).

[0064] In vitro experiments verify the effect of CCL8 on neutrophil migration ability

[0065] Bone marrow cells were collected from wild-type C57BL / 6 mice aged 18 months and above, and neutrophils were isolated using a neutrophil isolation kit. Take a new 15mL centrifuge tube, add 4mL of neutrophil separation kit reagent A to the centrifuge tube, and then carefully superimpose 2mL of separation kit reagent C on reagent A to form a clear gradient liquid surface. Slowly add the resuspended bone marrow cell suspension to the centrifuge tube, superimpose it on the surface of the separation liquid, and keep the liquid surface boundaries between reagent A and reagent C, and between reagent C and cell suspension clear. Centrifuge at room temperature, 1000g, and centrifuge for 30min. Use a pipette to draw the lower neutrophil buffy coat layer into a new centrifuge tube, add 10mL of cell washing solution to wash the cells, centrifuge at 250g for 10min, discard the supernatant, add 5mL of cell washing solution to resuspend the cells, centrifuge at 250g for 5min, discard the supernatant, resuspend the cells and count them, and use 200uL of serum-free RPMI1640 medium to count 2×10 5 The cells were resuspended and added to the upper chamber of the transwell plate, and 650ul of RPMI 1640 medium containing 10% fetal bovine serum and 0nM, 0.01nM, 0.1nM, 1nM, and 10nM CCL8 were added to the lower chamber. After 2 hours, the number of neutrophils that migrated to the lower chamber was counted and the migration index was calculated. It was found that CCL8 could enhance the migration ability of neutrophils ( Fig.10 A); Collect neutrophils that migrated to the lower chamber, extract RNA, and use qRT-PCR to detect the transcription levels of CCL8 main receptors CCR1, CCR2, CCR3, CCR5, and CCR8. It was found that compared with other receptors, CCR1 had the highest transcription level ( Fig.10 B).

[0066] In vitro assay for detecting CCR1 + Functional molecules of neutrophils

[0067] Neutrophils were isolated from wild-type mice aged 6-8 weeks and 18 months and above using a neutrophil isolation kit. Neutrophils were labeled with PE-anti-mouseCD191(CCR1)Antibody flow cytometry antibody. The antibody and cells were incubated on ice in the dark for 15 minutes, centrifuged at 4°C, 1800 rpm / min for 5 minutes, the supernatant was discarded, and the cells were resuspended in DPBS. CCR1 was isolated using a flow cytometer. + Neutrophils were counted and counted as 2 × 10 5 / mL concentration was plated in 24-well plates, and 0nM, 0.01nM, 0.1nM, 1nM, and 10nM CCL8 were added to RPMI1640 medium containing 10% fetal bovine serum and cultured for 6 hours. After the corresponding RNA was extracted, the expression of immunosuppressive molecule TGFβ1 was detected by qRT-PCR. It was found that compared with the 6-8 week old mouse group, the CCR1 of mice aged 18 months and above was + Neutrophils express higher levels of TGFβ1 ( Fig.11 A); CCR1 in mice aged 18 months and above was sorted out using the above-mentioned flow cytometry method +Neutrophils were added to RPMI1640 medium containing 10% fetal bovine serum and 0nM, 0.01nM, 0.1nM, 1nM, and 10nM CCL8, respectively, and cultured for 6 hours. The cells were washed with DPBS buffer and centrifuged at 4°C, 1800rpm / min for 5min. 50-100uL protein lysis buffer was added to each sample according to the cell volume, mixed evenly with a 100uL pipette, and the cells were broken with an ultrasonic disruptor. The supernatant was collected into a new 1.5mL EP tube, and the protein concentration of each sample was detected by the Coomassie brilliant blue method. Then, the protein concentration was adjusted to keep the protein concentration of each sample consistent. 5× loading buffer was added to each sample, and the metal bath was heated at 100°C for 15min. Prepare fast electrophoresis buffer with MOPS-SDS instant granules, then tear off the pink rubber strip at the bottom of the LabPAGE 4-20% 12Wells protein precast gel plate, push the comb out of the precast gel plate smoothly, and then insert the green silicone seal strip of the inner frame of the electrophoresis tank back into the groove of the inner frame with the flat side facing outward. Install the precast gel plate into the electrophoresis device, pour MOPS-SDS Running Buffer into the inner tank of the electrophoresis tank so that it can cover the sample well and exceed 5 to 7mm, and add the same electrophoresis buffer to the outer tank of the electrophoresis tank. Use a medical syringe to absorb an appropriate amount of electrophoresis buffer, gently rinse the sample well, remove bubbles and residual storage buffer, add an appropriate amount of sample and protein marker according to the concentration of sample protein, turn on the power, set the electrophoresis tank parameters to 80V, 30min, then set the parameters to 160V, 40min and run the program. Prepare ice-free rapid transfer buffer, then cut an 8cm×5cm NC membrane, two sponges, and six filter papers, soak them with transfer solution, and put three filter papers together. After taking out the gel, stack them in the transfer folder in the order of sponge-three-layer filter paper-protein gel-NC membrane-three-layer filter paper-sponge, remove bubbles with a small roller, clamp the transfer folder after confirming that the gel is not placed upside down, put it in the transfer tank, pour the transfer solution, turn on the power, and confirm that the positive and negative poles are correct. Constant current 400mA, 30min. After the transfer is completed, put the NC membrane on a small shaker, immerse the NC membrane with 1×TBST buffer, and quickly wash it 3 times at 70rpm / min, each time for 5min, then immerse the NC membrane with rapid blocking solution, and slowly block it at 40rpm / min on the shaker for 20min. After the blocking is completed, immerse the NC membrane with 1×TBST buffer again, and quickly wash it 3 times at 70rpm / min, each time for 5min. The primary antibody and secondary antibody solutions were prepared with universal antibody diluent, and the prepared TGF-β1 primary antibody solution was immersed in the NC membrane, and incubated overnight at 40 rpm / min on a small shaker at 4°C.The next day, the primary antibody dilution was recovered, the room temperature was restored, the NC membrane was immersed in 1×TBST buffer, and the membrane was quickly washed 3 times at 70rpm / min for 5min each time. Then the NC membrane was immersed in the secondary antibody solution, and the membrane was slowly washed at 40rpm / min at room temperature for 1 hour. The secondary antibody dilution was recovered, and the NC membrane was immersed in 1×TB ST buffer and quickly washed 3 times at 70rpm / min for 5min each time. After the NC membrane was slightly dried, it was placed on a black plate, and the prepared ECL luminescent liquid was added, and the image was collected in the instrument. After the image was collected, the NC membrane was immersed in the primary and secondary antibody removal solution, and the membrane was quickly washed at 70rpm / min for 15min, and then the NC membrane was immersed in 1×TBST buffer, and the membrane was quickly washed at 70rpm / min for 3min, and then the membrane was blocked with a fast blocking solution at a slow speed of 40rpm / min for 20min, and then the β-actin primary antibody and the corresponding secondary antibody were re-incubated, and the image was collected in the instrument. Combined with the western blot results, it was found that CCR1. + The expression level of TGFβ1 in neutrophils increased with the increase of CCL8 content ( Fig.11 B).

[0068] Based on the above results, it can be seen that the cytokine CCL8 screened in this application can recruit CCR1 + Neutrophils and promote the expression of TGFβ1. This change in the local tissue microenvironment is closely related to the occurrence and development of esophageal squamous cell carcinoma.

[0069] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of cytokine CCL8 as a marker in the preparation of a kit for early screening of esophageal cancer.

2. The use according to claim 1, characterized in that: The cytokine CCL8 is highly expressed in the serum of patients with esophageal squamous cell carcinoma over 55 years old.

3. The use according to claim 1, characterized in that: The esophageal cancer is esophageal squamous cell carcinoma.

4. A kit for preventing and early screening of esophageal squamous cell carcinoma, characterized in that: The kit is an ELISA detection kit for detecting the content of CCL8 cytokine.

5. A method for predicting the risk of esophageal squamous cell carcinoma, characterized in that: Based on the kit described in claim 4, comprising the following steps: a) collecting a serum sample from the individual to be tested, and using the ELISA detection kit to detect the content of CCL8 in the sample; b) comparing the CCL8 content detected in step a) with a preset threshold value for esophageal squamous cell carcinoma; c) If the CCL8 content detected in step b) is higher than a preset threshold value for esophageal squamous cell carcinoma, the individual is determined to have a risk of esophageal squamous cell carcinoma.

6. The prediction method according to claim 5, characterized in that: In step a), the CCR1 recruited by CCL8 in the serum sample is further analyzed. + Detection of neutrophils.

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