Application of cisplatin combined with CHI3L1 neutralizing antibody in preparation of medicine for treating ovarian cancer

Abstract

The invention provides a combined drug combination of a CHI3L1 neutralizing antibody and cis-platinum for treating cancers, especially ovarian cancer and / or ovarian cancer ascites. Belongs to the field of biomedicine application. The CHI3L1 neutralizing antibody not only has high affinity, but also can inhibit invasion and metastasis of tumor cells, more accurately attack the tumor cells, reduce damage to normal cells and help patients who are not sensitive to chemotherapy regimens any more or have poor treatment effects. The CHI3L1 neutralizing antibodies of the present invention provide a more effective, safer and economical treatment option for patients with ovarian cancer and / or ovarian cancer ascites, particularly those having limited response to conventional chemotherapy methods. In addition, the development of the combined strategy may inspire more similar researches and promote the further development of the cancer treatment field.

Description

Technical Field

[0001] The present invention relates to the field of biomedical applications, and in particular to the application of cisplatin combined with CHI3L1 neutralizing antibody in the preparation of a drug for treating ovarian cancer. Background Art

[0002] Ovarian cancer is a malignant tumor with a high mortality rate among female cancers worldwide. Since the early symptoms are not obvious, many patients are already in the advanced stage when diagnosed, which brings great challenges to treatment. Although there are many treatment methods for ovarian cancer, advanced ovarian cancer is often accompanied by ovarian cancer ascites, which is an important clinical manifestation of ovarian cancer progression to the late stage. Ovarian cancer ascites is significantly different from ovarian cancer itself. The formation of ovarian cancer ascites is often accompanied by extensive metastasis (stage III or IV), peritoneal implantation and peritoneal inflammatory response. A large amount of ascites accumulates in the patient's abdominal cavity, which can cause symptoms such as abdominal distension, dyspnea, loss of appetite and pain, seriously affecting the patient's quality of life. These symptoms not only limit the patient's life activities, but also cause psychological problems such as anxiety and depression, further increasing the patient's physical and mental burden. In addition, the presence of ascites can also affect the effect of chemotherapy. Ascites is rich in tumor cells and a variety of inflammatory factors, which together promote the survival and spread of tumor cells and increase resistance to chemotherapy drugs. Drug-resistant genes and extracellular vesicles in ascites can transmit drug-resistant signals, making tumor cells resistant to platinum drugs (such as cisplatin) and taxanes (such as paclitaxel). This drug-resistant phenomenon not only reduces the efficacy of chemotherapy, but also accelerates the progression of the disease, making treatment more complicated and difficult. However, the pathogenesis of ascites in ovarian cancer is not fully understood. Current studies have shown that its occurrence may be related to the following factors: 1. Obstruction of lymphatic return: extensive metastasis of tumor cells leads to obstruction of lymphatic return channels in the abdominal cavity, resulting in ascites formation; 2. Abnormal angiogenesis: abnormal angiogenesis in the tumor microenvironment leads to increased vascular permeability, fluid exudation into the abdominal cavity, and the formation of ascites; 3. Peritoneal inflammatory response: the peritoneum is stimulated by tumor cells to produce an inflammatory response, which further aggravates the accumulation of ascites. Although the above pathological mechanisms have been widely discussed, there is currently no effective treatment for targeted ascites formation. Clinically, abdominal puncture and drainage of ascites are mainly used to relieve symptoms, but this method cannot prevent the continuous formation of ascites and may cause complications such as infection. Therefore, developing effective drugs to inhibit or treat ascites in ovarian cancer is of great clinical significance.

[0003] Current technological status and existing problems: 1. Limitations of chemotherapy: Cisplatin is a commonly used chemotherapy drug for the treatment of ovarian cancer. Although the initial treatment effect is obvious, many patients will develop resistance to cisplatin in the long run. In addition, the use of cisplatin is often accompanied by serious side effects, such as nephrotoxicity, neurotoxicity, and hepatotoxicity, which limit its clinical application range and therapeutic dose; 2. Challenges of antibody therapy: Although antibody therapy provides a treatment method for specific cancer markers and has high targeting, it may have limited effects when used alone, especially in highly heterogeneous ovarian cancer. In addition, the cost of antibody therapy is relatively high, and more cost-effective usage strategies need to be found. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to propose a combined drug combination of CHI3L1 neutralizing antibody and cisplatin for the treatment of cancer, especially ovarian cancer and / or ovarian cancer ascites. The present invention aims to provide a more effective, safer and economical treatment option for patients with ovarian cancer and / or ovarian cancer ascites, especially for those patients with limited response to traditional chemotherapy methods. In addition, the development of this combination strategy may also inspire more similar studies and promote further development in the field of cancer treatment.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0006] The invention discloses application of a CHI3L1 inhibitor in preparing a medicine for preventing and / or treating ascites caused by ovarian cancer.

[0007] Furthermore, the medicine also includes chemotherapeutic drugs.

[0008] Furthermore, the chemotherapy drug is cisplatin.

[0009] The present invention also discloses a chemotherapy drug sensitizer, which is characterized by a CHI3L1 inhibitor.

[0010] The present invention also discloses a pharmaceutical composition, characterized in that the pharmaceutical composition comprises a CHI3L1 inhibitor and a chemotherapeutic drug.

[0011] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0012] Furthermore, the sensitizer and the pharmaceutical composition are used in drugs for treating ascites caused by ovarian cancer.

[0013] Furthermore, according to the pharmaceutical application, the sensitizer and the pharmaceutical composition, the CHI3L1 inhibitor includes a substance that inhibits the activity of CHI3L1, or a genetic tool that reduces the level of CHI3L1 protein.

[0014] Furthermore, the substance that inhibits the activity of CHI3L1 protein includes CHI3L1 neutralizing antibody.

[0015] Furthermore, the genetic tools for reducing CHI3L1 protein levels include RNA interference, gene editing or gene knockout.

[0016] The present invention includes using genetic engineering technology to construct a plasmid expressing CHI3L1 antigen. After the plasmid is expressed in Escherichia coli, the recombinant protein is purified by Ni-NTA column affinity chromatography. After the protein is purified, the purified protein is electrophoretically detected using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and Western Blot (protein blotting) is used to further verify the correct expression of the protein.

[0017] The present invention also includes obtaining high-specificity neutralizing antibodies against CHI3L1 by immunizing C57BL / 6 mice, and using ELISA to detect the antibody titer to screen antibodies with high affinity.

[0018] The present invention also includes purification of the antibody using Protein G column chromatography technology, followed by SDS-PAGE and HPLC (high performance liquid chromatography) to detect the purity of the antibody, to ensure that the antibody has sufficiently high specificity and purity.

[0019] The present invention also includes the use of bioinformatics databases (such as GEPIA, TCGA, GTEx) to analyze the expression of CHI3L1 in ovarian cancer tissues and normal ovarian tissues. The results showed that the expression of CHI3L1 in ovarian cancer was significantly increased, especially in advanced ovarian cancer and high-grade patients, where the expression level was significantly higher than that in early cases.

[0020] The present invention also includes detecting the expression of CHI3L1 mRNA in ovarian cancer tissue samples by RT-qPCR (real-time quantitative PCR), and detecting the distribution and expression intensity of CHI3L1 protein in different tissues by immunohistochemistry (IHC), to further confirm its overexpression in tumor tissues.

[0021] The present invention also includes in vivo experiments in which ovarian cancer cell line ID8 (5×10 6 Cells were injected intraperitoneally into C57BL / 6 mice to establish peritoneal implantation and metastasis of ovarian cancer. After cell injection, the weight and abdominal circumference of the mice were monitored every three days.

[0022] The present invention also includes a mouse model in which the mouse was treated by intraperitoneal injection of neutralizing anti-CHI3L1 antibody (2 mg / kg) twice a week for 6 weeks. The control group was injected with an equal amount of isotype control IgG antibody.

[0023] The invention also includes the tumor burden of mice, which is assessed by measuring the volume of ascites, and the mice are dissected at the end point to count the number and volume of metastatic foci. The experiments show that antibody treatment significantly reduces ascites generation and the formation of abdominal metastatic foci.

[0024] The present invention also includes using immunofluorescence (IF) to analyze the expression of CHI3L1 protein in mouse tumor tissue to evaluate the therapeutic effect. In addition, H&E staining and Annexin V / PI staining are used to detect the apoptosis of tumor cells.

[0025] The present invention also includes in vitro experiments using lentiviral infection to stably knock down the ID8 ovarian cancer cell line. Chi3l1 genes, and establish stable Chi3l1 Knockdown cell lines (ID8-Scramble, ID8-sh Chi3l1 ). The knockdown efficiency was detected by RT-qPCR and Western Blot to confirm Chi3l1 Significant downregulation at both mRNA and protein levels.

[0026] The present invention also includes evaluating knockdown by CCK-8 (cell proliferation detection kit) experiment Chi3l1 The sensitivity of the cells to cisplatin (DDP) was tested. Chi3l1 The IC50 value of cisplatin in ovarian cancer cells with gene knockdown was significantly reduced, indicating that the knockdown Chi3l1 Genes that enhance the sensitivity of cells to chemotherapy.

[0027] The present invention also includes using flow cytometry to detect the early apoptosis ratio of cells. Through Annexin V / PI double staining experiments, the results show that knockdown Chi3l1 After cisplatin treatment, the early apoptosis of cells increased significantly. In addition, DNA damage was detected by γH2A.X staining. Chi3l1 Cisplatin-induced DNA damage was significantly increased after treatment.

[0028] The present invention also includes using RT-qPCR to analyze the mRNA levels of multiple drug resistance genes in ovarian cancer cells before and after cisplatin treatment, including Abcb1 , Abcc1 , Abcg1 , Abcg2 , Slc31a2 and Cd44 The results showed that knockdown Chi3l1 The expression of these drug-resistant genes was significantly reduced, indicating Chi3l1 It may promote chemotherapy resistance in ovarian cancer cells by regulating these genes.

[0029] The present invention also includes treating an ovarian cancer mouse model by combining a neutralizing anti-CHI3L1 antibody with cisplatin (2 mg / kg). The treatment group received a combined injection of the antibody and cisplatin, while the control group received a single treatment or a control antibody. The changes in the weight, abdominal circumference and tumor burden of the mice were monitored weekly. Further analysis by immunohistochemistry (IHC) showed that the expression of apoptosis markers in the tumor tissues of the mice in the combined treatment group was significantly upregulated, indicating that the combined treatment can effectively promote tumor cell apoptosis.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0031] The present invention provides an innovative method for treating ovarian cancer and / or ovarian cancer ascites. This combined treatment strategy combines a traditional chemotherapy drug (cisplatin) with a novel biological agent (CHI3L1 neutralizing antibody) to enhance the therapeutic effect, reduce side effects, and provide solutions to the limitations of existing treatments.

[0032] Improved efficacy, dual attack mechanism: CHI3L1 antibody can specifically target the molecular mechanisms associated with the progression of ovarian cancer and / or ovarian cancer ascites, such as inhibiting the invasion and metastasis ability of tumor cells, while cisplatin can kill tumor cells. This dual action may complement and enhance each other to improve the overall therapeutic effect.

[0033] Reduced side effects, targeted action: Using CHI3L1 antibodies can more precisely attack tumor cells and reduce damage to normal cells, thereby potentially alleviating side effects associated with traditional chemotherapy, such as toxicity and damage to non-target tissues.

[0034] Overcoming drug resistance and avoiding or delaying the development of resistance: Resistance to chemotherapy drugs is common in patients with ovarian cancer. The use of CHI3L1 antibodies may help overcome resistance to cisplatin because it provides a different mechanism of action and can attack tumor cells that may escape the effects of cisplatin.

[0035] Increase treatment options and provide new hope for refractory or relapsed patients: For patients who are no longer sensitive to or have poor response to traditional chemotherapy regimens (such as single cisplatin treatment), this new combination therapy may provide an effective new option.

[0036] Potential cost-effectiveness and improved treatment efficiency: Although the development of new drugs is costly, if combination therapy can effectively prolong patient survival and reduce frequent treatment switching due to drug resistance, it may be highly cost-effective in the long run. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1Preparation, purification and identification of neutralizing antibody anti-CHI3L1. Among them, A is the structure diagram of recombinant mouse CHI3L1 protein (rmCHI3L1); B is the expression of rmCHI3L1 protein detected by SDS-PAGE; C is the protein expression detected by Western Blot; D is the expression of rmCHI3L1 protein detected by Western Blot; E is the expression of rmCHI3L1 protein detected by SDS-PAGE; F is the relative titer of anti-CHI3L1 antibody in the serum of immune mice detected by ELISA; G is the titer of anti-CHI3L1 antibody under multiple dilution conditions detected by ELISA; H is the antigen specificity detection of anti-CHI3L1 neutralizing antibody, the left figure is the result of Western Blot detection of rmCHI3L1 protein expression by CHI3L1 commercial antibody, 1 and 2 are rmCHI3L1 samples extracted by lysing DE3 Escherichia coli, the right figure is the result of Western Blot detection of rmCHI3L1 protein expression by prepared anti-CHI3L1 neutralizing antibody, 1 and 2 are rmCHI3L1 samples extracted by lysing DE3 Escherichia coli.

[0038] Figure 2 To analyze the correlation between CHI3L1 expression in ovarian cancer and stage and pathological grade. Among them, A is the expression of CHI3L1 in TCGA, GTEx ovarian cancer tissues and normal ovarian tissues in the GEPIA database; B is the analysis of the expression of CHI3L1 in ovarian cancer tissues and normal ovarian tissues in the Kaplan Meierplotter database; C is the prognosis of CHI3L1 expression in 1,400 ovarian cancer patients collected in the Kaplan Meierplotter database; D is the analysis of the correlation between CHI3L1 expression and patient prognosis in the main pathological type of ovarian cancer, serous ovarian cancer, by Kaplan Meierplotter; E is the division of ovarian cancer patients into four groups according to clinical stage, and the analysis of the expression of CHI3L1 in each group; F is the division of patients into four groups according to the degree of malignancy according to the cytopathological rating of ovarian cancer tissue, and the analysis of the expression of CHI3L1 in each group; G is the division of patients into early and late groups based on the above GEO data, and the analysis of the expression of CHI3L1 in their ovarian cancer tissues; H is the retrospective collection of ovarian cancer patients for analysis of the expression of CHI3L1 in each group.

[0039] Figure 3 The effect of knocking down Chi3l1 on ascites formation and metastasis of ovarian cancer. Chi3l1 Cell line construction of ovarian cancer peritoneal implantation metastasis ascites statistics; B is the weight test of mice every 3 days during the model construction; C uses ID8-Scramble, ID8-sh Chi3l1 Cell lines were used to construct a mouse ovarian cancer peritoneal implantation metastasis model, and the statistical results were as follows; D is ID8-Scramble and sh Chi3l1 Statistics of intraperitoneal metastatic lesions in the two groups of mice; E is Chi3l1 Statistics of the total number of intraperitoneal metastases, the number of peritoneal metastases, the weight of the largest intraperitoneal metastases, and the number of renal surface metastases after gene knockdown; F is the number of tumor cells Chi3l1 CHI3L1 protein level in metastatic lesions after gene knockdown; G is Chi3l1 H is the mRNA level of CHI3L1 in ascites.

[0040] Figure 4 Knockdown Chi3l1 It can promote anoikis and apoptosis of tumor cells in ovarian cancer ascites. Chi3l1 In the peritoneal tumor metastasis model constructed by intraperitoneal injection, FACS was used to detect the proportion and number of tumor cells in ascites cells; B was FACS detection of the expression of Annexin V and PI in tumor cells in ascites cells; C was Western Blot technology to detect ID8-Scramble, sh Chi3l1 Expression of tumor cell apoptosis-related genes in ascites of the two groups.

[0041] Figure 5 To evaluate the therapeutic effect of neutralizing antibody anti-CHI3L1 on mouse ovarian cancer model. A is the dosage and frequency pattern of neutralizing antibody treatment; B is the neutralizing antibody treatment effectively inhibiting the progression of ovarian cancer in mice; C is the neutralizing antibody treatment inhibiting the weight, abdominal circumference growth rate and ascites volume of mice; D is the neutralizing antibody treatment inhibiting the peritoneal implantation and metastasis of ovarian cancer mice.

[0042] Figure 6 Neutralizing antibodies combined with DDP treatment inhibited the formation of ascites and metastasis in mice with OC. A shows the dissection of mice to observe metastatic nodules such as the peritoneum and diaphragm in the abdominal cavity; B shows the statistics of the weight of mice and the formation of ascites during the model construction; C shows the tumor statistics of the greater omentum, the largest metastatic lesion.

[0043] Figure 7 This is an analysis of the correlation between CHI3L1 expression and staging and pathological grade in ovarian cancer. A is the detection of DNA damage changes before and after treatment with two stable cell chemotherapy drugs, cisplatin, by γH2A.X staining; B is the detection of the proportion of early cell apoptosis before and after treatment with two stable cell chemotherapy drugs, cisplatin, by flow cytometry AnnexinV, and the peak graph and bar graph are statistically analyzed; C is the detection of two stable cell chemotherapy drugs before and after treatment with cisplatin by RT-qPCR Abcb1 , Abcc1 , Abcg1 , Abcg2 , Slc31a2 and Cd44 Changes in mRNA of drug resistance genes.

[0044] Figure 8 CHI3L1 promotes drug resistance in ovarian cancer cells. A is a fluorescence microscope to detect the infection efficiency of stable cells, and mCherry is a tumor cell infected with lentivirus; B is RT-qPCR verification Chi3l1 C is the Western blot verification of the protein knockdown efficiency of CHI3L1; D is a bar graph showing the statistical results of Western blot; E is a CCK8 experiment to detect the changes in cell viability of the two stably transfected cell lines under different cisplatin concentrations; F is a bar graph showing the IC50 values ​​of the two stably transfected cell lines. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0046] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0047] Example 1. Preparation of Anti-CHI3L1 Antibody.

[0048] 1. Materials and methods.

[0049] 1. Materials

[0050] Reagents: complete adjuvant (Merck, USA); phosphatase inhibitor (Thermo Fisher Scientific, USA); GAPDH antibody (Proteintech, USA); SDS (Solarbio Technology Co., Ltd., Beijing, China); BCA protein quantification kit (Biyuntian Biotechnology Co., Ltd., Shanghai, China); skim milk powder (Bio-Toda Co., Ltd., Beijing, China); chemiluminescent developer (Wanlei Biotechnology Co., Ltd., Shenyang, China); RIPA lysis buffer (Biyuntian Biotechnology Co., Ltd., Shanghai, China); methanol (Beijing Chemical Reagent Factory); color pre-stained protein marker (ThermoFisher, USA) =Country); protein loading buffer (Solaibao Technology Co., Ltd., Beijing, China); ELISA kit (R&D Company, USA); 1.5mL centrifuge tube, 2mL centrifuge tube (Eppendorf Company, Germany); goat anti-mouse secondary antibody (Proteintech Company, USA); SDS-PAGE gel preparation kit (Wanlei Biotechnology Co., Ltd., Shenyang, China); protease inhibitor (Thermo Fisher Scientific Biotechnology Co., Ltd., USA); incomplete adjuvant (Merck Company, USA); PBS buffer (Hyclone Company, USA); PVDF membrane (Millipore Company, USA); Ni-aganose His tag protein purification kit (Kangwei Century Biotechnology Co., Ltd., Beijing, China); hematoxylin stain and eosin stain (Solaibao Technology Co., Ltd., Beijing, China).

[0051] Antibody: Commercial CHI3L1 antibody (Cat. No. 12036-1-AP, Proteintech).

[0052] Cell line: Mouse ovarian cancer cell line ID8 (Mingjin Biotechnology Development Co., Ltd., Shanghai, China).

[0053] Animal model: C57BL / 6 wild-type SPF female mice aged 6-8 weeks (Huafukang Technology Co., Ltd., Beijing, China).

[0054] pET32a-CHI3L1 vector: pET32a-CHI3L1 plasmid and pET32a (+) DE3 Escherichia coli were preserved by the Immunology Teaching and Research Department of Basic Medical College, China Medical University. The pET32a-CHI3L1 plasmid was double-digested and ligated with the 22-381 aa fragment of mouse CHI3L1 and the pET32a plasmid vector, with BamH I endonuclease at the 5' end and Not I endonuclease at the 3' end.

[0055] (2) Methods

[0056] 1. Measure the growth curve of E. coli DE3.

[0057] After thawing the DE3 E. coli, take 3 μL of the E. coli containing the recombinant plasmid and inoculate it into 3 mL LB culture medium containing three antibiotics: chloramphenicol, kanamycin, and ampicillin at a ratio of 1:1000. Place the centrifuge tube in a shaking incubator and culture it overnight at 37°C and 220 rpm. Check whether the bottle cap is loose to ensure that the E. coli can perform aerobic respiration. After the bacteria have been cultured overnight, take 30 μL of the bacterial solution into 3 mL LB culture medium at a ratio of 1:100, take 200 μL of bacteria into a 96-well plate every hour, and use the prepared LB culture medium as a control for 6 hours.

[0058] 2. Identification of the expression of CHI3L1 recombinant protein.

[0059] (1) After thawing the DE3 E. coli, take 3 μL of the E. coli containing the recombinant plasmid and inoculate it into 3 mL of LB culture medium containing three antibiotics: chloramphenicol, kanamycin, and ampicillin at a ratio of 1:1000. Place the centrifuge tube in a shaking incubator at 37°C and 220 rpm and culture overnight. Check whether the bottle cap is loose to ensure that the bacteria can perform aerobic respiration.

[0060] (2) After the bacteria have been cultured overnight, pipette 200 μL of the bacterial solution into 20 mL of LB culture medium at a ratio of 1:100. Place the centrifuge tube in a shaking incubator and culture it at 37°C and 220 rpm for 4 to 6 h. Check whether the bottle cap is loose to ensure that the E. coli can perform aerobic respiration. Check the OD600 value of the E. coli every hour and terminate the culture when the OD600 value is approximately 0.6 h.

[0061] (3) The experiment was divided into a control group and an experimental group. 3 mL of bacterial solution was taken into four 15 mL centrifuge tubes. No IPTG was added to the two centrifuge tubes of the control group, and 21 μL of IPTG was added to the two test tubes of the experimental group. The centrifuge tubes were then placed in a shaking incubator at 19°C and 160 rpm to induce expression for 10 h.

[0062] (4) Pipette 1 mL of the induction bacterial solution into a 1.5 mL EP tube, add 50 μL of lysis buffer directly to resuspend, sonicate the resuspended bacterial solution, with a reference power of 60% W, on for 3 seconds, off for 5 seconds, and place on ice for lysis for 30 minutes. After lysis, add 12.5 μL of loading buffer directly to the total bacterial protein in the EP tube, shake and mix, centrifuge, preheat the heater, and cook at 100°C for 8 minutes. After the sample returns to room temperature, take 10 μL for Western Blot detection, and freeze the remaining sample.

[0063] 3. Optimization of recombinant protein inducer concentration.

[0064] (1) Add 20 mL of LB medium to a 50 mL sterile centrifuge tube, then add 20 μL each of kanamycin, ampicillin, and chloramphenicol. Pipette 70 μL of thawed DE3 Escherichia coli into the centrifuge tube containing the three antibiotics and LB medium. Place the 50 mL centrifuge tube in a shaking incubator at 37°C and 220 rpm and culture overnight. Check that the bottle cap is loose to ensure that the bacteria can breathe.

[0065] (2) After overnight, take 3 mL of bacterial solution into 6 15 mL sterile centrifuge tubes, add 3 μL, 9 μL, 15 μL, 21 μL, 27 μL, and 33 μL of IPTG to the centrifuge tubes respectively, and then place the centrifuge tubes in a shaking incubator at 19°C and 160 rpm to induce expression for 10 h.

[0066] (3) Pipette 1 mL of the induction solution into a 1.5 mL EP tube and centrifuge at 8000 rpm and 20°C for 3 min. Discard the supernatant and keep the precipitate. Add 50 μL of lysis buffer to the retained cell precipitate for resuspending. Ultrasonicate the resuspended bacterial solution at a reference power of 60% W, on for 3 seconds, off for 5 seconds, and place on ice for lysis for 30 minutes.

[0067] (4) After lysis, add 12.5 μL of loading buffer directly to the total bacterial protein in the EP tube, shake and mix, centrifuge, preheat the heater, and cook at 100°C for 8 min. After the sample returns to room temperature, take 10 μL for Western Blot detection, and freeze the remaining sample.

[0068] 4. Optimization of recombinant protein induction time.

[0069] (1) Add 20 mL of LB medium to a 50 mL sterile centrifuge tube, then add 20 μL of kanamycin, ampicillin, and chloramphenicol respectively. Pipette 70 μL of thawed DE3 Escherichia coli into the centrifuge tube containing the three antibiotics and LB medium. Place the 50 mL centrifuge tube in a shaking incubator at 37°C and 220 rpm for overnight incubation. Check whether the bottle cap is loose to ensure that the bacteria can breathe.

[0070] (2) On the second day, 3 mL of bacterial solution was transferred to 6 15 mL sterile centrifuge tubes. 21 μL of IPTG was added to the centrifuge tubes to a final concentration of 0.7 mM. The centrifuge tubes were then placed in a shaking incubator at 19°C and 160 rpm for 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, respectively.

[0071] (3) Pipette 1 mL of the induction solution into a 1.5 mL EP tube and centrifuge at 8000 rpm and 20°C for 3 min. Discard the supernatant and keep the precipitate. Add 50 μL of lysis buffer to the retained cell precipitate for resuspending. Ultrasonicate the resuspended bacterial solution at a reference power of 60% W, on for 3 seconds, off for 5 seconds, and place on ice for lysis for 30 minutes.

[0072] (4) After lysis, add 12.5 μL of loading buffer directly to the total bacterial protein in the EP tube, shake and mix, centrifuge, preheat the heater, and cook at 100°C for 8 min. After the sample returns to room temperature, take 10 μL for Western Blot detection, and freeze the remaining sample.

[0073] (5) After exploring the above conditions, the final optimized conditions were determined to be an inducer IPTG concentration of 0.7 mM and an optimal induction time of 10 h.

[0074] 5. Induced expression and purification of CHI3L1 recombinant protein.

[0075] (1) After thawing the DE3 E. coli, take 3 μL of the E. coli containing the recombinant plasmid and inoculate it into 3 mL of LB culture medium containing three antibiotics: chloramphenicol, kanamycin, and ampicillin at a ratio of 1:1000. Place the centrifuge tube in a shaking incubator at 37°C and 220 rpm and culture overnight. Check whether the bottle cap is loose to ensure that the bacteria can perform aerobic respiration.

[0076] (2) After the bacteria have been cultured overnight, pipette 2.5 mL of the bacterial solution into 250 mL of LB culture medium at a ratio of 1:100, then add 250 μL of phenamycin, ampicillin, and chloramphenicol, respectively. Place the centrifuge tube in a shaking incubator at 37°C and 220 rpm and culture for 4 to 6 h.

[0077] (3) When the bacterial OD value reaches about 0.6, add 250 μL IPTG to the conical flask and place the centrifuge tube in a shaking incubator at 19°C and 180 rpm for 10 h. Pour the bacteria in the conical flask into a 50 mL round-bottom centrifuge tube that has been pre-autoclaved, incubate at 9000 rpm for 5 min, and discard the supernatant. Add natural binding buffer to the precipitated bacteria to resuspend them, and then ultrasonically lyse the bacteria at 37%W, on for 3S, off for 5S, for 20 min.

[0078] (4) After sonication, centrifuge the bacterial solution at 4°C and 12,000 rpm for 15 min, take the supernatant after centrifugation and add it to a centrifuge tube containing 1-2 mL nickel column, and place it in a 4°C cold room on a shaker for 2-3 h. After the recombinant protein is fully combined with the nickel column, add 2-4 mL of rinsing solution, shake and mix, and centrifuge at 4°C and 1,500 rpm for 10 min. After the end, discard the supernatant, and repeat this step twice.

[0079] (5) Add 2 mL of eluent to the nickel column and centrifuge at 4°C and 1500 rpm for 10 min. After centrifugation, transfer the supernatant to another 15 mL centrifuge tube and repeat this step four times. Cut the dialysis bag to a suitable size, pour the eluted supernatant into it, and place the dialysis bag in a beaker filled with PBS solution. Stir slowly in a cold room and change the PBS solution every two hours for a total of five changes, with the fourth change overnight.

[0080] (6) The dialysis bag was placed on polyethylene glycol for concentration. When the volume of the solution in the dialysis bag was concentrated to 1-2 mL, the concentration was stopped. The liquid in the dialysis bag was taken out and centrifuged at 4°C and 9000 rpm for 6 min. The supernatant after centrifugation was used as CHI3L1 recombinant protein for subsequent experiments.

[0081] (7) Western Blot identification after recombinant protein purification: The experiment was divided into three groups, namely, the sample without IPTG inducer, the sample with IPTG inducer induced expression and lysed and extracted protein, and the purified CHI3L1 recombinant protein. A small amount of sample was taken from each of the three groups for electrophoresis. A commercial His-tag monoclonal antibody was used as the primary antibody and incubated overnight in a cold room shaker. The next day, the sample was luminescent. After the luminescence ended, the antibody was stripped with stripping solution, and the sample was re-blocked and incubated with a commercial GAPDH as an internal reference antibody again overnight. The above steps were repeated to luminesce.

[0082] 6. Preparation and validation of anti-CHI3L1 neutralizing antibodies.

[0083] (1) C57BL / 6 wild-type 6-8 week old SPF female mice were purchased from Beijing Huafukang Technology Co., Ltd. and raised in the Experimental Animal Center of China Medical University. The temperature of the breeding environment was maintained at 20-24°C and the humidity was maintained at 50-60%. This experiment has been approved by the Animal Experiment Ethics Committee of China Medical University to ensure that all experimental operations comply with ethical standards and animal welfare requirements. Approval number: 2023653.

[0084] (2) Take 30 mice, mix the CHI3L1 recombinant protein and complete adjuvant in a volume ratio of 1:1, and then immunize the mice subcutaneously at a dose of 50 μg of CHI3L1 protein and 50 μg of complete adjuvant per mouse. The second immunization is performed in the third week, and 30 μg of CHI3L1 protein and 50 μg of incomplete adjuvant are subcutaneously injected per mouse for immunization. Repeat the second operation in the fifth week to immunize the mice again, and the dose is the same as the second time.

[0085] (3) The mice were anesthetized by intraperitoneal injection of polyaldehyde, and the whiskers of the mice were trimmed with scissors. The entire eyeball was pulled out by clamping the base of the eyeball with forceps, and the blood was collected in a 1.5 mL centrifuge tube. After the blood was collected, it was left to stand at room temperature for 20 minutes to coagulate, and finally centrifuged at 4000 rpm and 20°C for 3 minutes. The supernatant after centrifugation was the prepared anti-CHI3L1 neutralizing antibody.

[0086] (4) The purified CHI3L1 recombinant protein was prepared and subjected to SDS-PAGE gel electrophoresis. The experiment was divided into a control group and an experimental group. The control group used a commercial CHI3L1 antibody, and the experimental group used the serum dilution of immunized mice as the primary antibody. After incubation overnight for 12 to 14 hours, goat anti-mouse antibody was used as the secondary antibody for incubation for 1 hour, and finally luminescence was performed to detect the antigen specificity of the neutralizing antibody by comparison with the commercial CHI3L1 antibody.

[0087] 2. Experimental results.

[0088] The experiment successfully prepared and purified rmCHI3L1 protein, and its expression was detected by Western Blot and SDS-PAGE. By ELISA detection, high-titer neutralizing anti-CHI3L1 antibodies were successfully obtained, showing good immunoreactivity ( Figure 1 ).

[0089] Example 2: Analysis of the expression of CHI3L1 in ovarian cancer and prognosis.

[0090] 1. Materials and methods.

[0091] 1. Materials

[0092] Reagents: paraformaldehyde (Sino-pharmaceutical); anhydrous ethanol (analytical grade) (Beijing Chemical Reagent Factory); xylene (Beijing Chemical Reagent Factory); neutral gum (Solaibao Technology Co., Ltd., Beijing, China); chloroform (Beijing Chemical Reagent Factory); isopropanol (analytical grade) (Beijing Chemical Reagent Factory); DEPC water (Solaibao Technology Co., Ltd., Beijing, China); RNAiso plus (Takara); RNA later RNA Stabilization Sloution (Life); SYBR® PremixEXTaqTMII (TliRNaseHPlus) (Takara); PrimeScriptRTreagentkit (Takara); red blood cell lysis buffer (Solaibao Technology Co., Ltd., Beijing, China); Hanks' solution (Nissin); sodium Pyruvate (Life); Penicillin-Streptomycin Solution (×100) (Solerbao Technology Co., Ltd., Beijing, China); FITC-Dextran (Sigma-Aldrich); β-mercaptoethanol (Solerbao Technology Co., Ltd., Beijing, China); DMEM; Penicillin-Streptomycin Mixture (100*) (Solerbao Technology Co., Ltd., Beijing, China); Trypan Blue Staining Solution (Sigma-Aldrich); TMB (Amresco).

[0093] Antibodies: β-actin Rabbit mAb (Cat. No.: 81115-1-RR, proteintech); β-tubulinMousemAb (Cat. No.: M20005XS, Abmart); Cleaved Caspase 3 (Cat. No.: Asp175, CST); Cleaved Caspase 6 (Cat. No.: Asp162, CST); Cleaved Caspase 7 (Cat. No.: Asp198, CST); goat anti-rabbit secondary antibody (Cat. No.: WLA023A, Wanlei Biotechnology Co., Ltd.); goat anti-mouse secondary antibody (Cat. No.: SA00001-1, proteintech).

[0094] sh Chi3l1 -ID8 cell line was maintained by the Department of Immunology, School of Basic Medical Sciences, China Medical University.

[0095] (2) Methods

[0096] 1. Cell culture.

[0097] The experiment used mouse (C57BL / 6) OCID8 cell line, which was cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS), and 100U / mL of penicillin and streptomycin were added to prevent bacterial infection. The culture conditions were 37°C and 5% CO2. To ensure the quality of cell culture, all cell lines were regularly tested for mycoplasma-free to ensure that there was no bacterial and fungal contamination.

[0098] 1.1 Cell recovery.

[0099] (1) Place the pipette and cell culture dish in a clean bench and sterilize with UV for 15 min. Preheat the complete culture DMEM + 10% FBS + 1% double antibody in a 37°C water bath.

[0100] (2) Take out the cell line from the -80°C freezer and immediately place it in a 37°C water bath to thaw rapidly.

[0101] (3) Then centrifuge at 800 rpm for 5 min.

[0102] (4) Discard the supernatant, add 1 mL of complete culture medium and gently pipette to resuspend the cells into a single-cell suspension. Then add the resuspended liquid to the culture dish containing complete culture medium and gently shake to spread the cells evenly.

[0103] (5) The cells were then placed in a cell culture incubator at 37°C and 5% CO2.

[0104] (6) After 12 hours of culture, change the medium.

[0105] 1.2 Cell passaging.

[0106] (1) Disinfect culture dishes and pipettes in the clean bench with ultraviolet light for 20 minutes; preheat complete culture medium, PBS, trypsin and other reagents.

[0107] (2) Take out the cells and observe that the cell density is 80-90%. Spray the cells with alcohol and place them in a clean bench. Gently shake the culture dish to discard the culture medium.

[0108] (3) Add 2 mL of PBS and gently shake the culture dish to wash the cells, then discard the PBS.

[0109] (4) Add 1 mL of trypsin, shake gently to allow the trypsin to soak the entire dish, and leave to digest at room temperature for 2 min.

[0110] (5) Observe the digestion status. The cells gradually become round and some cells are suspended. Discard the trypsin and add 3 mL of complete culture medium to terminate the digestion.

[0111] (6) Gently pipette the cells into a suspended state, transfer them to a 15 mL centrifuge tube, and centrifuge at 800 rpm for 5 min.

[0112] (7) Discard the supernatant, add 3 mL of complete culture medium, and subculture the cells into new culture dishes at a ratio of 1:3.

[0113] (8) Place in an incubator for culture.

[0114] 1.3 Cell cryopreservation.

[0115] (1) Disinfect with ultraviolet light in a clean bench for 20 min; preheat complete culture medium, PBS, trypsin, and serum-free freezing solution.

[0116] (2) Shake gently to clean and discard the original culture medium; sterilize with ultraviolet light in a clean bench for 20 minutes.

[0117] (3) Add 3 mL of PBS and gently shake the culture dish to wash the cells, then discard the PBS.

[0118] (4) Add 1 mL of EDTA-containing pancreatic enzyme, shake gently to allow the pancreatic enzyme to penetrate the entire culture dish, and leave it to digest at room temperature for 2 min.

[0119] (5) Observe the digestion status. The cells gradually become round and some cells are suspended. Discard the trypsin and add 3 mL of complete culture medium to terminate the digestion.

[0120] (6) Use a 1 mL pipette to pipette the cells into a suspended state, transfer them into a 5 mL centrifuge tube, and centrifuge at 800 rpm / min for 5 min.

[0121] (7) Discard the supernatant and add an appropriate amount of serum-free medium to resuspend the cells into a single-cell suspension.

[0122] (8) Take 1 mL of the cell resuspension and place it in a cryovial. After sealing, quickly place it in a -80°C refrigerator. After 24 to 72 hours, move the cryovial to liquid nitrogen and record the storage location.

[0123] 1.4 Establishment of stable lentiviral knockdown cell lines.

[0124] (1) Use lentiviral shRNA expression vector (GV298, Shanghai Jima Pharmaceutical Technology Co., Ltd.) to establish a stable expression cell line of ID8: Design shRNA for the targeted region of CHI3L1 gene, the target sequence is: upstream primer: 5′-CAGCAAGCTACTGATGGGCAT-3′, downstream primer: 5′-ATGCCCATCAGTAGCTTGCTG-3′; use GV298 lentiviral vector, which contains U6 promoter (PU6) to drive shRNA expression, IRES-Cherry fluorescent label, and carries puromycin resistance gene. The designed shRNA sequence was cloned into the multiple cloning site MCS in GV298 vector by double restriction digestion of AgeI and BamHI to construct GV298-CHI3L1-shRNA lentiviral vector containing shRNA.

[0125] (2) The recombinant lentiviral vector GV298-CHI3L1-shRNA was co-transfected with the auxiliary plasmids pVSV-G and psPAX2 into 293T cells using the lipofectamine 3000 (Invitrogen). After 72 h of culture, the 293T cell culture supernatant was collected for virus titer detection and lentiviral particles containing CHI3L1-targeting shRNA were obtained.

[0126] (3) ID8 cell line was measured at 5×10 4 Cells were seeded in a 24-well plate and incubated overnight in an incubator. The cell density was 30-40% the next day.

[0127] (4) Replace the medium with complete medium containing infection enhancement medium (HiTransGA) and add virus. Virus volume = (MOIX number of cells) / virus titer.

[0128] (5) After 12 h of culture, the medium was replaced with complete culture medium and culture was continued in the cell culture incubator for 72 h.

[0129] (6) The cells were subcultured and cultured in a 37°C 5% CO2 incubator for 24 h.

[0130] (7) The medium was replaced with complete medium containing 5 μg / mL puromycin for 5 to 7 days until all uninfected control cells died and stable low expression was obtained. Chi3l1 ID8 cells and their negative controls (labeled as ID8-sh Chi3l1 and ID8-Scramble).

[0131] The construction and packaging of the lentiviral expression vector was completed by Shanghai Jima Pharmaceutical Technology Co., Ltd. The fluorescence microscope, RT-qPCR and Western Blot methods in Example 4 were used to detect the knockdown of ID8 cells. Chi3l1 The results are shown in Figure 8 A, B, C, D.

[0132] 2. Experimental animals.

[0133] The C57BL / 6 wild-type SPF female mice used in the experiment were 6 to 8 weeks old and were purchased from Beijing Huafukang Technology Co., Ltd. Our laboratory used the Cre-LoxP system to Il13ra2 flox / flox Mice were crossed with Cre mice to obtain whole-body conditional knockouts Il13ra2 Transgenic mice. The mice were raised in the Experimental Animal Center of China Medical University. The temperature of the breeding environment was maintained at 22±2℃ and the humidity was maintained at 55%±5%. The mice were raised and managed by full-time animal technicians to ensure that the animals had free access to food and water. All breeding facilities and environments were disinfected regularly to ensure the health of the experimental animals and the reliability of the experimental results. This experiment has been approved by the Animal Experiment Ethics Committee of China Medical University to ensure that all experimental operations comply with ethical standards and animal welfare requirements. Approval number: 2023653.

[0134] 3. Establish an ovarian cancer peritoneal implantation and metastasis model in C57BL / 6 mice.

[0135] (1) Prepare the cells needed for animal experiments in advance. When the cells grow to 80%, digest them with trypsin, count them under a microscope, and resuspend them in PBS to a single cell suspension. Quantify the cells to 5×10 6 200 μL of each cell was placed on ice until use.

[0136] (2) Before inoculation, observe the condition of the mice, weigh them, and mark their ears and record the results.

[0137] (3) Catch and restrain the experimental mice according to the standard "SOP-Lac030 Standard Operating Procedure for the Catch and Restraint of Mice and Rats". Place the mice with their abdomen facing up and their heads down to protect the abdominal organs and prevent damage. Disinfect the abdomen.

[0138] (4) Hold a 1 mL syringe containing 200 μL of cell suspension in your right hand and insert it obliquely into the right middle and lower abdomen.

[0139] (5) After reaching the subcutaneous tissue, advance the needle another 3 to 5 mm at a 45° angle into the abdominal cavity until a feeling of empty space is felt.

[0140] (6) Fix the needle position. Aspirate the needle until there is no blood or thrombus and then push it forward slowly.

[0141] (7) After removing the needle, disinfect it again to confirm that there is no leakage.

[0142] 4. Extract total protein from cell lines.

[0143] (1) Discard the culture medium, wash three times with PBS, and place the culture dish upside down on absorbent paper for 20 seconds.

[0144] (2) Place the culture dish on ice; add an appropriate amount of lysis buffer containing 1% PMSF to the culture dish and slowly shake to digest for 30 minutes.

[0145] (3) Use a sterilized cell scraper to gently and thoroughly scrape the cells into a 1.5 mL EP tube.

[0146] (4) Centrifuge at 12,000 rpm for 5 min at 4°C; transfer the supernatant to a new EP tube for quantification.

[0147] (5) Prepare a 30-fold dilution of the protein sample. Prepare the working solution of BCA reagent A and B at a ratio of 50:1.

[0148] (6) Add 20 μL of protein diluent to each well of a 96-well plate, and then add 200 μL of BCA working solution.

[0149] (7) Incubate at 37°C for 30 min. Detect the OD value at 562 nm using an ELISA reader. Draw a standard curve and calculate the protein sample concentration.

[0150] 5. Western Blot.

[0151] (1) Prepare 8% and 10% separation gels and a 5% upper concentrated gel respectively, place them in a ziplock bag containing electrophoresis buffer and store at 4°C overnight for later use.

[0152] (2) Prepare the sample protein according to the calculated protein sample concentration. Control the protein amount in each well to 30 μg. Add 3 μL of 5× loading buffer, make up to 15 μL with PBS, and denature at 100°C for 10 min. Place the precast gel in the electrophoresis tank, add electrophoresis solution above the sample loading port, and add markers and samples according to the grouping.

[0153] (3) First, the sample is passed through the stacking gel at a constant voltage of 80 V, and then the constant voltage is switched to 120 V for electrophoresis.

[0154] (4) Determine the stopping time of electrophoresis according to the molecular weight.

[0155] (5) Prepare electro-transfer solution, cut the gel as required, activate the PVDF membrane in methanol for 15 seconds, and then equilibrate it in the electro-transfer solution.

[0156] (6) Arrange the membrane transfer clip in the order of cathode-fiber towel-3 layers of filter paper-gel-PVDF membrane-3 layers of filter paper-fiber towel-anode. Note that the process should be carried out in an ice bath electrotransfer solution without bubbles.

[0157] (7) Place the above electroporation clip in the electroporation tank. Note that the electroporation tank needs to be in an ice-water mixture water bath and perform the membrane transfer. The constant current is 200mA and the time required is determined based on the molecular weight of the protein.

[0158] (8) After transfer, wash the membrane gently in TBST washing solution for 5 seconds and discard the washing solution.

[0159] (9) Then place the tube in 5% BSA blocking solution and shake gently on a shaker at room temperature for 2 h.

[0160] (10) Place the blocked PVDF membrane in TBST washing solution and gently wash for 5 seconds. Discard the washing solution, place it in the primary antibody dilution solution, and incubate it at 4°C overnight.

[0161] (11) The next day, wash the incubated PVDF membrane three times with TBST solution, each time for 5 min.

[0162] (12) Place the PVDF membrane in the corresponding species secondary antibody and incubate at room temperature for 1 h with gentle shaking on a shaker.

[0163] (13) Wash the incubated PVDF membrane three times in TBST buffer.

[0164] (14) Prepare the luminescent solution in a 1:1 ratio and develop the image using an ECL imaging system.

[0165] 6. Extract RNA from cells and perform reverse transcription.

[0166] (1) Discard the culture medium, wash three times with pre-cooled PBS, and invert the culture dish on absorbent paper for 20 seconds. Add 1 mL of TRIzol and gently beat the cells to make a cell suspension. Transfer the cells to an enzyme-free EP tube and place them on ice for 5 minutes.

[0167] (2) Add 200 μL of chloroform to each tube, shake for 15 seconds, and then place on ice for 10 minutes.

[0168] (3) Centrifuge at 12,000 rpm for 15 min at 4°C and transfer the supernatant to a new sterile enzyme-free EP tube.

[0169] (4) Add an equal volume of isopropanol, mix thoroughly, and place on ice for 10 min.

[0170] (5) Centrifuge at 12,000 rpm for 10 min at 4°C and discard the supernatant. Prepare 75% alcohol with anhydrous ethanol + DEPC water and use 1 mL to wash the RNA precipitate.

[0171] (6) Centrifuge at 7500 rpm for 5 min at 4°C and discard the supernatant.

[0172] (7) Wash again, same as above.

[0173] (8) Remove the supernatant, air-dry, and add 20 μL DEPC water to dissolve.

[0174] (9) After sufficient dissolution, determine the concentration and prepare for reverse transcription.

[0175] Prepare the reverse transcription solution according to the measured RNA concentration, and perform reverse transcription step 1 (removal of genomic DNA reaction) according to the following system: .

[0176] Note: *1: In a 20μL reverse transcription reaction system, a maximum of 1μg of Total RNA can be used in the TB Green qPCR method.

[0177] 42℃ 2min; 4℃.

[0178] Reverse transcription reaction: .

[0179] 37℃ 15min; 85℃ 5sec; 4℃.

[0180] 7. Real-time quantitative PCR (RT-qPCR).

[0181] The obtained cDNA was used for qPCR loading according to the following system, and then real-time RT-qPCR amplification was performed, and the following program was set: 95℃ pre-denaturation for 30s, followed by 40 cycles, each cycle including 95℃ denaturation for 5s, 60℃ annealing and extension for 30s. Finally, the specificity of the amplified product was verified by the melting curve. The expression level of the target gene was calculated by relative quantification. Chi3l1 : Upstream primer: 5′-GCCTACTACGAGATATGCGACTTCC-3′, downstream primer: 5′-TCCTGCCAGCTTCTTCTCCTTCA-3′; Abcb1 : Upstream primer: 5′-AATGTTTCGTTATGCAGGTTGGC-3′, downstream primer: 5′-TGGCTCTTTTATCGGCCTCAC-3′; Abcc1 : Upstream primer: 5′-GACCGGGGCTACATCCAGAT-3′, downstream primer: 5′-TGTTGGGCTGACCAGTAACAC-3′; Abcg1: Upstream primer: 5′-TCATTGGCCTGCTGTACCTG-3′, downstream primer: 5′-TCTCCAGGGGAAAGGTCAGA-3′; Abcg2 : Upstream primer: 5′-TCCCCATGAGGTTCTTGCCA-3′, downstream primer: 5′-AGAGGCCAGAAAAGAGCATCATAA-3′; Slc3la2 : Upstream primer: 5′-GGCAAAGCCAAATTGCTCCA-3′, downstream primer: 5′-ACACAAAAACCACCTGAGGC-3′; Cd44 : Upstream primer: 5′-TCGATTTGAATGTAACCTGCCG-3′, downstream primer: 5′-CAGTCCGGGAGATACTGTAGC-3′; Actb : Upstream primer: 5′-GTGACGTTGACATCCGTAAAGA-3′, downstream primer: 5′-GCCGGACTCATCGTACTCC-3′. All RT-qPCR primers were synthesized by Shanghai Bioengineering Co., Ltd.

[0182] .

[0183] 8. Extraction of ascites cells.

[0184] (1) Kill the mouse, spray the surface with alcohol, remove the skin to expose the peritoneum, and gently draw out the ascites with a 20 mL syringe. Centrifuge at 1500 rpm for 5 min, and draw out the supernatant and store it at -80°C.

[0185] (2) Add 3 mL of red blood cell lysis buffer and lyse at 4°C for 3 min. Add PBS to 14 mL to terminate lysis. Centrifuge at 1500 rpm for 5 min.

[0186] (3) Discard the supernatant, resuspend the cells in FACS-HANKS and filter them through a 200-mesh nylon membrane; adjust the volume to 2 mL FACS-HANKS and count.

[0187] (4) Aliquot 50 μL for antibody staining.

[0188] 9. Extraction of immune cells from peritoneal metastases of ovarian cancer.

[0189] (1) Anesthesia.

[0190] (2) Shave the hair and open the abdomen. Remove the greater omentum tissue below the greater curvature of the stomach, place it in a 10 cm culture dish, wash it with cold PBS, and cut it into pieces with ophthalmic scissors.

[0191] (3) Prepare a 2 mL EP tube ①, add 1.5 mL of serum-free DMEM culture medium (containing 0.4% collagenase I), add the shredded greater omentum to tube ①, and 1.6 g of collagenase I.

[0192] (4) Incubate at 37°C for 30 min with rotational mix at 250-300 rpm.

[0193] (5) Filter through a nylon membrane with a pore size of 200.

[0194] (6) Centrifuge at 250–800 g for 5 min at 4°C. Discard the supernatant.

[0195] (7) Add 1 mL of PBS to resuspend and count.

[0196] 10. Flow staining.

[0197] (1) Resuspend the cells to a concentration of 1×10 using FACS-HANKS. 6 Single cell suspension at 100 cells / mL.

[0198] (2) Dilute the antibody according to the flow cytometry antibody instructions and add it to the cell tube.

[0199] (3) Incubate at 4°C in dark for 30 min.

[0200] (4) Add 1 mL of FACS-HANKS, mix thoroughly, and centrifuge at 5500 rpm for 1 min to wash away unbound antibodies.

[0201] (5) Add 300 μL FACS-HANKS (adjust the buffer volume according to the number of cells) and gently resuspend the cells.

[0202] (6) Add 1 μL 7AAD to each tube 10 min before flow cytometry.

[0203] (7) Before testing, cells need to be gently blown and filtered through a 300-mesh filter.

[0204] (8) When testing on the machine, data can only be collected after the voltage stabilizes.

[0205] 11. Ascites tumor cell sorting.

[0206] (1) Turn on the flow cytometry sorting machine and prepare the sample cells to be sorted after it runs stably.

[0207] (2) The cell concentration was set at 1×10 7 / mL, stain according to the established staining protocol for the cells to be sorted; then resuspend in complete culture medium.

[0208] (3) Debug the machine until it passes quality control, select and sort the appropriate nozzles, clean them, and then install them.

[0209] (4) Obtain the template in the system, draw the corresponding scatter plot, peak plot, etc. according to the staining channel, and perform gating.

[0210] (5) The cells are gently mixed and filtered through a 300-mesh filter before being put into the machine. Some cells are collected and then compensation and voltage adjustment are performed.

[0211] (6) Apply the same voltage and supplement the selected cells from the same batch and place them in complete culture medium.

[0212] (7) Each tube of cells must be tested for purity after sorting, and the purity must be >95%.

[0213] (8) The cells are then cultured or stored in a -80°C freezer.

[0214] 12. Immunohistochemical staining of human tissues.

[0215] (1) Remove the tissue, fix it in 4% paraformaldehyde to denature the protein in the tissue, and place it in a -4°C refrigerator away from light.

[0216] (2) Rinse with water to remove excess fixative and dehydrate in different concentrations of alcohol.

[0217] (3) Transparent the specimen by soaking it in xylene to replace the alcohol in the tissue.

[0218] (4) Pour the preheated paraffin into the tissue module and completely immerse it in the wax melting box for 2 hours before cooling and solidifying.

[0219] (5) Place the wax block on a microtome and slice it. First, remove the uneven parts and take the appropriate section for slicing. The thickness is about 4-5 μm. Place it in a warm water bath, spread the slices, and then pick them up and bake them.

[0220] (6) After baking, store in a dry and cool place at room temperature for later use.

[0221] (7) Before dyeing again, the slides need to be baked at 55°C for 30 minutes.

[0222] (8) The sections were then placed in xylene (I) for 10 min and xylene (II) for 10 min. They were then immersed in anhydrous ethanol and different concentrations of alcohol in a gradient manner (anhydrous ethanol (I) - anhydrous ethanol (II) - 95% ethanol - 90% ethanol - 80% ethanol - 70% ethanol - distilled water) for 5 min each to dewax and hydrate.

[0223] (9) Place the slices in a 100°C water bath with citric acid buffer for 15 min, then heat them intermittently in a microwave oven for a total of 10 min; cool them naturally to room temperature, and gently wash them three times with PBS.

[0224] (10) Add 3% H2O2 for 15 min; block with 5% BSA at 37°C for 2 h. Keep the slides in a humidified box throughout the process to prevent them from drying out.

[0225] (11) Discard the blocking solution, circle the outer edge of the tissue with a histochemical pen, add the primary antibody dilution solution, and place in a humidified box at 4°C overnight to prevent the slides from drying out.

[0226] (12) Wash gently with PBS three times, add secondary anti-horseradish enzyme (reagent 2) and incubate in a 37°C wet box for 30 min.

[0227] (13) Wash gently with PBS three times for 5 min each time.

[0228] (14) Add DAB staining solution and observe the process under a microscope for about 50 s. Terminate the reaction and rinse gently with tap water.

[0229] (15) After staining the sections in hematoxylin for 5–6 min, rinse gently with tap water and observe the depth of staining under a microscope. Differentiate with 1% hydrochloric acid alcohol for 9 s and then rinse gently with tap water to restore the blue color.

[0230] (16) Dehydrate the slices in the reverse order of gradient alcohol and xylene (95% ethanol (I)-95% ethanol-(II)-absolute ethanol (I)-absolute ethanol (II)-xylene) for 5 min each. Remove the slices and air dry them; add neutral resin to seal the slices gently and slowly to avoid bubbles. After drying, take photos under a microscope.

[0231] 13. Bioinformatics analysis.

[0232] In the experiment, GEPIA was used to analyze the mRNA expression of CHI3L1 in different types of ovarian cancer tissues, and the Correlation module was used to analyze the correlation between CHI3L1 and molecules such as PD1 in OV. KM Plotter was used to analyze the correlation between the high and low expression of CHI3L1 in different types of ovarian cancer and the survival time of patients. The TCGA database was used to screen the differential genes between the high and low expression groups of CHI3L1, and enrich the signal pathways that may be involved. The GEO database ovarian cancer-related data sets GSE51373, GSE30161, GSE9891, GSE65986, GSE63885, GSE18520, GSE15622, GSE14764, GSE26712, GSE26193, GSE23554, GSE19829, and GSE27651 were collected, and the data were cleaned after merging. The prognosis was analyzed by R package, and the clustering and pathway enrichment were analyzed by WGCNA.

[0233] 14. Statistical analysis.

[0234] The data used in this study were statistically analyzed in SPSS version 25.0 using Student's t -test, One-way ANOVA and other methods were used to analyze whether the differences within and between groups were statistically significant. Pearson's Correlation Coefficients were used to analyze the correlation. All data were recorded in the form of mean ± standard deviation (x ± s), and the data distribution was tested for normality before statistical analysis. P <0.05 was used as the standard to determine whether the difference in experimental data was statistically significant.

[0235] 2. Experimental results.

[0236] Through big data analysis and database verification, we found that the expression of CHI3L1 in ovarian cancer tissues was significantly higher than that in normal ovarian tissues, and its expression level was closely related to the clinical stage of the tumor, pathological grade and patient prognosis. In particular, in patients with advanced and high-grade ovarian cancer, the expression of CHI3L1 was significantly increased, and the overall survival rate of patients with high expression was significantly reduced, suggesting that CHI3L1 may be used as a biomarker for the malignant progression of ovarian cancer ( Figure 2 ).

[0237] By constructing an ovarian cancer peritoneal implantation metastasis model in a mouse model, the functional role of CHI3L1 in ovarian cancer metastasis was further verified. Chi3l1 After knocking out the gene, the experiment showed that the volume of ascites in mice was significantly reduced, and the number and size of tumor metastases were also significantly reduced, indicating that CHI3L1 plays a key role in promoting the metastasis and spread of ovarian cancer. Chi3l1 The gene also reduced the implantation and spread of ovarian cancer cells in the peritoneum and on organ surfaces, further supporting its important role in tumor metastasis ( Figure 3 ).

[0238] It is worth noting that knockdown Chi3l1 The gene significantly promoted anoikis in ovarian cancer cells, indicating that CHI3L1 enhanced the survival of tumor cells by inhibiting apoptosis. Chi3l1 After knockdown of the gene, the activation levels of Caspase 3, Caspase 6, and Caspase 7 in tumor cells increased significantly, suggesting that CHI3L1 promotes the survival of tumor cells in a harsh microenvironment by blocking the apoptosis pathway. Chi3l1 The gene can significantly increase the proportion of Annexin V-positive cells in tumor cells, indicating that it promotes tumor cell anoikis ( Figure 4 ).

[0239] Example 3: Treatment of ovarian cancer ascites with CHI3L1 inhibitors.

[0240] 1. Materials and methods.

[0241] 1. Establish a C57BL / 6 mouse ovarian cancer peritoneal implantation metastasis model using the same method as in Example 2.

[0242] 2. Starting from the third week, the neutralizing antibody was injected intraperitoneally twice a week until the sixth week.

[0243] 3. At the end of the experiment, the weight, abdominal circumference, ascites volume, number of metastatic lesions in major organs, and weight of the largest metastatic lesion of the mice were recorded.

[0244] 2. Experimental results.

[0245] CHI3L1 neutralizing antibodies showed good therapeutic effects in ovarian cancer mouse models. Antibody treatment significantly reduced ascites production in mice, inhibited the growth of body weight and abdominal circumference, and effectively inhibited the implantation and metastasis of ovarian cancer cells in the abdominal cavity ( Figure 5 ).

[0246] Example 4: CHI3L1 inhibitors are used in combination with cisplatin to treat ovarian cancer.

[0247] 1. Materials and methods.

[0248] 1. Materials

[0249] Annexin V-FITC / PI double staining apoptosis test (Jiangsu KeyGen Biotech Kit); Cell Counting Kit-8 (CCK-8) (Biosharp); IHC blocking solution (specially for mouse tissue to avoid cross-reaction) (Abmart); anti-fluorescence attenuation mounting medium containing DAPI (Solabo Technology Co., Ltd., Beijing, China).

[0250] (2) Methods

[0251] 1. CCK8 resistance test.

[0252] (1) Grow the subcultured cells to a density of 80-85%. After trypsin digestion, gently pipette complete medium into a cell suspension. Take 3×10 cells. 4 The cells were inoculated at a density of 100 μL / mL in a 96-well culture plate, and 100 μL of the cell suspension was added to each well and placed in an incubator overnight.

[0253] (2) After the cells were observed to be attached to the wall, the medium was replaced with complete culture medium containing PBS, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL of cisplatin.

[0254] (3) Terminate the culture after 12 h, 24 h, and 36 h, respectively.

[0255] (4) Add 10 μL of CCK8 solution to each well, and reserve blank wells and medium-only wells.

[0256] (5) After incubation at 37°C for 2.5 h, measure the absorbance at 450 nm, and use 650 nm as a control wavelength.

[0257] 2. Cell Immunofluorescence.

[0258] (1) Add 50 μL of culture medium to a 24-well plate and place a sterile cell slide into the well.

[0259] (2) Take cells in the logarithmic growth phase and inoculate them on a cell slide. Add 500 μL / well of culture medium and wait for the cells to adhere for 12 hours.

[0260] (3) After the cells adhere to the wall, discard the culture medium and replace it with drug-containing culture medium for 24 hours.

[0261] (4) After drug treatment, fix the slides with 4% paraformaldehyde for 30 min. Discard the supernatant and wash the slides with PBS three times, 5 min each time.

[0262] (5) Add 200 μL of blocking solution to each well and incubate at room temperature for 1.5 h.

[0263] (6) Add primary antibody and incubate at 4°C overnight.

[0264] (7) Recover the primary antibody and wash the slides with PBS three times, 5 min each time.

[0265] (8) Add secondary antibody and incubate at room temperature for 2 h.

[0266] (9) Wash the slides three times with PBS, 5 min each time.

[0267] (10) DAPI was added for staining and the cells were observed and photographed under a fluorescence microscope.

[0268] 3. Flow cytometry apoptosis staining.

[0269] (1) After ID8 cells were treated with DDP, they were digested with trypsin without EDTA. After the digestion was terminated with culture medium, the cells were collected by centrifugation at 800 g for 5 min.

[0270] (2) Wash the cells twice with PBS and collect 1×10 5cells, add 500 μL Binding Buffer and mix well.

[0271] (3) Add 5 μL Annexin V-FITC, mix well, and react at room temperature in the dark for 10 min.

[0272] (4) After the reaction is completed, the cell suspension is filtered through a 300-mesh filter into a sterile flow cytometer for flow cytometry testing.

[0273] 4. Construct a C57BL / 6 mouse peritoneal implantation and transfer cisplatin combined with CHI3L1 neutralizing antibody treatment model.

[0274] (1) The abdominal implantation and metastasis model of ovarian cancer in C57BL / 6 mice was established in the same manner as in Method 3 of Example 2.

[0275] (2) Neutralizing antibodies and chemotherapeutic drugs were intraperitoneally injected twice a week starting from the third week after modeling and continued until the sixth week.

[0276] (3) At the end of the seventh week of the experiment, the weight, abdominal circumference, ascites volume, number of metastatic lesions in major organs, and weight of the largest metastatic lesion of the mice were recorded.

[0277] 2. Experimental results.

[0278] In in vitro experiments, Chi3l1 The gene knockdown of significantly increased the sensitivity of ovarian cancer cells to cisplatin. Chi3l1 After knockdown of the gene, DNA damage in ovarian cancer cells after cisplatin treatment increased significantly, suggesting that CHI3L1 may enhance chemotherapy resistance by protecting cells from DNA damage. Chi3l1 The gene significantly increased cisplatin-induced apoptosis, further demonstrating the negative regulatory role of CHI3L1 in chemotherapy sensitivity. RT-qPCR results further revealed that Chi3l1 After gene knockdown, multiple drug resistance genes (such as Abcb1 , Abcc1 , Abcg1 , Abcg2 The expression of CHI3L1, CHI3L1-associated gene, and CHI3L1-associated gene were significantly decreased in ovarian cancer. The downregulation of these genes may be one of the reasons for the enhanced sensitivity of cells to cisplatin chemotherapy. This result shows that CHI3L1 plays a key role in ovarian cancer chemotherapy resistance by regulating the expression of resistance-related genes. Figure 6 ).

[0279] In the in vivo experiment, anti-CHI3L1 antibody combined with cisplatin treatment showed significant therapeutic effects in the ovarian cancer mouse model. Compared with the control group treated with cisplatin or antibody alone, the ascites formation, weight gain and abdominal metastasis of the mice in the combined treatment group were significantly inhibited. The number of abdominal metastatic foci such as peritoneum and diaphragm in the combined treatment group was greatly reduced, indicating that neutralizing CHI3L1 can effectively inhibit the peritoneal implantation and metastasis of ovarian cancer cells ( Figure 7 ).

[0280] Cell viability assay (CCK8) and IC50 analysis results showed that knockdown Chi3l1 After knockdown of the gene, the sensitivity of ovarian cancer cells to cisplatin increased significantly, indicating that CHI3L1 may mediate chemotherapy resistance in ovarian cancer by enhancing cell survival and resisting DNA damage. Chi3l1 The gene also significantly inhibited the expression of multiple drug-resistant genes, further illustrating the core regulatory role of CHI3L1 in the drug resistance mechanism of tumor cells ( Figure 8 ).

[0281] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of CHI3L1 inhibitors in the preparation of drugs for preventing and / or treating ascites caused by ovarian cancer.

2. The use according to claim 1, characterized in that: The drugs also include chemotherapeutic drugs.

3. The use according to claim 2, characterized in that: The chemotherapy drug is cisplatin.

4. A chemotherapeutic drug sensitizer, characterized in that: CHI3L1 inhibitors.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a CHI3L1 inhibitor and a chemotherapeutic drug. The pharmaceutical composition according to claim 5 further comprises a pharmaceutically acceptable carrier.

7. Use of the sensitizer according to claim 4 and the pharmaceutical composition according to claim 5 in a drug for treating ascites caused by ovarian cancer.

8. The CHI3L1 inhibitor in the pharmaceutical use according to claim 1, the sensitizer according to claim 4, and the pharmaceutical composition according to claim 5 comprises a substance that inhibits CHI3L1 activity, or a genetic tool that reduces CHI3L1 protein levels.

9. The use according to claim 8, characterized in that: The substance that inhibits the activity of CHI3L1 protein includes CHI3L1 neutralizing antibody.

10. The use according to claim 8, characterized in that: The genetic tools for reducing CHI3L1 protein levels include RNA interference, gene editing or gene knockout.

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