Human liver primary undifferentiated pleomorphic sarcoma cell line HUPS-X1 and its application

Abstract

The human primary undifferentiated pleomorphic sarcoma cell line HUPS‑X1 and its application belong to the field of microbial animal cell lines. The human primary undifferentiated pleomorphic sarcoma cell line is named human primary undifferentiated pleomorphic sarcoma cell line HUPS‑X1, and the deposit number is CCTCC NO: C2024319. The human primary undifferentiated pleomorphic sarcoma cell line HUPS‑X1 can be used in establishing a cell model for the occurrence, development or metastasis of primary undifferentiated pleomorphic sarcoma of the liver; it can be used in studying the differentiation mechanism, cell morphology and functional abnormalities, tumor infiltration and metastasis mechanism of primary undifferentiated pleomorphic sarcoma of the liver, and in guiding the cell model for comprehensive clinical diagnosis and treatment; it can be used in studying the occurrence mechanism of primary undifferentiated pleomorphic sarcoma of the liver; it can be used in establishing an animal model of primary undifferentiated pleomorphic sarcoma of the liver.

Description

Technical Field

[0001] The present invention belongs to the field of microbial animal cell lines, and specifically relates to a Chinese human liver primary undifferentiated pleomorphic sarcoma cell line HUPS-X1 derived from a surgical resection specimen and an application thereof. Background Art

[0002] Undifferentiated pleomorphic sarcoma (UPS), formerly known as malignant fibrous histiocytoma, is a relatively common soft tissue sarcoma that originates from mesenchymal tissue and can occur in various parts of the body, most commonly in the limbs and retroperitoneum. It is extremely rare for it to occur in the liver, and the misdiagnosis rate is high.

[0003] Liver UPS is a rare soft tissue sarcoma of adult liver. Most scholars believe that its tissue origin is the result of bidirectional differentiation of primitive mesenchymal cells into histiocytes and fibroblasts. Weiss et al. classified it into five categories based on the composition and morphological characteristics of tumor cells, namely, storiform polymorphic type, myxoid type, giant cell type, inflammatory type and hemangioma type. In 2002, WHO re-evaluated and revised the terminology and classification method of malignant fibrous histiocytoma. Most scholars believe that the essence of malignant fibrous histiocytoma is UPS, and its histological origin and differentiation direction are still unclear, so myxoid type and hemangioma type were removed. In 2013, WHO replaced malignant fibrous histiocytoma with UPS in the latest soft tissue classification. The etiology and pathogenesis of this disease are still unclear, and may be related to trauma, burns, radiation / surgical injury, etc. Patients with liver UPS often have no obvious clinical symptoms in the early stage, and often seek medical treatment due to abdominal pain, abdominal mass or weight loss. Serological examinations are non-specific, and tumor markers such as AFP, CA19-9 and CEA are often negative. In terms of treatment, since liver UPS is insensitive to radiotherapy and chemotherapy, and the tumors mostly grow invasively and are prone to recurrence after surgery, early surgical extended resection is a more effective way to treat it.

[0004] Pathologically, the composition of this type of tumor cells is relatively complex, and can be composed of tissue cell fibroblasts, foam cells, multinucleated tumor giant cells, inflammatory cells or xanthomas, etc. The tumor cells are obviously heterogeneous, and nuclear division is easy to see. Immunohistochemical staining plays a great role in the diagnosis of this type of tumor. Tumor cells can express a variety of markers, but different tumors express different types of markers. Most tumors are positive for Vim, CD68, Actin, AAT, and AACT, and a small number of tumors are focally positive for Keratin, S100, Ki-67, CK, CD31, etc. Among them, Vim and CD68 are extremely important markers for diagnosing UPS. The simultaneous expression of these markers is more conducive to the diagnosis of the disease.

[0005] Due to the rarity of the tumor and the lack of clinical trials, the pathogenesis and treatment of primary undifferentiated pleomorphic sarcoma of the liver remain to be determined.

[0006] 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 a Chinese primary undifferentiated pleomorphic sarcoma cell line of the liver. Therefore, it is very necessary to establish a Chinese primary undifferentiated pleomorphic sarcoma cell line of the liver. Summary of the invention

[0007] The purpose of the present invention is to provide a novel human primary undifferentiated pleomorphic sarcoma cell line of the liver and its application in view of the lack of a primary undifferentiated pleomorphic sarcoma cell line of the liver in the prior art. The cell line has stable biological characteristics and can be used as a cell model for studying primary undifferentiated pleomorphic sarcoma of the liver.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The invention provides a human liver primary undifferentiated pleomorphic sarcoma cell line named as human liver primary undifferentiated pleomorphic sarcoma cell line (Homo sapiens) HUPS-X1, and the preservation number of the preservation center is: CCTCC NO: C2024319.

[0010] Establishment of cell line: A surgical specimen from a 32-year-old female was digested with a mixture of type II collagenase and neutral protease and then cultured for primary culture. A stable primary undifferentiated pleomorphic sarcoma cell line of the liver was established by cell culture technology and named HUPS-X1.

[0011] This cell line 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. The collection number of the collection center is CCTCC NO: C2024319.

[0012] The cell line has the following biological characteristics:

[0013] 1. Cell growth characteristics: HUPS-X1 cells grow adherently without contact inhibition and may show superimposed growth.

[0014] 2. Cell doubling time: 63h.

[0015] 3. Clone-forming ability: The cell line has strong clone-forming ability.

[0016] 4. Cell immunohistochemistry results: HUPS-X1 is a primary undifferentiated pleomorphic sarcoma cell line of the liver.

[0017] 5. Chromosome analysis: 9% of HUPS-X1 cells had a diploid karyotype, 71% had a triploid karyotype, and 20% had a quadruple karyotype. There were some differences in chromosome number and morphology. The representative karyotype was: 61,XX der(4),der(6),der(7),inv(9),der(17),der(18).

[0018] 6. Tumor-forming ability: After inoculation into NXG mice, HUPS-X1 can quickly form subcutaneous transplanted tumors with a tumor formation rate of 100%.

[0019] The human primary undifferentiated pleomorphic sarcoma cell line HUPS-X1 of 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 primary undifferentiated pleomorphic sarcoma of the liver, and guiding clinical comprehensive diagnosis and treatment.

[0020] The human primary undifferentiated pleomorphic sarcoma cell line HUPS-X1 can be used in establishing a cell model for the occurrence, development or metastasis of primary undifferentiated pleomorphic sarcoma of the liver.

[0021] The human primary undifferentiated pleomorphic sarcoma cell line HUPS-X1 can be used in studying the differentiation mechanism, cell morphology and functional abnormalities, tumor infiltration and metastasis mechanism of primary undifferentiated pleomorphic sarcoma of the liver, and in guiding the cell model of comprehensive clinical diagnosis and treatment.

[0022] The human primary undifferentiated pleomorphic sarcoma cell line HUPS-X1 can be used in studying the occurrence mechanism of primary undifferentiated pleomorphic sarcoma of the liver.

[0023] The human primary undifferentiated pleomorphic sarcoma cell line HUPS-X1 can be used in establishing an animal model of primary undifferentiated pleomorphic sarcoma of the liver.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention establishes a novel Chinese primary undifferentiated pleomorphic sarcoma cell line HUPS-X1, which has multiple stable biological characteristics and is suitable for the study of primary undifferentiated pleomorphic sarcoma of the liver, including its occurrence, development, metastasis mechanism and guidance of comprehensive clinical diagnosis and treatment. It provides a reliable cell model for in-depth study of primary undifferentiated pleomorphic sarcoma of the liver.

[0026] 2. The establishment of this cell line will help reveal the pathogenesis of primary undifferentiated pleomorphic sarcoma of the liver and provide a theoretical basis for developing new treatments and improving patient survival rates.

[0027] 3. The Chinese primary undifferentiated pleomorphic sarcoma cell line of the liver provided by the present invention can also be used for drug screening and efficacy evaluation, providing strong support for comprehensive clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Pathological results of tumor tissues derived from HUPS-X1 cells.

[0029] Figure 2 This is a morphological observation of HUPS-X1 cells under a microscope.

[0030] Figure 3 This is the growth curve of HUPS-X1 cells. Figure 3 In the figure, the horizontal axis is the cell culture time, and the vertical axis is the cell number.

[0031] Figure 4 These are the results of the HUPS-X1 cell organoid formation experiment.

[0032] Figure 5 The immunohistochemical results of HUPS-X1 cells. Figure 5 In the figure, A indicates positive expression of CD34 in the cells; B indicates positive expression of CD68 in the cells; C indicates positive expression of CK-pan in the cells; D indicates negative expression of GP-3 in the cells; E indicates that the positive expression rate of Ki67 in the cells is 80%; F indicates positive expression of Vimentin in the cells.

[0033] Figure 6 The results of chromosome analysis of HUPS-X1 cells.

[0034] Figure 7 The results of the in vivo tumorigenicity experiment of HUPS-X1 cells in immunodeficient mice. Figure 7 In the figure, AB shows that HUPS-X1 can form transplanted tumors after subcutaneous inoculation into NXG mice, with a tumor formation rate of 100%; C shows that no metastatic lesions were found in the lungs and liver of mice after 4 weeks. DETAILED DESCRIPTION

[0035] 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.

[0036] The experimental methods described in the following examples are conventional methods unless otherwise specified.

[0037] Establishment and characterization of a human primary undifferentiated pleomorphic sarcoma cell line, HUPS-X1.

[0038] 1. Establishment of the human primary undifferentiated pleomorphic sarcoma cell line HUPS-X1

[0039] Tumor tissues of patients with primary undifferentiated pleomorphic sarcoma of the liver were collected clinically and digested with mixed enzymes for primary culture. A primary undifferentiated pleomorphic sarcoma cell line of the liver 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 result of this patient was primary undifferentiated pleomorphic sarcoma of the liver. Figure 1 shown.

[0040] The present invention adopts a surgical resection specimen taken from a 32-year-old female, and then digested with type II collagenase / neutral protease for primary culture, and cultured by cell culture technology to establish a primary undifferentiated pleomorphic sarcoma cell line of the liver, named HUPS-X1. 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 is CCTCC NO: C2024319.

[0041] 2. Detection of biological characteristics of human primary undifferentiated pleomorphic sarcoma cell line HUPS-X1

[0042] 1. Cell morphology: After the cells have grown stably and have been passaged, 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, cells grow attached to the wall and there is an overlapping growth phenomenon. The morphological observation of HUPS-X1 cells under a microscope is shown in the figure below. Figure 2 shown.

[0043] 2. HUPS-X1 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 the primary tumor tissue to clarify the correlation between the cells and the primary tumor tissue. The results are shown in Table 1.

[0044] Table 1

[0045]

[0046] As can be seen from Table 1, the STR typing results of HUPS-X1 cells were highly consistent with those of primary tumor tissues, indicating that the HUPS-X1 cell line was derived from the tumor tissue and maintained its genetic characteristics during in vitro culture.

[0047] 3. Take HUPS-X1 cells in the logarithmic growth phase. During the logarithmic growth phase, cells divide actively and their number increases exponentially. Use 0.25% trypsin (VivaCell) to dissociate the cells and prepare them into a single cell suspension. Trypsin can decompose the proteins between cells and disperse the cells into a single state, which is convenient for subsequent inoculation and measurement. Determine the density of the cell suspension and add 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 Each cell was inoculated into 9 wells of a 96-well plate (NEST), and the liquid volume in each well was supplemented to 100 μL with complete medium 2. After 20 hours, 100 μL of diluted CCK8 solution [10% CCK8 (APE×BIO) + 90% RPMI-1640 (Gibco)] was added to each well. After 3.5 hours of adding CCK8 solution, the absorbance of each well 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. The growth curve of HUPS-X1 cells is shown in Figure 2. Figure 3 As shown, this curve can reflect the linear relationship between cell number and absorbance, and intuitively show the changing trend of cell number over time; based on this growth curve, the website https: / / doubling-time.com / was used to calculate the doubling time of the cells, and it was found that the average doubling time of HUPS-X1 cells was about 63h.

[0048] 4. Collect logarithmically growing HUPS-X1 cells, dissociate the cells from the culture dish or culture flask with 0.25% trypsin, and place 1.5×10 5 Cells were seeded into 6-well plates (ultra-low attachment plates). The design of the ultra-low attachment plates prevented the cells from attaching to the bottom of the plate, allowing them to grow and aggregate in a suspended state. RPMI-1640 complete medium was used for culture to provide the nutrients and growth factors required for cell growth. Organoid formation was monitored by microscopic observation on days 7, 10, and 14 after seeding.

[0049] The experimental results are as follows Figure 4 As shown. Figure 4 It can be seen that HUPS-X1 cells are able to form tumor organoids under ultra-low adsorption conditions, indicating that these cells have the ability to grow and aggregate in suspension.

[0050] By observing the growth and aggregation behavior of HUPS-X1 cells under ultra-low adsorption conditions, it is verified that these cells have the potential to form tumor organoids, proving that the HUPS-X1 of the present invention can provide strong support for disease research, drug screening, regenerative medicine and other fields.

[0051] 5. Take HUPS-X1 cells in the logarithmic growth phase, enzymatically dissociate the cells to disperse them into a single state, and inoculate the dissociated cells onto sterile glass slides for growth. After 60 hours, wash the coverslips with PBS to remove residual culture medium and impurities, and fix them with 4% paraformaldehyde (Servicebio) for 15 minutes to maintain the morphology and structure of the cells; air-dry the slides, treat them with 0.5% TritonX-100 (MCE) for 20 minutes to increase the permeability of the cell membrane, and then proceed to subsequent staining. Embed the original tumor tissue and xenograft tumor in paraffin, cut them into 4μm thick sections, dry them at 60℃ for 5 hours, remove the water and paraffin in the sections, and proceed to subsequent staining.

[0052] Immunohistochemical staining: After dewaxing and rehydrating the sections, the slides were immersed in sodium citrate solution (10 mMol / L, pH=6.0) and boiled (90 s) for antigen repair to improve the binding ability of antigen and antibody; then the sections were incubated with 3% hydrogen peroxide solution at 37°C for 15 min to remove endogenous peroxidase in the tissue to avoid interference with subsequent staining; 100 µL normal goat serum was added and blocked at 37°C for 15 min to reduce the occurrence of nonspecific staining. Then, the sections were incubated with anti-CD34, anti-CD68, anti-CK-pan, anti-GP-3, anti-CEA, anti-Ki67 and anti-Vimentin at 37°C for 12 hours, and then the secondary antibodies were added and incubated at room temperature for 50 minutes; the DAB staining solution kit (Dako) was used for color development. DAB can be oxidized into brown precipitate in the presence of antibody complexes, thereby showing the location and distribution of antigens; the sections were rinsed with running water for 5 minutes and counterstained with hematoxylin to make the cell nuclei appear blue; after gradient ethanol dehydration, xylene transparent treatment, and neutral resin sealing, the sections were placed under an inverted microscope (Olympus, IX73+DP74) for observation. The results are shown in Figure 2. Figure 5 shown. Figure 5 The staining of different antibodies in cells and tissues is shown in Figure 2.

[0053] Figure 5 A in the figure indicates that CD34 is positively expressed in the cells, indicating the presence of CD34 antigen in the cells; Figure 5B in the figure indicates that CD68 is positively expressed in the cells, indicating the presence of CD68 antigen in the cells; Figure 5 The C in the figure indicates that CK-pan is positively expressed in the cells, indicating the presence of keratin in the cells; Figure 5 The D in the figure indicates that GP-3 is negatively expressed in the cells, indicating that there is no GP-3 antigen in the cells; Figure 5 The E in the figure indicates that the positive expression rate of Ki67 in cells is 80%. Ki67 is a nuclear antigen associated with proliferating cells. Its high expression rate indicates active cell proliferation. Figure 5 The F in the figure indicates that Vimentin is positively expressed in the cell. Vimentin is an intermediate filament protein, and its positive expression indicates the presence of intermediate filament structures in the cell.

[0054] The experiment successfully detected the expression of multiple antigens in HUPS-X1 cells, original tumor tissues and xenograft tumors through immunohistochemical staining technology. The expression of these antigens is of great significance for understanding the biological characteristics of cells, the occurrence and development mechanism of tumors, and evaluating the efficacy of drugs.

[0055] Experiments have shown that the HUPS-X1 of the present invention can be used in studying the biological behavior of cells, exploring its potential clinical applications, and developing treatment methods for related diseases.

[0056] 6. Take HUPS-X1 cells in the logarithmic growth phase, add colchicine (Spectrum) to the culture medium and make the colchicine concentration 0.2ug / mL, incubate for 90min, digest the cells with 0.25% trypsin (Vivacell) to form a cell suspension; centrifuge and discard the supernatant, resuspend the precipitate with 0.56% KCl solution, and incubate at 37℃ for 30min to swell the cells. 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 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, and observe the stained cells under a 100x oil microscope after sealing. Karyotype analysis was performed using ImageJ imaging software and the ChromosomeJ plug-in. Figure 6 shown.

[0057] The HUPS-X1 cells have a nearly diploid karyotype of 9%, a nearly triploid karyotype of 71%, and a nearly quadruple karyotype of 20%. There are certain differences in chromosome number and morphology. The representative karyotype is: 61,XX der(4),der(6),der(7),inv(9),der(17),der(18)( Figure 6 ).

[0058] The above experiments prove that the present invention can be applied in cell models of the occurrence, development or metastasis of primary undifferentiated pleomorphic sarcoma of the liver, the differentiation mechanism of primary undifferentiated pleomorphic sarcoma of the liver, abnormal cell morphology and function, tumor infiltration and metastasis mechanism, and cell models for guiding comprehensive clinical diagnosis and treatment.

[0059] 7. Take HUPS-X1 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 0.1ml per mouse, and the weight changes and growth of transplanted tumors of NXG mice were monitored regularly. After 4 weeks, the mice were euthanized, and the xenograft tumors and liver and lung tissues of the mice were removed by dissection 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 A and B in the figure show that HUPS-X1 can form transplanted tumors when inoculated subcutaneously in NXG mice, with a tumor formation rate of 100%; Figure 7 C in the figure indicates that no metastatic lesions were found in the lungs and liver of mice after 4 weeks. The above experiments prove that HUPS-X1 cells have a strong tumor-forming ability in NXG mice, and the present invention can be used in establishing an animal model of primary undifferentiated pleomorphic sarcoma of the liver.

[0060] 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 primary undifferentiated pleomorphic sarcoma cell line of liver, characterized by The cell line was named as human primary undifferentiated pleomorphic sarcoma of the liver HUPS-X1, and its deposit number was CCTCC NO: C2024319.

2. Use of the human primary undifferentiated pleomorphic sarcoma cell line HUPS-X1 of claim 1 in establishing a cell model of the occurrence, development or metastasis of primary undifferentiated pleomorphic sarcoma of the liver.

3. Use of the human primary undifferentiated pleomorphic sarcoma cell line HUPS-X1 of claim 1 in studying the differentiation mechanism, cell morphology and functional abnormalities, tumor infiltration and metastasis mechanism of primary undifferentiated pleomorphic sarcoma of the liver, and in guiding the cell model for comprehensive clinical diagnosis and treatment.

4. Use of the human primary undifferentiated pleomorphic sarcoma cell line HUPS-X1 of claim 1 in studying the pathogenesis of primary undifferentiated pleomorphic sarcoma of the liver.

5. Use of the human primary undifferentiated pleomorphic sarcoma cell line HUPS-X1 of claim 1 in establishing an animal model of primary undifferentiated pleomorphic sarcoma of the liver.

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