Primary multidrug resistant human distal bile duct cancer cell line CBC3T-6 Homo sapiens and application thereof

Abstract

The invention belongs to the technical field of microbial medicine, and provides a primary multidrug resistance human far-end bile duct cancer cell line CBC3T-6 Homo sapiens and application thereof.The human far-end bile duct cancer cell line CBC3T-6 Homo sapiens is separated from differentiated adenocarcinoma in a far-end bile duct, the human far-end bile duct cancer cell line CBC3T-6 Homo sapiens is already subcultured to 50 generations at present, the long-term in-vitro culture character is stable, and on the premise that the biological characteristics of bile duct cancer are reserved, the human far-end bile duct cancer cell line CBC3T-6 Homo sapiens can be used for preparing the primary multidrug resistance human far-end bile duct cancer cell line CBC3T-6 Homo sapiens. The compound has the characteristics of short multiplication time, high mouse subcutaneous tumor formation rate and the like, and has different degrees of drug resistance to gemcitabine, 5-fluorouracil, oxaliplatin and cis-platinum in first-line anti-cancer drugs for bile duct cancer. The method not only enriches an extrahepatic bile duct cancer cell bank, provides a new cell model for basic research and clinical drug research and development of bile duct cancer, but also provides powerful scientific research data for research developed based on the genetic background of Chinese population. Therefore, the primary multidrug resistant cell line has higher scientific research and clinical application values.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial medicine, in particular to a primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6 Homo sapiens and application thereof. Background Art

[0002] Cholangiocarcinoma (CCA) is a highly heterogeneous malignant tumor that originates from the biliary system and can occur from the intrahepatic bile duct to the major extrahepatic bile duct. Based on the anatomical site, cholangiocarcinoma can be divided into intrahepatic cholangiocarcinoma (iCCA) and extrahepatic cholangiocarcinoma (eCCA) when it occurs in the proximal part of the secondary bile duct (subclassified as pCCA and dCCA). Cholangiocarcinoma is the most common biliary malignancy and the second most common primary liver cancer after hepatocellular carcinoma, accounting for about 3% of all gastrointestinal tumors. The incidence of different types of cholangiocarcinoma varies greatly, with pCCA accounting for 50-60% of all cholangiocarcinomas, followed by dCCA (20-30%) and iCCA (10-20%) being the rarest. The highest incidence is in northeastern Thailand, Southeast Asia, and China. Cholangiocarcinoma lacks specific clinical manifestations, and genetic and acquired drug resistance are the main problems that lead to treatment failure in the vast majority of CCA patients.

[0003] At present, the occurrence, development and drug resistance mechanisms of cholangiocarcinoma have not been fully elucidated, and cell line models are essential for basic research on cholangiocarcinoma. However, as of now, there are 138 cholangiocarcinoma cell lines worldwide, mainly intrahepatic cholangiocarcinoma cell lines, and there are only 6 extrahepatic cholangiocarcinoma cell lines in the Chinese population. During long-term in vitro culture, most cholangiocarcinoma cell lines lose some of the characteristics of primary tumor tissues due to human or environmental factors, resulting in large differences in research results, and most drug-resistant cells are acquired. Naturally resistant cells are more reliable for studying mechanisms related to drug resistance.

[0004] Therefore, the establishment of cholangiocarcinoma cell lines, especially primary drug-resistant cell lines of extrahepatic cholangiocarcinoma, is crucial for studying the occurrence, development and drug resistance mechanism of cholangiocarcinoma. Summary of the invention

[0005] The first purpose of the present invention is to provide a primary multidrug-resistant distal cholangiocarcinoma cell line, mainly for the lack of cholangiocarcinoma cell lines from the Chinese population in China and the unclear mechanism of cholangiocarcinoma drug resistance.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide a human distal cholangiocarcinoma cell line CBC3T-6 Homo sapiens isolated from a distal bile duct differentiated adenocarcinoma.

[0008] The second object of the present invention is to provide a progeny cell line of the primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6 Homo sapiens.

[0009] The present invention provides a primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6 Homo sapiens, which was deposited in the China Center for Type Culture Collection on March 20, 2024, with a deposit number of CCTCC NO: C202436.

[0010] The present invention provides a primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6 Homo sapiens, which is used for detecting the cell line CBC3T-6 Homo sapiens and specifically comprises the following steps: S1: removing fibroblasts and other miscellaneous cells by digesting the cells, and finally screening out tumor cells;

[0011] S2: H&E staining of tumor cells followed by immunohistochemical analysis;

[0012] S3: The cells were then inoculated into 96-well plates. After the cells adhered to the plate, different concentrations of first-line chemotherapy drugs for bile duct cancer were added to observe the drug resistance of the cells to various drugs.

[0013] Furthermore, the tumor cells in step S1 are cholangiocarcinoma cell line CBC3T-6 Homo sapiens.

[0014] Furthermore, the first-line chemotherapy drugs in step S3 are cisplatin, gemcitabine, 5-fluorouracil, paclitaxel and oxaliplatin.

[0015] The third object of the present invention is to provide the application of the primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6Homo sapiens for the following technical solutions:

[0016] a) Establishing an animal model of human distal cholangiocarcinoma;

[0017] b) Screening or evaluating drugs for the treatment of distal bile duct cancer;

[0018] c) Study the mechanism of drug resistance in distal cholangiocarcinoma;

[0019] d) Develop drugs to reverse tumor resistance.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention provides a primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6 Homosapiens, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C202436. The cell line has stable characteristics and has been propagated to 50 generations. The human distal cholangiocarcinoma cell line was diagnosed as adenocarcinoma of the bile duct by pathological tissue pathology, and the bile duct markers CK7 and CK19 were positive, retaining the biological characteristics of extrahepatic cholangiocarcinoma. In addition, the CBC3T-6 cell line has the characteristics of short doubling time (about 50h), high subcutaneous tumor formation rate in mice, good uniformity, etc., and has different degrees of resistance to gemcitabine, 5-FU, oxaliplatin and cisplatin in the first-line anticancer drugs for cholangiocarcinoma, enriching the extrahepatic cholangiocarcinoma cell library.

[0022] 2. The mycoplasma test of the present invention was negative, indicating that the cell line was not contaminated by mycoplasma; the CBC3T-6 cell line has a high degree of malignancy and can be used to study the occurrence and development mechanism of distal cholangiocarcinoma.

[0023] 3. The present invention uses a primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6 Homo sapiens mouse subcutaneous tumor formation rate, which can be used to prepare a cholangiocarcinoma animal model. The distal cholangiocarcinoma animal model can be used for basic research and anticancer drug screening, and provides strong scientific research data for research based on the genetic background of the Chinese population. The cell line is a primary multidrug-resistant cell line, which has a higher scientific research and production application value than the currently available acquired drug-resistant cell lines, and provides new experimental materials for the study of clinical anticancer drug resistance and the study of reversing drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a proliferation curve of the cell line CBC3T-6 Homo sapiens of the present invention and a morphological change diagram of cells of different generations;

[0025] Note: A is the proliferation curve of CBC3T-6; B is the morphological observation table of CBC3T-6 cells at 5, 25, and 50 generations under an optical microscope.

[0026] Figure 2 This is a picture of the real-time living cell imaging result of the cell line of the present invention;

[0027] Figure 3 It is a graph of the identification results of the cell line STR of the present invention;

[0028] Note: Table 1 shows the results of STR identification; A is the STR spectrum of CBC3T-6 cells; B is the STR spectrum of tumor tissue derived from CBC3T-6 cells.

[0029] Figure 4is a G-banded karyotype analysis diagram of the cell line of the present invention;

[0030] Note: A and B are the results of karyotype analysis.

[0031] Figure 5 This is a diagram showing the results of identification of subcutaneous tumor formation of the cell line of the present invention;

[0032] Note: A is the tumor image after dissection of subcutaneous tumor; B is the volume growth curve of subcutaneous tumor; C is the weight growth curve of mice; D is the HE, CK7, CK19 and Ki67 identification results of primary tumor tissue, CBC3T-6 cells and subcutaneous tumor tissue.

[0033] Figure 6 This is the sensitivity result of the cell line of the present invention to the first-line anticancer drug for cholangiocarcinoma.

[0034] Note: AEs are the drug-dose response curves of gemcitabine, oxaliplatin, cisplatin, 5-fluorouracil (5-Fu), and paclitaxel, respectively. DETAILED DESCRIPTION

[0035] Example:

[0036] 1. Establishment of CBC3T-6 cell line

[0037] The tumor tissue was washed 3 times with ice PBS buffer, then placed in a culture dish, the fibrous connective tissue was removed, and the tissue blocks were minced into 1 mm pieces after washing again. 3 Digest in a mixture of neutral protease and type IV collagenase for 15 minutes, centrifuge and place the precipitate in a 37°C cell culture incubator with 5% CO2, change the medium every other day, and regularly digest the cells to remove fibroblasts and other miscellaneous cells, and finally screen out tumor cells.

[0038] After the line was successfully established, it was named human distal cholangiocarcinoma cell line CBC3T-6 Homo sapiens, and was deposited in the China Center for Type Culture Collection on March 20, 2024, with the deposit number CCTCC NO: C202436, and the address is Wuhan University, Wuhan, China.

[0039] 2. Doubling time of human distal cholangiocarcinoma cell line CBC3T-6 Homo sapiens

[0040] The human distal cholangiocarcinoma cell line CBC3T-6 Homo sapiens in the logarithmic growth phase was digested with trypsin and resuspended in DMEM / F-12 complete medium containing 10% FBS. The cell suspension was counted with a counting plate and the concentration was adjusted to 6000 cells / 200 μl. The cells were inoculated in a 96-well plate, 200 μl per well, 5 replicates per group, and CCK-8 solution was added at a ratio of 10% at 0, 24, 48, and 72 hours after culture. The cells were incubated in a cell culture incubator at 37°C for 2 hours, and the absorbance at 450 nm was then measured with an enzyme marker.

[0041] The results are as follows Figure 1 As shown in A, its population doubling time is about 50h, the proliferation rate is fast, and the malignancy is high, which is conducive to the study of cholangiocarcinoma in vitro model.

[0042] 3. Cell morphology observation

[0043] The growth of CBC3T-6 cells was observed under an inverted fluorescence microscope and photographed. Figure 1 As shown in B, there are 5th, 25th, and 50th generation cytological morphological pictures. The cell morphology does not change significantly with the increase of generations, and can be stably subcultured. The cells grow adherently, have good refractive properties, and are irregular in shape, with some being elliptical. When the cells are full, they grow in an overlapping manner, and cell contact inhibition disappears.

[0044] The results are as follows Figure 1 As shown in BD, the morphology of cells at the 5th, 25th, and 50th generations changed significantly, but their properties were stable.

[0045] IV. Live Cell Imaging of Human Distal Cholangiocarcinoma Cell Line CBC3T-6 Homo sapiens Cells

[0046] CBC3T-6 cells were seeded in a 96-well plate at a cell density of 2000 cells / 200 μl; the 96-well plate was placed in a Cytation5 live cell imaging workstation and set to take pictures every 2 hours for 96 hours.

[0047] Figure 2 The growth trajectory of CBC3T-6 cells is shown in the figure below, which are the cell photos at 0, 12, 24, 36, 48, 60, 72, 84, and 96 hours. The cells grow fast and have a good growth trend.

[0048] 5. Chromosome Karyotype Analysis of Human Distal Cholangiocarcinoma Cell Line CBC3T-6 Homo sapiens

[0049] When the CBC3T-6 cells with good growth status are in the logarithmic growth phase, add complete medium mixed with 10μg / mL colchicine and continue to culture for 2h. After discarding the medium, digest the cells with trypsin, wash with PBS buffer, centrifuge and add 5ml preheated hypotonic KCl solution 0.075M dropwise, incubate in a 37℃ water bath for 30min; then add 1ml freshly prepared methanol and glacial acetic acid fixative (methanol: glacial acetic acid, 3:1, V / V) for 5min; after centrifugation, add 6-8ml fixative dropwise again, mix well and fix at room temperature for 30min. After fixation, stain with Giemsa working solution, obtain metaphase chromosome specimens after sealing, and observe and record.

[0050] Figure 3 Chromosome karyotype analysis showed that the cell line had a nearly triploid karyotype with complex abnormalities in chromosome number and structure.

[0051] 6. STR Identification of Human Distal Cholangiocarcinoma Cell Line CBC3T-6 Homo sapiens Cells

[0052] CBC3T-6 cells and primary tumor tissues were collected for STR identification, including 21 gene loci.

[0053] The results of STR loci identification of CBC3T-6 Homo sapiens cells by China Center for Type Culture Collection are shown in Table 1. The above sequence was compared with the databases of ATCC, CELLOSAURUS and DSMZ depositories, and no identical genetic map was found. The results showed that CBC3T-6 was a newly established extrahepatic cholangiocarcinoma cell line and no cross contamination occurred during the primary culture process.

[0054] Figure 4 A is the STR profile of CBC3T-6 cells, and B is the STR profile of primary tumor tissue.

[0055] Table 1 STR loci

[0056]

[0057]

[0058] VII. Establishment of Mouse Subcutaneous Tumor Model

[0059] (1) CBC3T-6 cells in the logarithmic growth phase were trypsinized and centrifuged (800 rpm, 5 min), and the cell pellet was washed twice with phosphate buffer;

[0060] (2) Resuspend the cells in serum-free culture medium and count the cells and adjust the concentration to 1×10 7 / ml;

[0061] (3) 200 μl of cell suspension was injected into the right subscapularis of 4-week-old NOD / SCID mice (n=4), and observation and recording were performed regularly;

[0062] (4) Six days after inoculation, tumors began to form and grow, and the diameter of the tumor was measured every 4 days for 28 consecutive days;

[0063] (5) After 28 days, the mice were euthanized, the tumors were dissected out, photographed, and measured and recorded.

[0064] Figure 5 A is the tumor formed by CBC3T-6 cells in NOD / SCID mice; B is the growth curve of tumor volume, and the subcutaneous tumor formation rate of mice is 100%; C is the weight growth curve of mice, and the weight of mice does not change much during the subcutaneous tumor formation period. The results show that CBC3T-6 cells can form tumors quickly and grow uniformly in NOD / SCID mice; this cell mouse model can be used as an animal model for extrahepatic bile duct cancer related research.

[0065] 8. H&E staining

[0066] (1) Baking: Place the CBC3T-6 primary tumor tissue slices and subcutaneous transplanted tumor tissue slices in an oven and bake at 70°C for 1 h.

[0067] (2) Dewaxing: Transfer the slides to three cylinders of xylene and immerse them in each cylinder for 10 min for dewaxing.

[0068] (3) Hydration: The dewaxed sections were sequentially immersed in 100% ethanol I, 100% ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol for 1 min each, and then washed three times with double distilled water for 2 min each.

[0069] (4) Stain with hematoxylin for 5 min, then rinse twice with purified water.

[0070] (5) Differentiate with hydrochloric acid alcohol solution for 3 seconds and rinse twice with purified water.

[0071] (5) Cytoplasmic staining: Stain with eosin for 5 min and rinse twice with purified water.

[0072] (6) Soak in 75% ethanol, 85% ethanol, 95% ethanol, and anhydrous ethanol for 1 min each, air-dry, and seal the slides.

[0073] After the cell slides are fixed, they are washed twice with PBS, and then stained with hematoxylin, hydrochloric acid alcohol differentiation, and eosin in sequence. After washing with tap water, the slides are blown dry and sealed.

[0074] IX. Immunohistochemical Analysis

[0075] (1) Baking: Place the CBC3T-6 primary tumor tissue slices and subcutaneous transplanted tumor tissue slices in an oven and bake at 70°C for 1 h.

[0076] (2) Dewaxing: Transfer the slides to three cylinders of xylene and immerse them in each cylinder for 10 min for dewaxing.

[0077] (3) Hydration: The dewaxed sections were sequentially immersed in 100% ethanol I, 100% ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol for 1 min each, and then washed three times with double distilled water for 2 min each time.

[0078] (4) Antigen repair: Heat 0.01 M citrate buffer in a pressure cooker to boiling and use high temperature to repair antigen sites.

[0079] (5) Wash the sections 2 to 3 times with PBS, each time for 2 min.

[0080] (6) Add endogenous peroxidase blocker to the slices, incubate at room temperature for 10 min, and then wash three times in PBS, each time for 2 min.

[0081] (7) Add goat serum to cover the tissue sections for 10 min to block nonspecific binding.

[0082] (8) After discarding the blocking solution, place the plate in a wet box and add anti-CK7, CK19, and Ki67 antibodies. Incubate the plate in a 4°C refrigerator overnight.

[0083] (9) The next day, discard the primary antibody, wash with PBS three times for 2 min each time, transfer to a wet box, and add biotinylated secondary antibody for incubation for 30 min.

[0084] (10) DAB staining: Wash with PBS three times, add DAB and react for 1-3 min. Observe the color development under a microscope and terminate the color development in time.

[0085] (11) Hematoxylin staining: After washing the color developing solution, soak in hematoxylin staining solution for 4 min, rinse with purified water, differentiate with hydrochloric acid alcohol differentiation solution for 3 seconds, and then place in running water to rinse and blue for 5 min.

[0086] (12) After blueing, place the sections in 75% ethanol, 85% ethanol, 95% ethanol, and anhydrous ethanol for 1 min each. After air drying, add a drop of neutral gum to seal the sections.

[0087] After the CBC3T-6 cell slides were fixed with 4% paraformaldehyde, the cell membrane was transparentized using the permeability agent TritonX-100 to facilitate the penetration of the antibody, and then steps (5) to (12) were performed in sequence.

[0088] Figure 5E is HE staining of primary tumor specimens, CBC3T-6 cell slides, and xenograft tumors, as well as the immunohistochemical results of Ki67, CK7, and CK19. The HE staining results show that the tumor is a poorly differentiated adenocarcinoma of the bile duct, and Ki67, CK7, and CK19 are all positively expressed, indicating that the tumor is derived from the bile duct epithelium and has strong proliferation. Therefore, the human distal cholangiocarcinoma cell line CBC3T-6 provided by the present invention has a good in vivo model.

[0089] 10. Screening of first-line chemotherapy drugs for cholangiocarcinoma

[0090] After digestion of cells, the concentration was adjusted to 5 × 10 4 / ml cell suspension was inoculated into 96-well plates, 5000 cells / well, and 3 replicates per group. The next day, after the cells adhered to the wall, different concentrations of first-line chemotherapy drugs for cholangiocarcinoma were added: cisplatin, gemcitabine, 5-fluorouracil, paclitaxel and oxaliplatin. After the drug was applied for 48 hours, CCK-8 solution was added and incubated for 2 hours. The OD value at 450nm was detected with an enzyme reader, and the curve was drawn using Graphpad software to calculate the IC50 value.

[0091] The results are as follows Figure 6 As shown in AF, among the chemotherapy drugs, gemcitabine (IC50 = 1584μM), 5-FU (IC50 = 810.9μM), cisplatin (IC50 = 237μM) and oxaliplatin (IC50 = 144.4μM) have different degrees of resistance to CBC3T-6 cells. Among the resistant drugs, gemcitabine is the most obvious, followed by 5-FU. Paclitaxel (IC50 = 0.083μM) is relatively sensitive to CBC3T-6 cells. The different sensitivity of CBC3T-6 cells to first-line chemotherapy drugs for cholangiocarcinoma is conducive to the study of resistance-related mechanisms and provides a good cell model for the clinical development of drugs that reverse resistance.

[0092] In summary, the present invention provides a primary multidrug-resistant distal cholangiocarcinoma cell line CBC3T-6. The cell line has a strong proliferation ability, stable properties during in vitro culture, can be passaged multiple times, and STR confirms that it is a newly established extrahepatic cholangiocarcinoma cell line, and has the ability to form subcutaneous tumors in mice, which is a good in vitro research model for cholangiocarcinoma. The most prominent feature of this cell line is its multidrug resistance. By detecting the OD value of 450nm with an ELISA instrument, and using Graphpad software to draw a curve and calculate the IC50 value, it is judged that the cell line has different degrees of tolerance to the first-line chemotherapy drugs for the treatment of cholangiocarcinoma, which can be used to study the mechanism of chemotherapy resistance of extrahepatic cholangiocarcinoma, and further develop drugs related to reversing tumor resistance. At present, most of the research on tumor cell resistance lines is acquired resistance, and the primary resistance line is closer to the real environment of the human body, which has more advantages in the study of resistance and has high scientific research and clinical value.

[0093] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6 Homo sapiens, characterized in that: The human distal cholangiocarcinoma cell line CBC3T-6 was isolated from a moderately differentiated adenocarcinoma of the distal bile duct.

2. A primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6 Homosapiens according to claim 1, characterized in that: Included are progeny cell lines of the primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6.

3. The primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6 Homosapiens according to claim 1, characterized in that: The assay for the cell line CBC3T-6 specifically includes the following steps: S1: Remove fibroblasts and other miscells by digesting cells, and finally screen out tumor cells; S2: H&E staining of tumor cells followed by immunohistochemical analysis; S3: The cells were then inoculated into 96-well plates. After the cells adhered to the plate, different concentrations of first-line chemotherapy drugs for bile duct cancer were added to observe the drug resistance of the cells to various drugs.

4. A primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6 Homosapiens according to claim 3, characterized in that: The tumor cells in step S1 are cholangiocarcinoma cell line CBC3T-6 Homo sapiens.

5. A primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6 Homosapiens according to claim 4, characterized in that: The first-line chemotherapy drugs in step S3 are cisplatin, gemcitabine, 5-fluorouracil, paclitaxel and oxaliplatin.

6. A use of a primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6 Homo sapiens, characterized in that: Applied to the primary multidrug-resistant human distal cholangiocarcinoma cell line CBC3T-6Homo sapiens described in any one of claims 1 to 5, and applied to the following technical solutions: a) Establishing an animal model of human distal cholangiocarcinoma; b) Screening or evaluating drugs for the treatment of distal bile duct cancer; c) Study the mechanism of drug resistance in distal cholangiocarcinoma; d) Develop drugs to reverse tumor resistance.

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