A malignant pleomorphic adenoma tissue block MPA9H-1 and its use

Abstract

The present invention relates to the field of cell engineering, and in particular to a human malignant pleomorphic adenoma tissue block MPA9H-1 and its use. The malignant pleomorphic adenoma tissue block MPA9H-1 provided by the present invention can be used as a research platform, and has the following uses: studying the disease pathogenesis of malignant pleomorphic adenoma; studying the gene function related to malignant pleomorphic adenoma; studying the mechanism of action of drugs related to malignant pleomorphic adenoma; through the above research, further clarifying the molecular mechanism of the onset of malignant pleomorphic adenoma, discovering new therapeutic targets through the tissue pathogenesis mechanism, facilitating drug research and development, such as the efficacy study of chemically synthesized drugs, screening and identification of effective ingredients of traditional Chinese medicine, etc.

Description

Technical Field

[0001] The invention relates to the field of cell engineering, in particular to a human malignant pleomorphic adenoma tissue block MPA9H-1 and a use thereof. Background Art

[0002] Pleomorphic adenoma (PA) is the most common benign salivary gland tumor. PA that persists for a long time or recurs repeatedly often undergoes malignant transformation and turns into malignant pleomorphic adenoma (MPA). According to statistics, MPA accounts for about 12% (2.8% to 42.4%) of all salivary gland malignancies. Among the salivary gland malignancies in the eastern population of China, MPA accounts for about 8%, making it the third most common salivary gland malignancy. Currently, the main treatment for MPA is surgical resection, combined with postoperative radiotherapy and chemotherapy, but the prognosis of patients is not good.

[0003] The development of new therapeutic drugs and the exploration of new diagnostic and treatment options have become key issues to be addressed in MPA research. In vitro and in vivo tumor models that retain the genetic background and biobehavioral characteristics of patient tumors and can simulate the heterogeneity of clinical patient tumors are effective tools for tumor research. Patient-derived xenograft models (PDX) and human-derived primary cells (PDC) cultured on their basis are gradually providing a stable and reliable preclinical platform for studying tumor biology and evaluating anti-tumor drugs, as well as for tumor molecular pathological diagnosis, molecular typing, targeted drug development, biomarker discovery and translational research. In recent years, a large number of tissue-derived xenograft models have been established in colorectal cancer, breast cancer, lung cancer, bladder cancer, and oral squamous cell carcinoma. In comparison, MPA preclinical models are particularly lacking, and there are no PDX reports yet, which has hindered the progress of its research to a certain extent. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a malignant pleomorphic adenoma tissue block MPA9H-1 and cells derived from the tissue block and uses thereof, so as to solve the problems in the prior art of few malignant pleomorphic adenoma transplant tumor models and few drug sensitivity studies, and lack of research models.

[0005] To achieve the above-mentioned purpose and other related purposes, the present invention provides a human malignant pleomorphic adenoma tissue block MPA9H-1 and cells derived from the tissue block. The human malignant pleomorphic adenoma tissue block MPA9H-1 is deposited in the China Center for Type Culture Collection CCTCC, the preservation date is 2022-11-30, the preservation number is CCTCC No: C2022358, and the preservation address is Wuhan University, Wuhan, China.

[0006] The malignant pleomorphic adenoma tissue block MPA9H-1 is derived from a primary human tumor.

[0007] Preferably, compared with other tumor cells or tumor cells, the malignant pleomorphic adenoma tissue block MPA9H-1 or tumor cells express one or more selected from the following: RNA, protein or polypeptide corresponding to AE1 / AE3, CK7, CK14, P63, S-100, Vimentin or Ki-67.

[0008] The present invention also provides a method for constructing a malignant pleomorphic adenoma PDX animal model: inoculating a malignant pleomorphic adenoma tissue block MPA9H-1 or tumor cells into a model animal.

[0009] The present invention also provides a malignant pleomorphic adenoma PDX animal model constructed by the aforementioned construction method.

[0010] The present invention also provides the use of the malignant pleomorphic adenoma tissue block MPA9H-1 or tumor cell or the malignant pleomorphic adenoma PDX animal model in developing tumor treatment targets and in screening tumor treatment products.

[0011] The present invention also provides the use of the malignant pleomorphic adenoma tissue block MPA9H-1 or tumor cell or the malignant pleomorphic adenoma PDX animal model in the screening of malignant pleomorphic adenoma biomarkers or the study of the occurrence and development mechanism of malignant pleomorphic adenoma.

[0012] The present invention also provides use of malignant pleomorphic adenoma tissue block MPA9H-1 or tumor cells in preparing a malignant pleomorphic adenoma PDX animal model construction product.

[0013] As described above, the malignant pleomorphic adenoma tissue block MPA9H-1 and its use of the present invention have the following beneficial effects:

[0014] Malignant pleomorphic adenoma cells can be used as a research platform for the following purposes:

[0015] 1. Basic research

[0016] (1) The pathogenesis of malignant pleomorphic adenoma;

[0017] (2) gene functions associated with malignant pleomorphic adenoma;

[0018] (3) Mechanism of action of drugs related to malignant pleomorphic adenoma.

[0019] 2. Disease diagnosis and treatment

[0020] Through basic research, the molecular mechanism of the pathogenesis of malignant pleomorphic adenoma will be further clarified, and new therapeutic targets will be discovered through tissue development mechanisms, which will facilitate drug research and development, such as efficacy research on chemically synthesized drugs, screening and identification of effective ingredients in traditional Chinese medicine, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a mouse model of malignant pleomorphic adenoma model P1 and P2 generation transplanted tumors.

[0022] Figure 2 Flowchart for constructing a malignant pleomorphic adenoma model.

[0023] Figure 3 This is the growth curve of the oral salivary gland malignant pleomorphic adenoma xenograft model one month after inoculation. This is the growth curve of the primary tumor model from P1 to P3.

[0024] Figure 4 This is the histopathological verification of the oral salivary gland malignant pleomorphic adenoma transplant tumor model. A. HE section of the primary tumor model, HE magnification is ×20; B. HE section of the primary tumor, HE magnification is ×20; C. HE section of the primary tumor model, HE magnification is ×40; D. HE section of the primary tumor, HE magnification is ×40.

[0025] Figure 5 The results of immunohistochemical staining of the oral salivary gland malignant pleomorphic adenoma xenograft model. The IHC magnification is ×40.

[0026] Figure 6 The figure shows the FISH staining results of the oral salivary gland malignant pleomorphic adenoma transplanted tumor model.

[0027] Figure 7 This is a copy number variation result diagram of the oral salivary gland malignant pleomorphic adenoma transplant model. In the figure, compared with normal cells, the red part is the part with increased copy number, the green part is the part with consistent copy number, and the blue part is the part with decreased copy number.

[0028] Figure 8 This is a graph showing the single nucleotide variation results of the oral salivary gland malignant pleomorphic adenoma transplant model.

[0029] Fig. 9 This is a microscopic image of the cell morphology of a malignant pleomorphic adenoma of a human salivary gland. The magnification is ×20.

[0030] Fig.10 The PDC cells (generation P5) of human salivary gland malignant pleomorphic adenoma expressed malignant pleomorphic adenoma-related markers. The IF magnification was ×20. DETAILED DESCRIPTION

[0031] The present invention provides a human malignant pleomorphic adenoma tissue block MPA9H-1 and tumor cells derived from the tissue block. The human malignant pleomorphic adenoma tissue block MPA9H-1 is deposited in China Center for Type Culture Collection CCTCC, with a preservation date of 2022-11-30, a preservation number of CCTCC No: C2022358, and a preservation address of Wuhan University, Wuhan, China.

[0032] The malignant pleomorphic adenoma tissue block MPA9H-1 is derived from a primary human tumor.

[0033] Compared with other tumor tissue blocks or tumor cells, the malignant pleomorphic adenoma tissue block MPA9H-1 or tumor cells derived from MPA9H-1 express one or more selected from the following: RNA, protein or polypeptide corresponding to AE1 / AE3, CK7, CK14, P63, S-100, Vimentin or Ki-67.

[0034] The HER-2 gene of the malignant pleomorphic adenoma tissue block MPA9H-1 or the tumor cells derived from MPA9H-1 is amplified, with an amplification ratio of 25-35%, and the PLAG1 gene is broken. However, the HER-2 gene of the healthy salivary gland tissue block or the healthy salivary gland cells is not amplified, and the PLAG1 gene is not broken.

[0035] The gene copy number of chromosome 1, chromosome 2, chromosome 3, chromosome 6, chromosome 9, chromosome 11 or chromosome 14 in the malignant pleomorphic adenoma tissue block MPA9H-1 or in the tumor cells derived from MPA9H-1 is increased compared with that in the healthy salivary gland tissue block or healthy salivary gland cells; the gene copy number of chromosome 10, chromosome 16, chromosome 17 or chromosome 22 is decreased compared with that in the healthy salivary gland tissue block or healthy salivary gland cells.

[0036] The malignant pleomorphic adenoma tumor cells in the malignant pleomorphic adenoma tissue block MPA9H-1 of the present invention are slender spindle-shaped or contain a small amount of irregular polygonal cells.

[0037] The present invention also provides a method for establishing tumor cells derived from the aforementioned malignant pleomorphic adenoma tissue block MPA9H-1, the method comprising the following steps:

[0038] 1) Tissues obtained from the primary tumor lesions of patients with primary oral salivary gland malignant pleomorphic adenoma were processed into tissue fragments after the connective tissue and necrotic tissue were removed;

[0039] 2) culturing the above tissue fragments;

[0040] 3) Culture and obtain malignant pleomorphic gonadal tumor cells.

[0041] In some embodiments, the tissue obtained from the primary tumor in step 1) is washed with 5% penicillin-streptomycin-containing PBS and then the connective tissue and necrotic tissue are removed. Preferably, the volume of the tissue obtained from the primary tumor is 0.5-1.8 cm 3 .

[0042] In some specific embodiments, the volume size of the tissue fragments in step 1) is 0.8-1.2 mm*0.8-1.2 mm*0.8-1.2 mm.

[0043] In some embodiments, the culturing method in step 3) comprises one or more of the following steps:

[0044] 1a) The aforementioned tissue fragments are evenly transferred to the bottom of a T25 culture flask;

[0045] 1b) adding 1-3 ml of the culture medium of the malignant pleomorphic adenoma tissue piece MPA9H-1 to the culture flask in step 1a);

[0046] 1c) placing the culture flask in step 1b) upside down in a cell culture incubator for 5-9 hours, and then culturing it upright for 3-5 days;

[0047] 1d) Add 3-6 of the above culture medium from step 1c) and continue culturing until the actual cell area is larger than the field of view of the 10x objective lens and 10x eyepiece combination.

[0048] In some specific embodiments, the culture medium of the malignant pleomorphic adenoma tissue block MPA9H-1 comprises one or more of DMEM, serum or antibiotics. Preferably, the serum is fetal bovine serum; more preferably, the fetal bovine serum accounts for 15-25% of the volume of the culture medium.

[0049] The present invention also provides a method for constructing the aforementioned malignant pleomorphic adenoma PDX animal model, the method comprising inoculating a malignant pleomorphic adenoma tissue block MPA9H-1 or tumor cells derived from MPA9H-1 into the back of a mouse. The malignant pleomorphic adenoma tissue block MPA9H-1 is derived from tissue fragments obtained from the primary focus of a malignant pleomorphic adenoma.

[0050] In some specific embodiments, the size of the tissue fragments is 0.4-1.6 mm*0.4-1.6 mm*0.4-1.6 mm.

[0051] The present invention also provides a malignant pleomorphic adenoma PDX animal model constructed by the aforementioned construction method, wherein the malignant pleomorphic adenoma animal model is transplanted with a malignant pleomorphic adenoma tissue block MPA9H-1 or tumor cells derived from MPA9H-1. Preferably, the malignant pleomorphic adenoma animal model is a mouse model.

[0052] In a specific embodiment, the aforementioned sections of the malignant pleomorphic adenoma have one or more of the following characteristics: the tumor cells are densely arranged; compared with healthy salivary gland cells, the nuclear-to-cytoplasmic ratio of malignant pleomorphic adenoma cells is larger; compared with healthy salivary gland cells, more nuclear division phases are observed in malignant pleomorphic adenoma cells; and hyalinization is observed in the tumor stroma of the malignant pleomorphic adenoma.

[0053] In one specific embodiment, the aforementioned mouse is a nude mouse.

[0054] The aforementioned malignant pleomorphic adenoma animal model is a BALB / c Nude mouse model. Preferably, the aforementioned mouse is a male mouse with an age of 3-7 weeks and a body weight of 12-25 grams.

[0055] The present invention also provides the use of the malignant pleomorphic adenoma tissue block MPA9H-1, the tumor cells derived from MPA9H-1 or the malignant pleomorphic adenoma PDX animal model in developing tumor therapeutic targets or screening tumor therapeutic products. Preferably, the aforementioned tumor is a malignant pleomorphic adenoma. The development of tumor therapeutic targets or screening of tumor therapeutic products are all performed in vitro.

[0056] The aforementioned tumor treatment product is selected from drugs. The aforementioned use is, for example, to study the drug metabolism, efficacy or safety of the treatment of malignant pleomorphic adenoma using the malignant pleomorphic adenoma tissue block MPA9H-1, tumor cells derived from MPA9H-1 or PDX animal models. Another example is to screen drugs for the treatment of malignant pleomorphic adenoma using the malignant pleomorphic adenoma tissue block MPA9H-1, tumor cells derived from MPA9H-1 or PDX animal models.

[0057] In some embodiments, the drug is selected from one or more of ponatinib, carboplatin, cisplatin, paclitaxel or pembrolizumab.

[0058] The present invention also provides the use of the aforementioned malignant pleomorphic adenoma tissue block MPA9H-1, tumor cells derived from MPA9H-1 or PDX animal model in the screening of malignant pleomorphic adenoma biomarkers or the study of the occurrence and development mechanism of malignant pleomorphic adenoma. The mechanism study is a study conducted in vitro.

[0059] The present invention also provides the use of the aforementioned malignant pleomorphic adenoma tissue block MPA9H-1 and tumor cells derived from MPA9H-1 in preparing a malignant pleomorphic adenoma PDX animal model construction product.

[0060] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0061] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0062] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0063] Example 1 Material preparation for construction of malignant pleomorphic adenoma PDX

[0064] Tumor specimen: obtained from a 74-year-old female patient who underwent surgical resection in the Department of Oral and Maxillofacial-Head and Neck Tumors of the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Pathological diagnosis was primary oral salivary gland malignant pleomorphic adenoma. The patient was a newly diagnosed patient with the primary lesion located at the right skull base. The patient signed an informed consent form.

[0065] Experimental animals: Male BALB / c nude mice, 4 - 6 weeks old, weighing about 15 - 20 g, were purchased from the Experimental Animal Business Department of Shanghai Institute of Planned Parenthood Research [SCXK(Shanghai) 2018 - 0006] and adaptively fed in the specific pathogen-free (SPF) animal house of the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine [SYXK(Shanghai) 2012 - 0007] for 1 week. The temperature of the animal house breeding environment was controlled at 22℃ - 26℃, the relative humidity was 40% - 60%, the breeding density did not exceed 3 animals per cage, and the related cages, drinking water, bedding and feed were all sterilized. The nude mice could move and eat freely in the cage. The welfare of experimental animals and the specific experimental procedures involved in this research all followed the breeding regulations, use guidelines of the Experimental Animal Center of the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine and the medical ethics rules and regulations of the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine.

[0066] Tumor sample collection: Tumor specimens were collected during the operation, and the ex vivo time was less than 30 minutes. Typical areas in the tumor lesions were selected, avoiding areas such as liquefaction and necrosis. The tumor tissue was cut into several small pieces with a size of about 3mm×3mm×3mm. 2 - 3 small pieces were reserved for constructing the PDX model and placed in a centrifuge tube containing PBS solution (added with 500 IU / mL penicillin and 500 mg / mL streptomycin), stored at 4℃, and sent to the animal house within 1 hour for the experiment; another 2 - 3 small pieces were placed in 10% neutral buffered formalin solution and fixed for 12 - 24 hours for subsequent histopathological and molecular characterization experiments.

[0067] Example 2 Construction method of malignant pleomorphic adenoma PDX

[0068] Tumor tissue samples of malignant pleomorphic adenoma were quickly placed in centrifuge tubes containing commercial tissue preservation solution (MACS, Germany). The centrifuge tubes were placed in ice boxes and transported to the laboratory. In a biosafety cabinet, the tissue was transferred to a culture dish and soaked in PBS containing 5% penicillin-streptomycin for about 10 minutes. Transferred to a culture dish containing a newly prepared serum-free culture medium (the main component is 5% penicillin-streptomycin), and mucosal and other tissues were removed. The tissue was cut into tissue fragments of about 1mm×1mm×1mm in size. The tissue fragments were transferred to a new culture dish containing MPA culture medium. The main component of the culture medium is DMEM+20% fetal bovine serum. Using a 1 ml pipette tip, transfer the tissue dipped in the culture medium to the bottom of a T25 culture flask and evenly distribute the tissue. Add 2 ml of MPA culture medium, tighten the bottle mouth, and invert it into a cell culture incubator. After 6 to 8 hours, the culture flask was placed upright. After 4 days, gently remove it, add 4 ml of MPA culture medium, and continue to culture. During this period, observe the cell confluence under the microscope as needed. When the cell migration area is about the field of view of the microscope combined with the 10x objective lens and the 10x eyepiece, the trypsin digestion method is used for subculture.

[0069] Example 3 Immunohistochemical staining of primary tumor and corresponding transplanted tumor model

[0070] Histological observation and analysis of malignant pleomorphic adenoma transplanted tumor sections: Primary and subcultured tumor-bearing mice were sacrificed 7-8 weeks after inoculation, and tumor tissues were removed from all tumor-bearing mice ( Figure 1 ), fixed overnight in 10% neutral buffered formaldehyde solution (generally no more than 24h), embedded in paraffin, sliced ​​at 3μm, stained with hematoxylin & eosin (HE), observed routinely, and compared with the primary tumor. Immunohistochemistry (IHC) detection and analysis: The paraffin-embedded tumor tissue was sliced ​​into several 3μm continuous slices and baked at 70℃ for 1h; after dewaxing and hydration, antigen retrieval was performed in EDTA buffer with a pH of 8.0; the slices were blocked with 3% H2O2 solution to eliminate the activity of endogenous catalase in the tissue. After rinsing with PBS for 3 times, primary antibodies (AE1 / AE3, Catalog No. Kit-0009, CK7, Catalog No. Kit-0021, CK14, Catalog No. MAB-0169, S-100, Catalog No. Kit-0007, Ki-67, Catalog No. RMA-0731, Vimentin, Catalog No. Kit-0019, P63, Catalog No. MAB-0694, all of which were purchased from Fuzhou Maixin Biotechnology Development Co., Ltd.) were added and incubated at room temperature for 1 hour. After rinsing with PBS for 3 times, secondary antibodies were added and incubated at room temperature for 30 minutes. After rinsing with PBS for 3 times, DAB was used for color development and hematoxylin counterstaining, and the slides were sealed with neutral gum and observed.

[0071] Immunohistochemical staining was used to detect the expression of malignant pleomorphic adenoma markers AE1 / AE3, CK7, CK14, P63, S-100, Vimentin and Ki-67, which reflects cell proliferation activity, in transplanted tumors and corresponding primary tumors. The immunohistochemical results of positive cell localization and staining intensity in transplanted tumors and corresponding patients' primary tumors are detailed in ( Figure 5 ), showing a high degree of consistency in the results.

[0072] Example 4 Identification of primary tumor and corresponding transplanted tumor model by FISH staining

[0073] Fluorescence in situ hybridization (FISH) analysis of malignant pleomorphic adenoma transplanted tumor sections: Paraffin-embedded tumor tissue was sliced ​​several times at 3 μm and baked at 70°C for 1 hour; after dewaxing and hydration, the sections were pretreated in 90°C distilled water for 30 minutes. The tissue was digested in a 54°C pepsin digestion solution for 15-20 minutes, and the slides were dehydrated and air-dried with gradient ethanol. Under light-proof conditions, 5-10 μL of probe (HER-2, item number F.01360-01 and PLAG1 item number F.01252-01, all of which were purchased from Guangzhou Anping Pharmaceutical Technology Co., Ltd.) was added dropwise, and the slide was covered with rubber cement to seal the slide. Denaturation of the hybridizer: 76°C, 6 minutes, hybridization temperature 42°C, 16 hours 30 minutes. The next day, the slides were taken out, the rubber cement was removed, and the slides were placed in a 2×SSC / 0.3% NP-40 (pH 7.0-7.5) solution preheated to 72°C for 2 minutes, and then the sections were placed in a 2×SSC / 0.1% NP-40 solution at room temperature for 2 minutes. Dehydrated with gradient ethanol, dried naturally in the dark, 10 μL of DAPI counterstaining solution was added to the corresponding area of ​​probe hybridization, covered with a coverslip, and observed under a fluorescence microscope. Tumor cells were searched for with three objective lenses of 10x, 40x, and 100x oil objective lens, and the red and green signals in 20 cells were counted under a 100x oil objective lens.

[0074] FISH staining was used to detect the amplification of HER-2 and PLAG1 in malignant pleomorphic adenoma in transplanted tumors and corresponding primary tumors. FISH results showed that the amplification ratio of HER-2 gene in transplanted tumors and primary tumors was 25-35%; PLAG1 gene was broken ( Figure 6 ), the results remain consistent.

[0075] Example 5 Identification of primary tumors and corresponding transplanted tumor models by sequencing

[0076] Sequencing analysis of malignant pleomorphic adenoma xenografts: Genomic DNA was extracted from primary tumors and xenografts using the QIAGEN German DNA extraction kit. DNA quantification and integrity were determined by Nanodrop spectrophotometer and 1% agarose electrophoresis, respectively. Genomic DNA samples of primary tumors and xenografts were captured using the Agilent SureSelect Human Whole Exon v8 Library (Agilent Technologies, USA) according to the manufacturer's protocol. Whole exome sequencing was used to analyze whether the copy number variation and single nucleotide variation of the PDX model were consistent with the corresponding primary tumor.

[0077] Whole exome sequencing was used to detect copy number variation and single nucleotide variation in malignant pleomorphic adenoma in transplanted tumors and corresponding primary tumors. The results showed that the copy number variation and single nucleotide variation in primary tumors and transplanted tumors were highly consistent and highly consistent ( Figure 7 , Figure 8 ).

[0078] Example 6 Establishment of a malignant pleomorphic adenoma transplant model

[0079] Malignant pleomorphic adenoma tissue was inoculated subcutaneously in nude mice under sterile conditions according to the following steps: Before the experiment, the surface of the biosafety cabinet was sprayed and disinfected with 75% ethanol, and the inoculated nude mice were numbered and weighed by ear clipping. According to the weight of the nude mice, tribromoethanol was extracted at 0.02mL / 10g and anesthetized by intraperitoneal injection, and the maximum injection volume did not exceed 0.1mL; then the fresh tumor tissue specimens were trimmed into 1mm×1mm×1mm size and washed 3 times with PBS solution (500IU / mL penicillin and 500mg / mL streptomycin were added); the back skin of the anesthetized nude mice was disinfected with 75% ethanol, and a small incision of about 5mm was opened subcutaneously; microsurgical forceps were used for blunt separation, the trimmed tissue was inoculated subcutaneously, the incision was sutured, and 75% ethanol was used for disinfection and hemostasis. According to the sample size, 3-4 nude mice were generally inoculated.

[0080] Tumor formation in nude mice: According to the inoculation time and growth rate of the transplanted tumor, one week after inoculation, the maximum long and short diameters of the transplanted tumor were measured twice a week with a vernier caliper, and its volume was estimated (formula: transplanted tumor volume = long diameter × (short diameter^2) / 2), and the tumor volume time growth curve was drawn. Method for judging whether the transplantation is successful: the transplanted tumor volume is 1000-2000mm 3 Or after 2 months of observation, the transplanted tumor volume is greater than 200mm 3 If the transplant continues to grow, the transplant is considered successful.

[0081] After the primary transplanted tumor has grown stably, it is serially passaged. The specific process of constructing the malignant pleomorphic gonadal transplanted tumor model is shown in Figure 2. Construction of the nude mouse malignant pleomorphic tumor model: A primary malignant pleomorphic adenoma model nude mouse was randomly selected for serial passaging (the experimental steps were the same as the primary construction) and subsequent experiments. The primary constructed transplanted tumor was defined as the P0 generation, and the subsequent transplanted tumor constructed based on the P0 generation was the P1 generation, and so on.

[0082] The growth of transplanted tumors can be observed under the naked eye of the experimental nude mice (about 1 week after surgery). During this period, the mice's vital signs were stable, and there were no significant changes in appetite and mental state. According to the naked eye observation and long and short diameter measurement of transplanted tumors twice a week, there were obvious differences in transplanted tumors of different generations of the same patient. Generally, as the number of transplanted tumors increases, the tumor growth rate slightly accelerates ( Figure 3 ).

[0083] Example 7 Histopathological Observation of Primary Tumor and Transplanted Tumor Model

[0084] In order to verify whether the stable sequence transplanted tumors retain the characteristics of the primary tumor in histopathology, HE sections of the transplanted tumors and their corresponding primary tumor tissues were observed, and it was found that the transplanted tumor tissues retained the histopathological morphological characteristics of their corresponding primary tumors. The primary tumor cells of malignant pleomorphic adenoma are densely arranged, with a large nuclear-cytoplasmic ratio, and many nuclear division phases can be seen. A small amount of hyaline degeneration can be seen in the tumor stroma. These pathological characteristics are also seen in the transplanted tumors ( Figure 4 ).

[0085] Example 8 Identification of cell slides of malignant pleomorphic adenoma PDC

[0086] Immunofluorescence (IF) method: After 48 hours of cell climbing, take out the cells, wash twice with PBS, and then fix them with 4% neutral buffered formaldehyde at room temperature for 30 minutes, and use membrane permeabilization solution for 15 minutes. Then, after blocking the cells with blocking buffer (PBS containing 5% skim milk powder) for 1 hour, add antibodies (AE1 / AE3, CK7, Synaptophysin, S-100, Ki-67, Vimentin, P63) and incubate overnight at 4°C. After washing with PBS, add Anti-mouse IgG (H+L), F(ab')2Fragment (Alexa Fluor 500, IgG400, IgG500, IgG600, IgG700, IgG800, IgG900, IgG1000, IgG1100, IgG1200, IgG1300, IgG1400, IgG1500, IgG1600, IgG1700, IgG1800, IgG1900, IgG2100, IgG1900, IgG2200, IgG1100, IgG1100, IgG1200, IgG1500, IgG1600, IgG1700, IgG1800, IgG19 ... 488Conjugate) #4408 secondary antibody for 30 min, wash 3 times with PBS, add DAPI mounting medium containing antifade agent and cover the cells, and observe under fluorescence microscope.

[0087] The cell morphology of subcultured PDCs is as follows Fig. 9 As shown in (P3 generation), the tumor cells are slender spindle-shaped, containing a small number of irregular polygonal cells. After continuous passage, the cells still maintain a stable cell morphology and a stable cell proliferation rate.

[0088] Immunofluorescence was used to identify the markers of malignant pleomorphic adenoma PDC. The results of immunofluorescence showed that the cell slides (P5 generation) of malignant pleomorphic adenoma PDC all stably expressed the markers of malignant pleomorphic adenoma ( Fig.10 ).

[0089] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. Human malignant pleomorphic adenoma tissue block MPA9H-1 with the deposit number of CCTCC No: C2022358; The malignant pleomorphic adenoma tissue block MPA9H-1 was derived from a human tumor primary located at the right skull base.

2. The malignant pleomorphic adenoma tissue block MPA9H-1 according to claim 1, characterized in that: Compared with other tumor tissue blocks, the malignant pleomorphic adenoma tissue block MPA9H-1 expresses one or more of the following: RNA, protein or polypeptide corresponding to AE1 / AE3, CK7, CK14, P63, S-100, Vimentin or Ki-67.

3. The malignant pleomorphic adenoma tissue block MPA9H-1 according to claim 1, characterized in that: Compared with healthy salivary gland tissue blocks, the HER-2 gene in the malignant pleomorphic adenoma tissue block MPA9H-1 is amplified with an amplification ratio of 25-35%, and the PLAG1 gene is broken; and / or, the copy number of chromosome 1, chromosome 2, chromosome 3, chromosome 6, chromosome 9, chromosome 11 or chromosome 14 in the malignant pleomorphic adenoma tissue block MPA9H-1 is increased compared with that in healthy salivary gland tissue blocks; the copy number of chromosome 10, chromosome 16, chromosome 17 or chromosome 22 is decreased compared with that in healthy salivary gland tissue blocks.

4. A method for constructing a malignant pleomorphic adenoma PDX animal model, characterized in that: The malignant pleomorphic adenoma tissue block MPA9H-1 according to any one of claims 1 to 3 is inoculated into the model animal.

5. The construction method according to claim 4, characterized in that: The size of the tissue block is 0.4-1.6 mm*0.4-1.6 mm*0.4-1.6 mm.

6. Use of the malignant pleomorphic adenoma tissue block MPA9H-1 according to any one of claims 1 to 3 or the malignant pleomorphic adenoma PDX animal model constructed by the method according to claim 4 or 5 in studying the in vitro mechanism of the occurrence and development of malignant pleomorphic adenoma.

7. Use of the malignant pleomorphic adenoma tissue block MPA9H-1 according to any one of claims 1 to 3 in preparing a malignant pleomorphic adenoma PDX animal model construction product.

Images