Human extrahepatic bile duct cancer cell line EBC-X1 and application thereof

Abstract

The invention discloses a human extrahepatic bile duct cancer cell line EBC-X1 and application thereof, and belongs to the field of microbial animal cell lines. The human extrahepatic bile duct cancer cell line is named as a human extrahepatic bile duct cancer cell line EBC-X1, and the preservation number of the human extrahepatic bile duct cancer cell line EBC-X1 is CCTCC No: C2024318. The human extrahepatic bile duct cancer cell line EBC-X1 can be applied to establishment of a cell model for occurrence, development or metastasis of extrahepatic bile duct cancer. The human extrahepatic bile duct cancer cell line EBC-X1 can be applied to research on differentiation mechanism, cell morphology and dysfunction and tumor infiltration and metastasis mechanism of extrahepatic bile duct cancer and cell models for guiding clinical comprehensive diagnosis and treatment. The human extrahepatic cholangiocarcinoma cell line EBC-X1 can be applied to research on the occurrence mechanism of extrahepatic cholangiocarcinoma and screening of drugs for preventing and treating extrahepatic cholangiocarcinoma. The extrahepatic bile duct cancer cell line EBC-X1 can be applied to establishment of an extrahepatic bile duct cancer animal model.

Description

Technical Field

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

[0002] Cholangiocarcinoma is a highly heterogeneous primary malignant tumor originating from the biliary tree. According to the anatomical site, cholangiocarcinoma can be roughly divided into intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma. Extrahepatic cholangiocarcinoma can be divided into hilar cholangiocarcinoma and distal cholangiocarcinoma according to the location of the cystic duct. Embryologically, intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma have different origins. The former originates from bipotent hepatoblasts, and the latter originates from the ventral foregut region close to the liver and ventral pancreas. Due to the different origins of cholangiocarcinoma subtypes, they show significant clinical, epidemiological, molecular and genetic heterogeneity. The incidence and mortality of cholangiocarcinoma are generally increasing worldwide. Since most patients with cholangiocarcinoma are already in the advanced stage when diagnosed, 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% to 20%. Therefore, cholangiocarcinoma remains a major public health problem worldwide, and more basic research is needed 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 complete tumor cell line library should reflect the diversity of tumor phenotypes and provide cell lines with different tumor heterogeneity. At the same time, due to racial and regional differences, the continuous establishment of new cell lines with different backgrounds has important scientific research value. However, there are few Chinese extrahepatic cholangiocarcinoma cell lines reported in the literature so far. Therefore, it is very necessary to establish a Chinese extrahepatic cholangiocarcinoma cell line. Summary of the invention

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

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

[0006] The present invention provides a human extrahepatic cholangiocarcinoma cell line named as human extrahepatic cholangiocarcinoma cell line EBC-X1, which has been deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: C2024318.

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

[0008] Establishment of cell line: The present invention uses a surgical resection specimen of extrahepatic bile duct cancer taken from a 58-year-old male, and digests it with a mixture of type II collagenase / neutral protease for primary culture. An extrahepatic bile duct cancer cell line is established using standard cell culture techniques. After multiple passages, a stable extrahepatic bile duct cancer cell line is successfully established and named EBC-X1.

[0009] The human extrahepatic cholangiocarcinoma cell line EBC-X1 Homo sapiens was deposited in the China Center for Type Culture Collection (CCTCC) on September 16, 2024. The address of the collection center is: Wuhan University, Wuhan, China, and the collection number of the collection center is CCTCC NO: C2024318.

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

[0011] 1. Cell growth characteristics: Cells grow adherently without contact inhibition, and superimposed growth may occur.

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

[0013] 3. The results of cell immunohistochemistry showed that EBC-X1 was a moderately or poorly differentiated extrahepatic cholangiocarcinoma cell line.

[0014] 4. Chromosome analysis showed that EBC-X1 cells had a complex karyotype, of which 91% were subdiploid and 9% were subtriploid. The representative karyotype was: 35,X der(4),del(5)(q35),der(8),inv(9),der(11),rob(13,15).

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

[0016] 6. Tumor-forming ability: After inoculation into NXG mice, EBC-X1 can quickly form subcutaneous transplanted tumors with a tumor formation rate of 100%.

[0017] The human extrahepatic cholangiocarcinoma cell line EBC-X1 can be used as a cell model for studying the differentiation mechanism, cell morphology and functional abnormalities, tumor infiltration and metastasis mechanism of extrahepatic cholangiocarcinoma and guiding comprehensive clinical diagnosis and treatment.

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

[0019] The human extrahepatic cholangiocarcinoma cell line EBC-X1 can be used 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.

[0020] The human extrahepatic cholangiocarcinoma cell line EBC-X1 can be used in studying the occurrence mechanism of extrahepatic cholangiocarcinoma and screening drugs for preventing and treating extrahepatic cholangiocarcinoma.

[0021] The extrahepatic cholangiocarcinoma cell line EBC-X1 can be used in establishing an extrahepatic cholangiocarcinoma animal model.

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

[0023] The present invention establishes a new Chinese extrahepatic cholangiocarcinoma cell line EBC-X1, which provides a new tool for the study of extrahepatic cholangiocarcinoma; the cell line has multiple biological characteristics, and has significant application value in the study of extrahepatic cholangiocarcinoma, including its occurrence, development, metastasis mechanism research, animal model construction, guidance of clinical comprehensive diagnosis and treatment guidance, and drug screening, which helps to promote the comprehensive development of extrahepatic cholangiocarcinoma research. The Chinese extrahepatic cholangiocarcinoma cell line provided by the present invention helps to promote the research progress of extrahepatic cholangiocarcinoma in China and provides new ideas and methods for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the pathological results of tumor tissue derived from EBC-X1 cells.

[0025] Figure 2 This is a morphological observation of EBC-X1 cells under a microscope.

[0026] Figure 3 This is the growth curve of EBC-X1 cells, where the horizontal axis is the cell culture time and the vertical axis is the cell number.

[0027] Figure 4 The immunohistochemical results of EBC-X1 cells. A indicates positive expression of CK7 in cells; B indicates positive expression of CK19 in cells; C indicates positive expression rate of Ki67 in cells is 70%; D indicates positive expression of CA 19-9 in cells; E indicates focal positive expression of CEA in cells.

[0028] Figure 5 The results of chromosome analysis of EBC-X1 cells.

[0029] Figure 6The drug sensitivity results of EBC-X1 cells, among which A is resistant to oxaliplatin, B is resistant to albumin-bound paclitaxel, C is sensitive to gemcitabine, and D is resistant to fluorouracil.

[0030] Figure 7 These are the results of the tumorigenicity experiment of EBC-X1 cells in vivo in immunodeficient mice. AB shows that EBC-X1 cells can form transplanted tumors subcutaneously in NXG mice, with a tumor formation rate of 100%; CD shows that no metastatic lesions were found in the lungs and liver of mice after 4 weeks. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, 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 intended to limit the present invention. On the contrary, the present invention covers any substitution, modification, equivalent method and scheme made on the essence and scope of the present invention as defined by the claims.

[0032] The experimental methods described in the following examples are conventional methods unless otherwise specified.

[0033] Establishment and identification of human extrahepatic cholangiocarcinoma cell line EBC-X1.

[0034] 1. Establishment of the human extrahepatic cholangiocarcinoma cell line EBC-X1

[0035] Tumor tissues of patients with extrahepatic cholangiocarcinoma were collected clinically and digested with mixed enzymes for primary culture. A cell line of extrahepatic cholangiocarcinoma cells that can be continuously subcultured was successfully established. Currently, it has been subcultured for more than 50 generations, and the cell characteristics remain stable. The pathological results of this patient were moderately or poorly differentiated extrahepatic cholangiocarcinoma. Figure 1 shown.

[0036] The present invention uses a surgical resection specimen taken from a 58-year-old male, digested with a mixture of type II collagenase / neutral protease, and then cultured for primary culture. An extrahepatic bile duct cancer cell line named EBC-X1 was established by cell culture technology. It has been deposited on September 16, 2024, and the depository unit is 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 collection center is CCTCC NO: C2024318.

[0037] 2. Detection of biological characteristics of human extrahepatic cholangiocarcinoma cell line EBC-X1

[0038] 1. Cell morphology: After the cells have grown stably and have been cultured, live cell observation is performed on the cultured cells; the monolayer cells grown on the coverslip are fixed with 95% ethanol by volume, stained with H&E, and observed under a light microscope. The results show that under a phase contrast microscope, the cells are arranged in an epithelial-like pattern, and the cells grow attached to the wall with overlapping growth. The morphological observation of EBC-X1 cells under a microscope is shown in the figure below: Figure 2 shown.

[0039] 2. Take EBC-X1 cells in the logarithmic growth phase, collect the cells after enzymatic dissociation with pancreatic enzyme (VivaCell), and send them together with the primary tumor tissue 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 were highly consistent with those of primary tumor tissues, indicating that the EBC-X1 cell line was derived from the tumor tissue and maintained its genetic characteristics during in vitro culture.

[0043] 3. Accurate determination of cell doubling time: EBC-X1 cells in the logarithmic growth phase were selected, and trypsin (VivaCell, concentration of 0.25%) was used to enzymatically dissociate the cells. The cells were incubated in a cell culture incubator at 37°C for 5 minutes, and the supernatant was discarded by centrifugation. The cells were resuspended with pre-cooled PBS buffer, and centrifuged and washed again. The washed cell pellet was resuspended with an appropriate amount of complete culture medium (composed of RPMI-1640 culture medium, 10% fetal bovine serum, and 1% double antibody (penicillin-streptomycin mixture, concentrations of 100U / mL and 100μg / 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 4Each cell was inoculated in 9 different wells of a 96-well plate (NEST). After inoculation, the liquid volume in each well was supplemented to 100 μL with complete culture medium and incubated in a cell culture incubator at 37°C and 5% CO2. After incubation for 20 hours, 100 μL of diluted CCK8 solution [10% CCK8 (APE×BIO) + 90% RPMI-1640 (Gibco)] was added. After incubation for another 3.5 hours, the absorbance of each well at a wavelength of 450 nm was measured using an enzyme reader (BioTek, synergy H1). The growth curve of EBC-X1 cells was drawn using GraphPad Prism 8.0.2 software, with the time after cell inoculation as the horizontal axis and the corresponding number of cells as the vertical axis. Based on the growth curve, the cell doubling time was calculated using the website. The cell doubling time is approximately 64.5 hours. The growth curve of EBC-X1 cells is shown in the figure. Figure 3 shown. Figure 3 Clearly display 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 for growth. After 60 hours, wash the coverslips with PBS to remove non-adherent cells and residual culture medium. Soak the slides in 4% paraformaldehyde (Servicebio) solution and fix them at room temperature for 15 minutes to stably preserve the cell morphology and antigen components. After fixation, place the slides in a ventilated place to air dry naturally. After air drying, treat them with 0.5% Triton X-100 (MCE) solution for 20 minutes to increase the permeability of the cell membrane, and then proceed to subsequent staining. The original tumor tissue and xenograft tumor were embedded in paraffin and cut into 4μm thick slices. The slices were flattened on slides and dried at 60℃ for 5 hours before subsequent staining.

[0045] Immunohistochemical staining: After dewaxing and rehydration, the slides were immersed in sodium citrate solution (10mmol / L, pH=6.0), boiled (90s) for antigen retrieval, incubated in 3% hydrogen peroxide solution at 37°C for 15min, 100μL normal goat serum was added, and the slides were blocked in a 37°C incubator for 15min to reduce nonspecific antibody binding and background staining. Then, they were incubated with anti-CK7, anti-CK19, anti-Ki67, anti-CA19-9, and anti-CEA at 37°C for 12h, and then the secondary antibody was added and incubated at room temperature for 50min; the DAB staining solution kit (Dako) was used for color development, the slides were rinsed with running water for 5min, counterstained with hematoxylin, dehydrated with gradient ethanol, transparentized with xylene, sealed with neutral resin, and observed under an inverted microscope (Olympus, IX73+DP74). The results are shown in Figure 4 shown. Figure 4 A in the figure indicates that CK7 is positively expressed in the cells, which is manifested by obvious brown staining in the cytoplasm or cell membrane; Figure 4 B in the figure indicates that CK19 is positively expressed in the cells, and the staining site is also located in the cytoplasm or cell membrane; Figure 4 The C in the figure indicates that the positive expression rate of Ki67 in cells is 70%, which is obtained by counting the ratio of positively stained cells to the total number of cells. Positive cells are mainly characterized by brown-stained nuclei. Figure 4 D in the figure indicates that CA19-9 is positively expressed in cells, showing brown staining of the cytoplasm or cell membrane; Figure 4 E in the figure indicates focally positive expression of CEA in cells, that is, obvious brown staining appears in some cell areas, while other areas are weakly stained or not stained.

[0046] 5. EBC-X1 cell chromosome karyotype analysis and application association: Take EBC-X1 cells in the logarithmic growth phase, add colchicine (Spectrum) to the culture medium and make the colchicine concentration 0.2µg / mL, incubate for 90 minutes, 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 incubate at 37℃ for 30 minutes. 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 thoroughly, centrifuge and discard the supernatant, and resuspend with fixative 1 again to obtain a chromosome suspension. Drop a drop of the suspension onto a glass slide and put it in an 80℃ oven to dry for 3h. Digest the dried slide in trypsin for 1min, then put it in Giemsa stain (BIOSIC) for 8min, take it out and rinse off the stain with running water, and observe it under a 100x oil microscope after sealing. Karyotype analysis was performed using ImageJ imaging software and the ChromosomeJ plug-in. Figure 5 shown.

[0047] EBC-X1 cells present a complex karyotype, of which 91% of the cells have a subdiploid karyotype, that is, the number of chromosomes is lower than the normal diploid number; 9% of the cells have a subtriploid karyotype, with the number of chromosomes 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 characteristic indicates that EBC-X1 cells have multiple abnormalities at the chromosomal level, including chromosome deletion, inversion, translocation, etc. These abnormalities may be closely related to the occurrence and development of extrahepatic bile duct carcinoma.

[0048] The above experiments prove that the present invention can be applied in cell models of the occurrence, development or metastasis of extrahepatic bile duct cancer, as well as in cell models for the differentiation mechanism, abnormal cell morphology and function, tumor infiltration and metastasis mechanism of extrahepatic bile duct cancer, and for guiding comprehensive clinical diagnosis and treatment.

[0049] 6. EBC-X1 cell drug sensitivity test and result analysis: Prepare EBC-X1 cell suspension after 0.25% trypsin digestion. Inoculate 10,000 cells / 100 μL per well into a 96-well plate. After the cells adhere well to the wall, perform drug treatment. The experiment sets up an experimental group and a control group. The experimental group is added with anti-tumor drugs of different concentration gradients, 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 solution. After 72 h of drug action, the complete medium was replaced with 100 μL serum-free medium containing 10% (v / v) CCK8. After 2 h, the OD value at 450 nm was measured. The drug dose-response curve was drawn using GraphPad Prism 8.0.2 software (GraphPad Inc., San Diego, CA, USA), and the drug IC50 was calculated. This analysis was repeated three times. The results are shown in Figure 6 shown.

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

[0056] The above experiments prove that the present invention can be used in studying the occurrence mechanism of extrahepatic bile duct cancer and screening drugs for preventing and treating extrahepatic bile duct cancer.

[0057] 7. Experiment and application verification of EBC-X1 cell NXG mouse subcutaneous transplant tumor: EBC-X1 cells in the logarithmic growth phase were enzymatically dissociated to prepare cell suspension and the cell density was adjusted to 1×10 7 / mL. The mixed cell suspension was inoculated into the subcutaneous part of the middle and posterior part of the right axilla of three NXG mice at a volume of 0.1 mL per mouse, and the weight changes and growth of transplanted tumors of the NXG mice were monitored regularly. The mice were euthanized after 4 weeks, and the xenograft tumors and 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 shown in Figure 7 As shown, Figure 7 AC in the figure is that EBC-X1 can form transplanted tumors when inoculated subcutaneously in NXG mice, with a tumor formation rate of 100%; Figure 7 D in the figure means that no metastatic lesions were found in the lungs and liver of mice after 4 weeks. The above experiments prove that the present invention can be applied in establishing an animal model of extrahepatic bile duct cancer.

[0058] The above embodiments are only preferred embodiments of the present invention and cannot be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope 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 EBC-X1 as claimed in claim 1 in establishing a cell model of the occurrence, development or metastasis of extrahepatic cholangiocarcinoma.

3. Use of the human extrahepatic cholangiocarcinoma cell line EBC-X1 as described 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 to guide comprehensive clinical diagnosis and treatment.

4. Use of the human extrahepatic cholangiocarcinoma cell line EBC-X1 as claimed in 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 EBC-X1 as claimed in claim 1 in establishing an extrahepatic cholangiocarcinoma animal model.

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