Human-derived ampulla cancer cell line DPC-X3 and application thereof

Abstract

The invention discloses a human-derived ampulla cancer cell line DPC-X3 and application thereof, and belongs to the field of microbial animal cell lines. The human-derived kettle belly cancer cell line is named as a human-derived kettle belly cancer cell line DPC-X3, and the preservation number is CCTCC (China Center For Type Culture Collection): C2024317. The human-derived ampulla cancer cell line DPC-X3 can be applied to establishment of a cell model for occurrence, development or metastasis of ampulla cancer. The human-derived ampulla cancer cell line DPC-X3 can be applied to cell models for researching the differentiation mechanism, cell morphology and dysfunction and tumor infiltration and metastasis mechanism of ampulla cancer and guiding clinical comprehensive diagnosis and treatment. The human-derived ampulla cancer cell line DPC-X3 can be applied to research on the occurrence mechanism of the ampulla cancer and screening of drugs for preventing and treating the ampulla cancer. The ampulla cancer cell line DPC-X3 can be applied to establishment of an ampulla cancer animal model.

Description

Technical Field

[0001] The present invention belongs to the field of microbial animal cell lines, and in particular relates to a Chinese ampullary cancer cell line and application thereof. Background Art

[0002] Ampullary carcinoma is a relatively rare tumor, accounting for 0.2% of gastrointestinal cancers, but 20% of all periampullary cancers. The incidence of ampullary carcinoma has gradually increased in recent years. Ampullary carcinoma can occur at any age, but it is more common between 60 and 65 years old; the male-female ratio is about 3:2. Because the symptoms of biliary obstruction appear early, the surgical resection rate of ampullary carcinoma is much higher than that of other periampullary malignancies, and about 50% of patients have the opportunity for surgery when they are diagnosed. Its surgical volume exceeds one-third of the total number of pancreaticoduodenectomy surgeries, and the prognosis is good. The 5-year survival rate after surgery exceeds 52.8%. Despite the high potential curative resection rate, most patients with ampullary carcinoma will eventually die from tumor recurrence. In 1994, Kimura et al. first divided ampullary carcinoma into intestinal and pancreaticobiliary types based on histological characteristics, among which the prevalence of pancreaticobiliary type was 72%, much higher than that of intestinal type. In 2010, the World Health Organization revised the pathological diagnostic criteria for ampullary carcinoma into three different histopathological subtypes based on morphological and immunohistochemical features: intestinal ampullary carcinoma, pancreaticobiliary ampullary carcinoma, and mixed ampullary carcinoma. Due to the rarity of the tumor and the lack of clinical trials, standardized adjuvant treatment options for ampullary carcinoma remain to be determined.

[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 be able to 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, no literature has reported Chinese ampullary carcinoma cell lines so far. Therefore, it is very necessary to establish a Chinese ampullary carcinoma cell line. Summary of the invention

[0004] The purpose of the present invention is to provide a new ampullary cancer cell line DPC-X3 of Chinese origin and its application in view of the shortcomings of the prior art such as the lack of ampullary cancer cell line.

[0005] The invention provides a human ampullary cancer cell line named as human ampullary cancer cell line DPC-X3, with a deposit number of CCTCC No: C2024317.

[0006] The human ampullary carcinoma cell line DPC-X3 can be used as a cell model for studying the occurrence, development or metastasis mechanism of ampullary carcinoma. The human ampullary carcinoma cell line DPC-X3 can also be used to establish an animal model of ampullary carcinoma.

[0007] The present invention uses a surgical resection specimen taken from a 72-year-old male, which is digested with a mixture of type II collagenase / neutral protease for primary culture, and then cultured using cell culture technology to establish an ampullary cancer cell line, named DPC-X3; it was preserved on September 16, 2024, and the preservation 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 preservation center's preservation number is: CCTCC, C2024317.

[0008] This cell line has the following biological characteristics:

[0009] 1. The cells grow by adhering to the wall without contact inhibition, and superimposed growth may occur.

[0010] 2. The cell doubling time is 79h.

[0011] 3. The cell line has strong clone-forming ability.

[0012] 4. The results of cell immunohistochemistry showed that DPC-X3 is a moderately differentiated ampullary carcinoma cell line.

[0013] 5. Chromosome analysis showed that 80% of DPC-X3 cells were subdiploid and 20% were subtriploid. There were certain differences in chromosome number and morphology. The representative karyotype was: 44,XY inv(9),-18,-20,-22,+mar.

[0014] 6. DPC-X3 cells are resistant to oxaliplatin, paclitaxel, gemcitabine and 5-FU.

[0015] 7. After inoculation into NXG mice, DPC-X3 can quickly form subcutaneous transplanted tumors with a tumor formation rate of 100%.

[0016] 8. The human ampullary carcinoma cell line DPC-X3 described in the present invention can be used as a cell model for studying the differentiation mechanism, cell morphology and functional abnormalities, tumor infiltration and metastasis mechanism of ampullary carcinoma, and guiding clinical comprehensive diagnosis and treatment.

[0017] The human ampullary cancer cell line DPC-X3 can be used in establishing a cell model for the occurrence, development or metastasis of ampullary cancer.

[0018] The human ampullary carcinoma cell line DPC-X3 can be used in studying the differentiation mechanism of ampullary carcinoma, abnormal cell morphology and function, and the mechanism of tumor infiltration and metastasis, as well as in guiding the cell model for comprehensive clinical diagnosis and treatment.

[0019] The human ampullary cancer cell line DPC-X3 can be used in studying the occurrence mechanism of ampullary cancer and screening drugs for preventing and treating ampullary cancer.

[0020] The human ampullary cancer cell line DPC-X3 can be used in establishing an ampullary cancer animal model.

[0021] The present invention establishes a novel Chinese ampullary cancer cell line DPC-X3, which has multiple biological characteristics and is suitable for the study of ampullary cancer, including its occurrence, development, metastasis mechanism and guidance of comprehensive clinical diagnosis and treatment. The Chinese ampullary cancer cell line provided by the present invention is helpful to promote the research progress of Chinese ampullary cancer and provide new ideas and methods for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Pathological results of tumor tissue derived from DPC-X3 cells.

[0023] Figure 2 This is a morphological observation of DPC-X3 cells under a microscope.

[0024] Figure 3 is the growth curve of DPC-X3 cells. Figure 3 In the figure, the horizontal axis is the cell culture time, and the vertical axis is the cell number.

[0025] Figure 4 These are the results of the DPC-X3 cell organoid formation experiment.

[0026] Figure 5 The immunohistochemical results of DPC-X3 cells. Figure 5 In the figure, A indicates positive expression of CK7 in the cells; B indicates positive expression of CK20 in the cells; C indicates negative expression of CDX2 in the cells; D indicates that the positive expression rate of Ki67 in the cells is 40%; E indicates focal positive expression of CEA in the cells.

[0027] Figure 6 The results of chromosome analysis of DPC-X3 cells.

[0028] Figure 7 The results of drug sensitivity of DPC-X3 cells are shown in Figure 2. Figure 7 Among them, A is resistant to oxaliplatin, B is resistant to paclitaxel, C is resistant to gemcitabine, and D is resistant to fluorouracil.

[0029] Figure 8 The results of the in vivo tumorigenicity experiment of DPC-X3 cells in immunodeficient mice. Figure 8 In the figure, AB shows that DPC-X3 can form transplanted tumors after subcutaneous inoculation into 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

[0030] 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.

[0031] The experimental methods described in the following examples are conventional methods unless otherwise specified.

[0032] Establishment and identification of human ampullary carcinoma cell line DPC-X3.

[0033] 1. Establishment of human ampullary carcinoma cell line DPC-X3

[0034] Tumor tissues of patients with ampullary carcinoma were collected clinically and digested with mixed enzymes for primary culture. A continuous-generation ampullary carcinoma cell line was successfully established. Currently, it has been passed for more than 70 generations, and the cell characteristics remain stable. The pathological result of this patient was moderately differentiated ampullary carcinoma. Figure 1 shown.

[0035] The present invention adopts a surgical resection specimen taken from a 72-year-old male, digested with a mixture of type II collagenase / neutral protease, and then cultured for primary culture. A ampullary cancer cell line named DPC-X3 was established by cell culture technology. It was 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 is CCTCC, C2024317.

[0036] 2. Detection of biological characteristics of human ampullary carcinoma cell line DPC-X3

[0037] 1. Cell morphology: After the cells grew stably and were passaged, live cell observation of DPC-X3 cells was performed. The results showed that under a phase contrast microscope, the cells were arranged in an epithelial-like pattern, adhered to the wall, and had overlapping growth. The morphological observation of DPC-X3 cells under a microscope is shown in the figure below: Figure 2 As shown. This arrangement and growth pattern are consistent with the characteristics of malignant tumor cells, indicating that DPC-X3 cells maintain their malignant biological characteristics under in vitro culture conditions. In addition, the monolayer cells grown on the coverslip were fixed with 95% ethanol by volume, stained with H&E, and observed under a light microscope. Under a light microscope, it was found that the cell morphology was consistent with the observation results under a phase contrast microscope, further confirming the epithelial-like arrangement and adherent growth characteristics of DPC-X3 cells.

[0038] 2. In order to clarify the correlation between DPC-X3 cells and primary tumor tissues, DPC-X3 cells in the logarithmic growth phase were collected after enzymatic dissociation with pancreatic enzyme (VivaCell) and sent to Suzhou Jianda Biotechnology Co., Ltd. for STR analysis together with primary tumor tissues. The results are shown in Table 1.

[0039] Table 1

[0040]

[0041] As can be seen from Table 1, the STR typing results of DPC-X3 cells were highly consistent with those of primary tumor tissues, indicating that the DPC-X3 cell line was indeed derived from the tumor tissue and maintained its genetic characteristics during in vitro culture.

[0042] 3. Take the DPC-X3 cells in the logarithmic growth phase and use 0.25% trypsin (VivaCell) to dissociate them into single cell suspension. After measuring the density of the cell suspension, 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 wells of a 96-well plate (NEST), and the volume of the liquid in each well was supplemented to 100 μL using complete medium 2 (RPMI-1640 + 10% FBS + 1% penicillin-streptomycin, BI). After 20 hours, 100 μl of diluted CCK8 solution [10% CCK8 (APE×BIO) + 90% RPMI-1640 (Gibco)] was added. After 3.5 hours, the absorbance at 450 nm was measured using a microplate reader (BioTek, synergy H1), and a standard curve was drawn with absorbance as the x-axis and cell number as the y-axis. Based on this growth curve, the standard curve was plotted using the website The cell doubling time was calculated and the average doubling time was about 79h. The growth curve of DPC-X3 cells is shown in Figure 3 shown.

[0043] 4. Collect the logarithmically growing DPC-X3 cells and, after digestion, add 1.5×10 5 The cells were seeded into 6-well plates (ultra-low attachment plates) and cultured in RPMI-1640 complete medium. Organoid formation was monitored on days 7, 10, and 14 after seeding. Figure 4 As shown. Figure 4 It can be seen that DPC-X3 cells are able to form tumor organoids under ultra-low adsorption conditions, indicating that these cells have the ability to grow and aggregate in a suspended state and can simulate the growth environment of tumors in vivo.

[0044] 5. Take DPC-X3 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, fix them with 4% paraformaldehyde (Servicebio) for 15 minutes, air-dry them, and treat them with 0.5% Triton X-100 (MCE) for 20 minutes before subsequent staining. Embed the original tumor tissue and xenograft tumor in paraffin and cut them into 4μm thick sections, dry them at 60℃ for 5 hours, and then proceed to subsequent staining.

[0045] Immunohistochemical staining: After dewaxing and rehydration, the slides were immersed in sodium citrate solution (10 mMol / L, pH=6.0), boiled (90s) for antigen retrieval, incubated in 3% hydrogen peroxide solution at 37°C for 15min, and 100 µl normal goat serum was added, and blocked at 37°C for 15 min. Then, they were incubated with anti-CK7, anti-CK20, anti-CDX2, anti-Ki67, and anti-CEA at 37°C for 12h, and then the secondary antibody was added and incubated at room temperature for 50min; DAB staining solution kit (Dako) was used for color development, washed 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 5 As shown, the expression of CK7, CK20, CDX2, Ki67 and CEA in cells is shown. Figure 5 In the figure, A indicates positive expression of CK7 in the cells; B indicates positive expression of CK20 in the cells; C indicates positive expression of CDX2 in the cells; D indicates that the positive expression rate of Ki67 in the cells is 40%; E indicates focal positive expression of CEA in the cells.

[0046] 6. Take DPC-X3 cells in the logarithmic growth phase, add colchicine (Spectrum) to the complete medium and make the colchicine concentration 0.2ug / mL, incubate for 90min, 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 30min. Add fixative 1 (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 dry it in an 80℃ oven for 3h. Digest the dried slide in trypsin for 1min, then stain it in Giemsa stain (BIOSIC) for 8min, take it out and rinse off the stain with running water, seal the slide and observe it under a 100x oil microscope. Karyotype analysis was performed using ImageJ imaging software and the ChromosomeJ plug-in. Figure 6 shown.

[0047] DPC-X3 cells are 80% hypodiploid and 20% hypotriploid, with some differences in chromosome number and morphology. The representative karyotype is: 44,XY inv(9),-18,-20,-22,+mar( Figure 6 ); This result reveals the genetic variation of DPC-X3 cells.

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

[0049] 7. Take DPC-X3 cells in the logarithmic growth phase and prepare a single cell suspension after enzymatic dissociation. Then adjust the cell density in the suspension to 1.2×10 5 / mL, and after thorough mixing, add 100 μL / well to a 96-well plate (NEST). After 24 hours, anti-tumor drugs (such as oxaliplatin, paclitaxel, gemcitabine, and 5-fluorouracil) were diluted into drug solutions of different concentrations with complete medium 2, and the medium in the 96-well plate was discarded. Drug solutions of different concentrations were added to the drug treatment group, and complete medium 2 was added to the control group. Four replicate wells were set up for both the drug treatment group and the control group, and the amount of drug solution or complete medium 2 added to each well was 170 μL. After 72 hours of drug action, the drug solution and complete medium in the 96-well plate were discarded, and 100 μL of diluted CCK8 solution [10% CCK8 (APE×BIO) + 90% RPMI- 1640 (Gibco)] was added to each well. At the same time, 100 μL of diluted CCK8 solution was added to each of the four blank wells without cell suspension as background correction. After incubation at 37°C for 3.5 h, the absorbance at 450 nm was measured using a microplate reader (BioTek, synergy H1). The drug dose-response curve was plotted 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 7 As shown. By treating DPC-X3 cells with different concentrations of anti-tumor drugs, the cell survival rate was measured using the CCK8 method, the drug dose-response curve was drawn, and the drug IC50 was calculated. Figure 7 It can be seen that DPC-X3 is resistant to oxaliplatin (IC50 = 51.21 µmol / L), paclitaxel (IC50 = 24.66 µmol / L), gemcitabine (IC50 >600 µmol / L), and 5-fluorouracil (IC50 >3840 µmol / L). The experimental results show that DPC-X3 cells are resistant to drugs such as oxaliplatin, paclitaxel, gemcitabine, and 5-fluorouracil.

[0050] The above experiments prove that the present invention can be used in studying the occurrence mechanism of ampullary cancer and screening drugs for preventing and treating ampullary cancer.

[0051] 8. Take DPC-X3 cells in the logarithmic growth phase, enzymatically dissociate them to make a cell suspension and adjust the cell density 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 two NXG mice at a volume of 0.1 mL per mouse. The weight changes and growth of transplanted tumors of NXG mice were monitored regularly, and an animal model of ampullary carcinoma was successfully established. 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 8 As shown: A and B show that DPC-X3 can form transplanted tumors after being inoculated subcutaneously in NXG mice, with a tumor formation rate of 100%; C and D show 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 used in establishing an animal model of ampullary carcinoma.

[0052] 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 ampullary cancer cell line, characterized in that The cell line was named as human ampullary carcinoma cell line DPC-X3, and its deposit number was CCTCC NO: C2024317.

2. Use of the human ampullary carcinoma cell line DPC-X3 as claimed in claim 1 in establishing a cell model for the occurrence, development or metastasis of ampullary carcinoma.

3. Use of the human ampullary carcinoma cell line DPC-X3 as claimed in claim 1 in studying the differentiation mechanism, cell morphology and functional abnormalities, tumor infiltration and metastasis mechanism of ampullary carcinoma and in guiding the cell model for comprehensive clinical diagnosis and treatment.

4. Use of the human ampullary cancer cell line DPC-X3 as claimed in claim 1 in studying the pathogenesis of ampullary cancer and screening drugs for preventing and treating ampullary cancer.

5. Use of the human ampullary cancer cell line DPC-X3 as claimed in claim 1 in establishing an ampullary cancer animal model.

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