Human extrahepatic bile duct cancer cell line EBC-X1 and its applications

Abstract

Human extrahepatic bile duct cancer cell line EBC-X1 and its applications belong to the field of microbial animal cell lines. The human extrahepatic bile duct cancer cell line is named human extrahepatic bile duct cancer cell line EBC-X1, and the preservation number is CCTCC No: C2024318. The human extrahepatic bile duct cancer cell line EBC-X1 can be used in the establishment of cell models for the occurrence, development or metastasis of extrahepatic bile duct cancer. The human extrahepatic bile duct cancer cell line EBC-X1 can be used in cell models for studying the differentiation mechanism, abnormal cell morphology and function, tumor invasion and metastasis mechanism of extrahepatic bile duct cancer and guiding clinical comprehensive diagnosis and treatment. The human extrahepatic bile duct cancer cell line EBC-X1 can be used in studying the pathogenesis of extrahepatic bile duct cancer and screening drugs for the prevention and treatment of extrahepatic bile duct cancer. The extrahepatic bile duct cancer cell line EBC-X1 can be used in the establishment of animal models of extrahepatic bile duct cancer.

Description

Technical Field

[0001] The present invention belongs to the field of microbial animal cell lines, and particularly relates to a Chinese-derived extrahepatic bile duct cancer cell line and its application. Background Art

[0002] Cholangiocarcinoma is a highly heterogeneous primary malignant tumor originating from the biliary tree. According to the anatomical location, cholangiocarcinoma can be roughly divided into intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma can be further divided into hilar cholangiocarcinoma and distal cholangiocarcinoma according to the position of the cystic duct. Embryologically, intrahepatic bile duct cells and extrahepatic bile duct cells have different origins. The former originates from bipotential hepatoblasts, and the latter originates from the ventral foregut region near the liver and ventral pancreas. Due to the different origins of cholangiocarcinoma subtypes, they exhibit significant clinical, epidemiological, molecular, and genetic heterogeneity. The incidence and mortality of cholangiocarcinoma show an overall upward trend globally. Since most cholangiocarcinoma patients are in the advanced stage at the time of diagnosis, the prognosis is very poor. Only about 30% of patients have the opportunity for surgical resection, and the 5-year survival rate is only 7% - 20%. Therefore, cholangiocarcinoma remains a major public health problem globally and requires more basic research to support the progress of clinical diagnosis and treatment.

[0003] Tumor cell lines can reflect the characteristics of tumors to a certain extent and are the most widely used models in tumor research. In the past few decades, tumor cell lines have greatly promoted the understanding of tumor biology and the development of anti-tumor drugs. A perfect tumor cell line library should reflect the diversity of tumor phenotypes and be able to provide cell lines with different tumor heterogeneities. At the same time, due to racial and geographical differences, it is of great scientific research value to continuously establish new cell lines with different backgrounds. However, so far, there are few reports in the literature on Chinese-derived extrahepatic bile duct cancer cell lines. Therefore, it is very necessary to establish a Chinese-derived extrahepatic bile duct cancer cell line. Summary of the Invention

[0004] The purpose of the present invention is to provide a novel Chinese-derived extrahepatic bile duct cancer cell line EBC-X1 and its application in view of the deficiency of the existing technology in lacking extrahepatic bile duct cancer cell lines, so as to provide a more solid theoretical basis and practical tool for clinical practice.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides a human-derived extrahepatic bile duct cancer cell line named human extrahepatic bile duct cancer cell line EBC-X1, which has been deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC No: C2024318.

[0007] The human extrahepatic bile duct cancer cell line EBC-X1 can be used as a cell model for studying the mechanisms of the occurrence, development, or metastasis of extrahepatic bile duct cancer. The human extrahepatic bile duct cancer cell line EBC-X1 can also be used to establish an animal model of extrahepatic bile duct cancer.

[0008] Establishment of the cell line: In this invention, a surgical resection specimen of extrahepatic bile duct cancer from a 58-year-old male was taken, and after digestion with a mixture of type II collagenase / neutral protease, primary culture was performed. Through standard cell culture techniques, an extrahepatic bile duct cancer cell line was established. After multiple passages, a stable extrahepatic bile duct cancer cell line was successfully established and named EBC-X1.

[0009] The human extrahepatic bile duct cancer cell line EBC-X1 Homo sapiens was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC) on September 16, 2024. The address of the deposit center is: Wuhan University, Wuhan, China. The deposit number of the deposit center is CCTCC NO: C2024318.

[0010] This cell line has the following biological characteristics:

[0011] 1. Cell growth characteristics: The cells grow adherently, without contact inhibition, and can show superposition growth.

[0012] 2. Cell doubling time: 64.5 h.

[0013] 3. The results of cell immunohistochemistry show that: EBC-X1 is a moderately and poorly differentiated extrahepatic bile duct cancer cell line.

[0014] 4. Chromosome analysis indicates that: The EBC-X1 cells have a complex karyotype, among which 91% are hypodiploid karyotypes and 9% are hypertriploid karyotypes; its representative karyotype is: 35,X der(4),del(5)(q35),der(8),inv(9),der(11),rob(13,15).

[0015] 5. Drug sensitivity: The EBC-X1 cells show resistance to albumin-bound paclitaxel, fluorouracil, and oxaliplatin, and are sensitive to gemcitabine.

[0016] 6. Tumorigenic ability: After inoculation into NXG mice, EBC-X1 can rapidly form subcutaneous xenografts, and the tumorigenicity rate is 100%.

[0017] The human extrahepatic bile duct cancer cell line EBC-X1 can be used as a cell model for studying the differentiation mechanism of extrahepatic bile duct cancer, abnormal cell morphology and function, tumor invasion and metastasis mechanism, and guiding clinical comprehensive diagnosis and treatment, etc.

[0018] The human extrahepatic bile duct cancer cell line EBC-X1 can be used in the establishment of a cell model for the occurrence, development or metastasis of extrahepatic bile duct cancer.

[0019] The human extrahepatic bile duct cancer cell line EBC-X1 can be used in a cell model for studying the differentiation mechanism, abnormal cell morphology and function, tumor invasion and metastasis mechanism of extrahepatic bile duct cancer and guiding clinical comprehensive diagnosis and treatment.

[0020] The human extrahepatic bile duct cancer cell line EBC-X1 can be used in the study of the pathogenesis of extrahepatic bile duct cancer and the screening of drugs for the prevention and treatment of extrahepatic bile duct cancer.

[0021] The extrahepatic bile duct cancer cell line EBC-X1 can be used in the establishment of an animal model of extrahepatic bile duct cancer.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention establishes a novel Chinese-derived extrahepatic bile duct cancer cell line EBC-X1, providing a brand-new tool for the study of extrahepatic bile duct cancer; this cell line has various biological characteristics and has significant application value in the study of extrahepatic bile duct cancer, including the research on its occurrence, development, and metastasis mechanisms, the construction of animal models, guiding clinical comprehensive diagnosis and treatment, and drug screening, etc., which helps to promote the comprehensive development of the research on extrahepatic bile duct cancer. The Chinese-derived extrahepatic bile duct cancer cell line provided by the present invention helps to promote the research progress of Chinese extrahepatic bile duct cancer and provides new ideas and methods for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the pathological result of the tumor tissue derived from EBC-X1 cells.

[0025] Figure 2 It is the morphological observation diagram of EBC-X1 cells under the microscope.

[0026] Figure 3 It is the growth curve of EBC-X1 cells. Among them, the abscissa is the cell culture time, and the ordinate is the cell number.

[0027] Figure 4 It is the immunohistochemical result of EBC-X1 cells. Among them, A shows the positive expression of CK7 in the cells; B shows the positive expression of CK19 in the cells; C shows that the positive expression rate of Ki67 in the cells is 70%; D shows the positive expression of CA 19-9 in the cells; E shows the focal positive expression of CEA in the cells.

[0028] Figure 5 It is the chromosome analysis result of EBC-X1 cells.

[0029] Figure 6These are the drug sensitivity results of EBC-X1 cells. Among them, A indicates resistance to oxaliplatin, B indicates resistance to albumin-bound paclitaxel, C indicates sensitivity to gemcitabine, and D indicates resistance to fluorouracil.

[0030] Figure 7 These are the results of the tumorigenicity experiment of EBC-X1 cells in immunodeficient mice. Among them, A - B indicates that EBC-X1 can form transplanted tumors when inoculated subcutaneously in NXG mice, and the tumorigenicity rate is 100%; C - D indicates that no metastatic lesions are seen in the lungs and livers of the mice after 4 weeks. Detailed implementation manners

[0031] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following embodiments will further illustrate the present invention in conjunction with the accompanying drawings. 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. On the contrary, the present invention covers any alternatives, modifications, equivalent methods and solutions made within the spirit and scope of the present invention defined by the claims.

[0032] In the following embodiments, the experimental methods are all conventional methods unless otherwise specified.

[0033] Establishment and identification of the human extrahepatic bile duct cancer cell line EBC-X1.

[0034] I. Establishment of the human extrahepatic bile duct cancer cell line EBC-X1

[0035] Tumor tissues of patients with extrahepatic bile duct cancer were clinically collected, subjected to primary culture after digestion with a mixed enzyme, and then a continuously passaged extrahepatic bile duct cancer cell line was successfully established. Currently, it has been passaged more than 50 generations, and the cell characteristics remain stable. The pathological result of this patient was moderately and poorly differentiated extrahepatic bile duct cancer, as Figure 1 shown.

[0036] The present invention uses a surgical resection specimen from a 58-year-old male. After digestion with a mixture of type II collagenase / neutral protease, primary culture was carried out, and an extrahepatic bile duct cancer cell line was established through cell culture technology and named EBC-X1. It was deposited on September 16, 2024, with the depositary institution: China Center for Type Culture Collection (abbreviated as CCTCC, address: Bayi Road, Hongshan District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Wuhan University), and the deposit number of the depositary center: CCTCC NO: C2024318.

[0037] II. Detection of the biological characteristics of the human extrahepatic bile duct cancer cell line EBC-X1

[0038] 1. Cell morphology: After the cells grew stably and were passaged, living cells were observed. In addition, monolayer cells grown on cover slips were fixed with 95% ethanol by volume and stained with H&E, and observed under a light microscope. The results showed that under a phase contrast microscope, the cells were arranged in an epithelial-like pattern, adhered to the wall and grew with overlapping. The morphological observation diagram of EBC-X1 cells under the microscope is as Figure 2 shown.

[0039] 2. EBC-X1 cells in the logarithmic growth phase were collected after being enzymatically dissociated with trypsin (VivaCell), and together with the primary tumor tissue, they were sent to Suzhou Jianda Biotechnology Co., Ltd. for STR analysis to clarify the correlation between the cells and the primary tumor tissue. The results are shown in Table 1.

[0040] Table 1

[0041]

[0042] As can be seen from Table 1, the STR typing results of EBC-X1 cells and the primary tumor tissue were highly consistent, indicating that the EBC-X1 cell line was derived from this tumor tissue and maintained its genetic characteristics during in vitro culture.

[0043] 3. Precise determination of cell doubling time: EBC-X1 cells in the logarithmic growth phase were selected, enzymatically dissociated with trypsin (VivaCell, concentration 0.25%), incubated in a cell culture incubator at 37 °C for 5 min, centrifuged to discard the supernatant, and the cells were resuspended with pre-cooled PBS buffer, centrifuged and washed again. The washed cell pellet was resuspended with an appropriate amount of complete medium (composed of RPMI-1640 medium, 10% fetal bovine serum, 1% double antibody (penicillin-streptomycin mixture, concentrations 100 U / mL and 100 μg / mL respectively)) to prepare a single cell suspension (labeled as P40). The prepared single cell suspension was counted, and after measuring the cell suspension density, 6×10 3 , 8×10 3 , 1×10 4 , 1.2×10 4 , 1.5×10 4 , 1.8×10 4 , 2.1×10 4 , 2.4×10 4 , 2.8×10 4 cells were inoculated into 9 different wells of a 96-well plate (NEST). After inoculation, the liquid volume in each well was made up to 100 μL with complete medium and placed at 37 °C, 5% CO 2Incubate in a cell incubator. After 20 h of incubation, add 100 μL of diluted CCK8 solution [10% CCK8 (APE×BIO) + 90% RPMI-1640 (Gibco)], and continue to incubate for 3.5 h. Then measure the absorbance of each well at a wavelength of 450 nm using a microplate reader (BioTek, synergy H1). Using the time after cell inoculation as the abscissa and the corresponding cell number as the ordinate, use GraphPad Prism 8.0.2 software to plot the growth curve of EBC-X1 cells. Based on the growth curve, use a website to calculate the doubling time of the cells. The cell doubling time is approximately 64.5 h. The growth curve of EBC-X1 cells is as Figure 3 shown. Figure 3 It clearly shows the growth trend of cells at different time points, providing intuitive data support for in-depth understanding of cell proliferation characteristics.

[0044] 4. Immunohistochemical staining and result analysis: Take EBC-X1 cells in the logarithmic growth phase, enzymatically dissociate them and inoculate them on sterile glass slides to grow. After 60 h, wash the coverslips with PBS to remove non-adherent cells and residual culture medium. Immerse the slides in a 4% paraformaldehyde (Servicebio) solution and fix them at room temperature for 15 min to stably preserve cell morphology and antigen components. After fixation, place the slides in a ventilated place to air dry naturally. After air drying, treat the slides with a 0.5% Triton X-100 (MCE) solution for 20 min to increase cell membrane permeability, and then subsequent staining can be carried out. Embed the original tumor tissue and xenograft tumors in paraffin and cut them into 4-μm thick sections. Place the sections flat on glass slides, dry them at 60 °C for 5 h, and then carry out subsequent staining.

[0045] Immunohistochemical staining: After dewaxing and rehydration, immerse the glass slides in a sodium citrate solution (10 mmol / L, pH = 6.0), boil (90 s) for antigen retrieval, incubate in a 3% hydrogen peroxide solution at 37 °C for 15 min, add 100 μL of normal goat serum, and incubate in a 37 °C incubator for 15 min to reduce non-specific antibody binding and reduce background staining. Then incubate with anti-CK7, anti-CK19, anti-Ki67, anti-CA19-9, and anti-CEA at 37 °C for 12 h, and then add the secondary antibody and incubate at room temperature for 50 min; use a DAB staining kit (Dako) for color development, rinse the glass slides with running water for 5 min, then counterstain with hematoxylin, dehydrate with gradient ethanol, clear with xylene, and mount with neutral resin. Observe under an inverted microscope (Olympus, IX73 + DP74). The results are as Figure 4 shown. Figure 4 In A, it indicates the positive expression of CK7 in cells, showing obvious brown staining in the cytoplasm or cell membrane; Figure 4B in it indicates the positive expression of CK19 in cells, and the staining site is also located in the cytoplasm or cell membrane; Figure 4 C in it indicates that the positive expression rate of Ki67 in cells is 70%, which is obtained by counting the ratio of the number of positively stained cells to the total number of cells. The positive cells are mainly characterized by the nucleus being stained brown; Figure 4 D in it indicates the positive expression of CA19-9 in cells, showing brown staining in the cytoplasm or cell membrane; Figure 4 E in it indicates the focal positive expression of CEA in cells, that is, obvious brown staining appears in some cell regions, while the staining in other regions is weak or absent.

[0046] 5. Chromosome karyotype analysis of EBC-X1 cells and application correlation: Take EBC-X1 cells in the logarithmic growth phase, add colchicine (Spectrum) to the culture medium to make the colchicine concentration 0.2 μg / mL, and after incubating for 90 min, use 0.25% trypsin (Vivacell) to digest the cells into a cell suspension; centrifuge and discard the supernatant, resuspend the precipitate with 0.56% KCl solution, and place it at 37 °C for incubation for 30 min. Add fixative 1 (fixative 1 is a mixed solution of formic acid and glacial acetic acid, where formic acid: glacial acetic acid = 3:1), mix well, centrifuge and discard the supernatant, and resuspend again with fixative 1 to obtain a suspension of chromosomes. Drop a drop of this suspension onto a glass slide, place it in an 80 °C oven and dry it for 3 h. Put the dried slide into trypsin for digestion for 1 min, then put it into Giemsa stain (BIOSIC) for staining for 8 min, take it out, rinse off the stain with running water, and observe it under an oil immersion microscope at 100 times magnification after sealing. Use ImageJ imaging software and ChromosomeJ plug-in for karyotype analysis. The results are as Figure 5 shown.

[0047] EBC-X1 cells present a complex karyotype. Among them, 91% of the cells have a hypodiploid karyotype, that is, the chromosome number is lower than the normal diploid number; 9% of the cells are hypotriploid karyotypes, and the chromosome number is between diploid and triploid. Its representative karyotype is: 35, X der(4), del(5)(q35), der(8), inv(9), der(11), rob(13,15) ( Figure 5 ). This karyotype feature indicates that there are multiple abnormalities in EBC-X1 cells at the chromosome level, including chromosome deletion, inversion, translocation, etc. These abnormalities may be closely related to the occurrence and development of extrahepatic cholangiocarcinoma.

[0048] The above experiments prove that the present invention can be applied in cell models of the occurrence, development or metastasis of extrahepatic cholangiocarcinoma and cell models of the differentiation mechanism, cell morphology and functional abnormalities, tumor invasion and metastasis mechanism of extrahepatic cholangiocarcinoma and guiding clinical comprehensive diagnosis and treatment.

[0049] 6. EBC-X1 cell drug sensitivity experiment and result analysis: After digestion with 0.25% trypsin, an EBC-X1 cell suspension was prepared. The cells were seeded into a 96-well plate at a density of 10,000 cells / 100 μL per well. After the cells adhered well, drug treatment was carried out. The experiment was set up with experimental groups and control groups. In the experimental groups, anti-tumor drugs with different concentration gradients were added, specifically including:

[0050] Gemcitabine: 600 μmol / L, 150 μmol / L, 30 μmol / L, 6 μmol / L, 1.5 μmol / L, 0.3 μmol / L, 0.06 μmol / L, 0.015 μmol / L, 0.003 μmol / L, 0.0006 μmol / L; its plasma peak concentration: 19.01 μmol / L;

[0051] Albumin-bound paclitaxel: 50 μmol / L, 25 μmol / L, 12.5 μmol / L, 2.5 μmol / L, 0.5 μmol / L, 0.1 μmol / L, 0.025 μmol / L, 0.005 μmol / L, 0.001 μmol / L; plasma peak concentration: 16.2 μmol / L;

[0052] Oxaliplatin: 400 μmol / L, 200 μmol / L, 100 μmol / L, 40 μmol / L, 20 μmol / L, 10 μmol / L, 5 μmol / L, 2.5 μmol / L, 1.25 μmol / L, 0.625 μmol / L, 0.3125 μmol / L, 0.15625 μmol / L; plasma peak concentration: 11.33 μmol / L;

[0053] Fluorouracil: 3840 μmol / L, 960 μmol / L, 240 μmol / L, 60 μmol / L, 15 μmol / L, 3 μmol / L, 0.6 μmol / L, 0.15 μmol / L, 0.03 μmol / L, 0.006 μmol / L; plasma peak concentration: 76.92 μmol / L;

[0054] The control group was added with the corresponding drug dissolution solution. After the drug acted for 72 h, 100 μL of serum-free medium containing 10% (v / v) CCK8 was used to replace the complete medium. After 2 h, the OD value at 450 nm was measured. GraphPad Prism 8.0.2 software (GraphPad Inc., San Diego, CA, USA) was used to plot the drug dose-response curve, and the drug IC50 was calculated. This analysis was repeated three times. The results are as Figure 6 shown.

[0055] It can be seen from Figure 6 that EBC-X1 is resistant to oxaliplatin (IC50 = 33.6 μmol / L) ( Figure 6 A in), albumin-bound paclitaxel (IC50 > 40 μmol / L) ( Figure 6 B in) and fluorouracil (IC50 = 165.1 μmol / L) ( Figure 6 D in), and sensitive to gemcitabine (IC50 = 0.011 μmol / L) ( Figure 6 C in).

[0056] The above experiments prove that the present invention can be applied in the study of the pathogenesis of extrahepatic cholangiocarcinoma and the screening of drugs for the prevention and treatment of extrahepatic cholangiocarcinoma.

[0057] 7. EBC-X1 cell NXG mouse subcutaneous xenograft tumor experiment and application verification: EBC-X1 cells in the logarithmic growth phase were taken, enzymatically dissociated to prepare a cell suspension, and the cell density was adjusted to 1×10 7 / mL. The mixed cell suspension was inoculated subcutaneously into the mid-back part of the right axilla of 3 NXG mice at a dose of 0.1 mL per mouse, and the body weight changes of the NXG mice and the growth of the xenograft tumors were regularly monitored. After 4 weeks, the mice were euthanized, and the xenograft tumors and the liver and lung tissues of the mice were dissected for observation. The xenograft tumors were fixed with 4% paraformaldehyde (Servicebio) for subsequent H&E staining and immunohistochemical staining. The results are as Figure 7 shown, Figure 7 A-C in show that EBC-X1 inoculated subcutaneously into NXG mice can form xenograft tumors, and the tumor formation rate is 100%; Figure 7 D in shows that no metastatic lesions were found in the lungs and livers of the mice after 4 weeks. The above experiments prove that the present invention can be applied in the establishment of an animal model of extrahepatic cholangiocarcinoma.

[0058] The above embodiments are only preferred embodiments of the present invention and cannot be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made in accordance with the scope of the present invention application shall still fall within the scope covered by the patent of the present invention.

Claims

1. Human extrahepatic cholangiocarcinoma cell line, characterized by It was named as the human extrahepatic cholangiocarcinoma cell line EBC-X1, and the deposit number was CCTCC NO: C2024318.

2. Use of the human extrahepatic cholangiocarcinoma cell line as claimed in claim 1 in establishing a cell model for the occurrence, development or metastasis of extrahepatic cholangiocarcinoma.

3. Use of the human extrahepatic cholangiocarcinoma cell line as claimed in claim 1 in studying the differentiation mechanism, cell morphology and functional abnormalities, tumor infiltration and metastasis mechanism of extrahepatic cholangiocarcinoma and as a cell model for guiding comprehensive clinical diagnosis and treatment.

4. Use of the human extrahepatic cholangiocarcinoma cell line according to claim 1 in studying the pathogenesis of extrahepatic cholangiocarcinoma and screening drugs for preventing and treating extrahepatic cholangiocarcinoma.

5. Use of the human extrahepatic cholangiocarcinoma cell line according to claim 1 in establishing an extrahepatic cholangiocarcinoma animal model.

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