Chest adenoma B3 type source cell strain as well as construction and application thereof

Abstract

The invention discloses a breast adenoma B3 type source cell strain as well as construction and application thereof, the breast adenoma B3 type source cell strain is named as human breast adenoma cell Hthy2301 Homo sapiens and is preserved in China Center for Type Culture Collection on March 14, 2024, and the preservation number is CCTCC NO: C202478. The human thymoma cell line is stable in character, can be passaged in vitro for a long time, and has high proliferation activity. The human thymoma cell line disclosed by the invention can be used as an effective cell model, and a new experimental model can be provided for deeply researching the occurrence, development and transfer mechanisms of thymoma, the drug resistance mechanism, drug screening, drug effect evaluation and the like.

Description

Technical Field

[0001] The invention belongs to the field of microbial cells, and in particular relates to a human thymoma cell line and an application thereof. Background Art

[0002] The thymus is located in the anterior mediastinum region behind the sternum and in front of the pericardium. It is an important lymphatic organ in the human body and the central organ of the immune system, guiding the development, differentiation and maturation of T lymphocytes. The thymus is mainly composed of epithelial cells and lymphocytes. Under normal circumstances, it gradually shrinks with age and is replaced by adipose tissue; however, epithelial cells, stimulated by genetic and environmental factors, will abnormally proliferate or invade surrounding tissues and develop into thymic epithelial tumors (TETs). The incidence of TETs is 1.3 to 3.2 / 1 million cases, and the average age at diagnosis is 50 to 60 years old. What is more special is that about 25% of TETs patients will have associated autoimmune clinical symptoms, including myasthenia gravis, pure red aplastic anemia, systemic lupus erythematosus, etc. Most patients will eliminate symptoms after surgical resection; but sometimes patients will worsen after resection and develop myasthenia gravis crisis, which is life-threatening. The cause of TETs formation and the mechanism of associated autoimmune dysfunction are still unclear.

[0003] There are many pathological subtypes of thymic epithelial tumors. The most widely used classification system is the WHO classification system, which subdivides TETs into thymoma (TM) and thymic carcinoma (TC) with squamous epithelial malignancy as the main type. Thymoma includes type A TMs, type AB TMs, type B1 TMs, type B2 TMs, and type B3 TMs. Studies at the molecular level have reported that type A TMs and type AB TMs have more frequent GTF2I and HRAS mutations; type B has a T cell signaling spectrum; TC has the highest molecular complexity among TETs, with frequent CDKN2A and TP53 changes and mutation loads, while the frequency of genetic abnormalities in KIT, PTEN / PI3K / mTOR, ATM, ALK, ERBB2, and CYLD1 is low. In recent years, there have been many attempts at precision therapy based on molecular genetic information in TETs. For example, tyrosine kinase inhibitors and immune checkpoints targeting KIT, mTOR, and VEGFR have been adopted as second-line systemic treatments. However, multiple clinical trials have shown that more than half of patients need to stop taking the drug early due to concerns about efficacy, toxicity, response prediction factors, and adverse reactions. At present, there are still no clearly recommended molecular targeted drugs and immunomonoclonal antibody inhibitors in the NCCN guidelines. In-depth in vivo and in vitro studies of key molecular markers and molecular regulatory mechanisms can not only provide diagnostic information for the precise diagnosis and prognosis prediction of TETs, but also provide target information for decision-making on adjuvant therapy.

[0004] In order to study the biological characteristics and molecular mechanisms of tumors and evaluate the adaptability of in vitro models, tumor cell lines have become an important research tool. The establishment of human tumor cell lines provides rich experimental materials for understanding the biological characteristics of tumors, studying their carcinogenesis, molecular genetics, and metastasis evolution mechanisms, thereby helping to establish standard treatment models. In recent decades, a large number of human tumor cell lines have been isolated and used for long-term culture. These cell lines provide important models for molecular level research of tumors. Due to the low incidence of TETs and the abundance of lymphocytes, the number of human thymoma cell lines reported so far is very small compared to other tumors; and cell lines such as TY-82, ThyL-6, 1U-TAB-1, T1889, and MP57 are all from abroad, and there is only one Thy0517 cell line in China. In addition, there is almost no cell line based on different pathological types of TETs, especially type B. As for key issues such as whether the origin epithelial cells of different subtypes such as B1, B2, and B3 are the same, and the differences and commonalities in molecular markers, there is a lack of in vitro cell experimental research, which seriously restricts the selection of preclinical drug research experiments.

[0005] In summary, due to the lack of in vitro research models for TETs, especially the lack of TETs cell lines, the in vitro functional detection of TETs can only be limited to limited short-term primary culture. Most primary cells have limited in vitro culture and passaging time. After a certain number of passaging, they will enter the senescence phase and can no longer continue to proliferate. At the same time, repeated passaging of primary cells is not only time-consuming and laborious, but also there are differences between batches of cells, and the stability of cell physiological indicators cannot be maintained. Therefore, establishing a reliable TETs cell line model that can be used repeatedly for a long time is a top priority for thymoma research. Summary of the invention

[0006] In view of this, the present invention provides a thymoma B3 type derived cell line and its application, which provides a reliable TETs cell line model that can be used repeatedly for a long time for studying the pathogenesis of thymoma and drug screening.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The human thymoma cell line, named human thymoma cell Hthy2301 Homo sapiens, was deposited in the China Center for Type Culture Collection (CCTCC) on March 14, 2024, with the deposit number CCTCC NO: C202478.

[0009] The present invention also provides a progeny cell line of the human thymoma cell line described above.

[0010] The human thymoma cell line or its progeny cell line provided by the present invention is derived from B3 type thymic epithelial tumor tissue.

[0011] In some embodiments of the present invention, the human thymoma cell line or its progeny cell line described above is positive for SV40T expression.

[0012] In some embodiments of the present invention, CK is positively expressed in the human thymoma cell line or its progeny cell line as described above.

[0013] In some embodiments of the present invention, CK8 is positively expressed in the human thymoma cell line or its progeny cell line as described above.

[0014] In some embodiments of the present invention, CK18 is positively expressed in the human thymoma cell line or its progeny cell line as described above.

[0015] In some embodiments of the present invention, CK19 is positively expressed in the human thymoma cell line or its progeny cell line as described above.

[0016] In addition, the present invention also provides a method for constructing the above-mentioned human thymoma cell line or its progeny cell line, comprising the following steps:

[0017] The thymoma specimen tissue of the thymoma type B3 patient is obtained, and primary culture is performed by enzyme digestion to obtain primary thymoma cells; the primary thymoma cells are transfected with SV40 to obtain immortalized cells, and then single cell suspension is cloned and purified, and amplified and cultured for at least 50 generations.

[0018] In some specific embodiments of the present invention, the construction method comprises the following steps:

[0019] After obtaining a thymoma specimen tissue from a thymoma type B3 patient, the thymoma specimen tissue is repeatedly rinsed with PBS buffer for at least 2 times, the thymoma specimen tissue is cut into small tissue blocks, placed in a centrifuge tube, PBS buffer is added, mixed, centrifuged and the supernatant is discarded for washing, the mixing, centrifugation and washing are repeated at least 2 times, and the precipitated tissue cells are collected;

[0020] Add cell culture medium to the centrifuge tube containing the precipitated tissue cells for resuspending, transfer to a culture dish, and culture in a 37°C, 5% CO2 incubator for 24 hours; then, replace with a new cell culture medium to continue culturing, and then replace the cell culture medium every 48 to 72 hours. When the cells growing on the wall around the tissue fuse into a sheet, digest with trypsin for 1 to 2 minutes, add cell culture medium to terminate digestion, centrifuge, and obtain tumor cell precipitation, which is the primary thymoma cell; subculture at a ratio of 1:2 or 1:3, and repeat the digestion and subculture process until the third generation of primary thymoma cells is obtained;

[0021] The thymoma primary cells of passage 3 were resuspended in cell culture medium and the number of cells per well was 5×10 4Inoculate 24-well plates, culture in a 37°C, 5% CO2 incubator, and incubate overnight; after the cells adhere to the wall, use a new cell culture medium containing SV40 overexpression lentivirus for transfection, and the amount of SV40 overexpression lentivirus is set at an MOI of 40. After 24 hours, use a new cell culture medium to continue culturing, and then replace the fresh cell culture medium every 2 to 3 days for culturing; observe the number of surviving cells, and when the volume in the well increases to 80 to 90%, select the successfully transfected cells, use the cell culture medium for monoclonal screening and culture, select the monoclonal cells with good growth potential for expansion culture, and the expanded cells are the P1 generation; then subculture at a ratio of 1:2 or 1:3, and freeze each subculture, and expand and culture for at least 50 generations.

[0022] The present invention also provides use of the human thymoma cell line or its progeny cell line as described above in a cell model of a human thymoma cell line.

[0023] The present invention also provides the use of the human thymoma cell line or its progeny cell line as described above as a cell model for the occurrence, development and metastasis of human thymoma.

[0024] The present invention also provides the use of the human thymoma cell line or its progeny cell line as described above in studying the drug resistance mechanism of human thymoma, screening drugs for treating human thymoma, and predicting drug sensitivity to human thymoma.

[0025] In the present invention, it is found through observation that the human thymoma cell line generally presents an epithelial-like morphology or spindle shape under an optical microscope, the cell nucleus is large and irregular, and multiple nucleoli appear; transmission electron microscopy shows that the cell nucleus is large and the outline is irregular. The human thymoma cell line is an immortalized cell with stable cell traits and can be stably propagated in vitro for a long time. At present, the in vitro propagation exceeds 50 generations. By performing genetic material STR analysis on the human thymoma cell line and searching for cell line DNA typing in the database, no cell line matching the cell was found, and it was identified as a new single cell line without other cell contamination; and the cell line was identified to be consistent with the source of the tissue specimen. The results of immunohistochemical analysis showed that the human thymoma cell line was positive for CK expression, CK8 expression, CK18 expression, and CK19 expression, and the results were consistent with the immunohistochemical detection of the tumor tissue from the patient.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The human thymoma cell line of the present invention is a new single cell line without contamination by other cells; the cell properties are stable and can be passaged multiple times; it has strong proliferation activity and can be amplified in large quantities and passaged and cultured in vitro for a long time; and it can be used as an effective cell model.

[0028] (2) The present invention overcomes the problem in the prior art that thymoma cell lines are difficult to obtain due to the particularity of thymoma tissue, the lack of effective and available thymoma specimens, and the technical difficulties in establishing continuous cell culture. The human thymoma cell line of the present invention is a B3 human thymoma cell line, which enriches the human thymoma cell line library.

[0029] (3) The human thymoma cell line of the present invention is positive for SV40T expression, which means that the cell immortalization has been successfully achieved, which means that the cell has acquired the characteristics of sustainable growth and proliferation ability, and can survive long-term passage. Moreover, it can also retain the differentiation phenotype of its original cell, and can reflect the biological characteristics of its original cell at the cellular level, and has the characteristics of high lineage rate and repeatability. The human thymoma cell line of the present invention is the first human thymoma immortalized cell line established using SV40 at home and abroad.

[0030] (4) The human thymoma cell line of the present invention is CK-positive, CK8-positive, CK18-positive, and CK19-positive. The human TETs cell line can provide a new experimental model for in-depth research on the occurrence, development, and metastasis mechanism of thymoma, as well as research on drug resistance mechanism, drug screening, and drug efficacy evaluation.

[0031] Deposit of biological materials

[0032] The human thymoma cell line of the present invention is named human thymoma cell Hthy2301 Homo sapiens, and was deposited in the China Center for Type Culture Collection (CCTCC for short, address: Wuhan University, Wuhan, China, Postal Code: 430072) on March 14, 2024, with a deposit number of CCTCC NO: C202478. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1A to Figure 1D The optical morphological pictures are respectively of the thymoma primary cells just crawling out (generation 0), the first generation primary cells, the second generation primary cells and the third generation primary cells obtained in Example 1, with a magnification of 40 times.

[0034] Figure 2 This is an optical morphological picture of the successful introduction of SV40 into the primary thymoma cells of Example 1, with a magnification of 40 times.

[0035] Figure 3A to Figure 3D The optical morphological pictures are respectively the monoclonal cells screened in Example 1, P1 cells, P30 cells, and P50 cells, with a magnification of 40 times.

[0036] Figure 4AThe morphological results of human thymoma cells Hthy2301 observed under a transmission electron microscope (TEM). Figure 4B This is a partial enlarged view.

[0037] Figure 5A To verify the expression of SV40T in human thymoma cells Hthy2301 using immunohistochemistry.

[0038] Figure 5B To verify the expression of SV40T in thymoma tissues by immunohistochemistry.

[0039] Figure 6A to Figure 6D Immunohistochemistry was used to verify the expression of CK, CK8, CK18 and CK19 in human thymoma cells Hthy2301.

[0040] Figure 7A to Figure 7D Immunohistochemistry was used to verify the expression of CK, CK8, CK18 and CK19 in thymoma tissues.

[0041] Figure 8 This is the growth curve of human thymoma cell line Hthy2301.

[0042] Fig.9A and Fig. 9B These are the cell observation results at 0h and 24h in the scratch experiment of human thymoma cell Hthy2301.

[0043] Fig. 10A and Fig. 10B They are the Transwell experiment results (invasion and migration) of human thymoma cells Hthy2301.

[0044] Fig.11 These are optical morphological images of thymoma primary cells at passage 5.

[0045] Fig.12 This is an optical morphological picture of unsuccessful introduction of SV40 into primary thymoma cells, with a magnification of 40 times. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The specific materials used and their sources listed in the following examples are merely exemplary and are not intended to limit the present invention.

[0047] The experimental methods in the following examples without specifying specific conditions can adopt conventional methods in the art, methods described in the open literature, or conditions recommended by suppliers. Various instruments, raw materials, and reagents not specifically described in the following examples are commercially available products well known in the art and can be obtained through commercial channels.

[0048] Source of human thymic tumor tissue specimens:

[0049] On February 23, 2023, a 28-year-old male patient came to the hospital for examination due to "left chest pain". He underwent chest CT enhancement, which showed: a large mass in the anterior superior mediastinum and multiple nodular masses in the pleura of the left lower lobe of the lung. Malignant tumors and invasive thymoma with possible pleural metastasis were considered. The patient signed the informed consent form before the operation. Postoperative pathological diagnosis: thymoma type B3.

[0050] The composition of the cell culture medium includes: DMEM complete culture medium (purchased from Gibco), 10% (v / v) fetal bovine serum (FBS) (purchased from Gibco), 100 U / mL penicillin + 100 μg / mL streptomycin double antibody. The cell culture medium is a sterile cell culture medium.

[0051] 1 mL of lentivirus-containing culture medium included 990 μL of cell culture medium and 10 μL of SV40 overexpression lentivirus (pLenti-III-CMV-SV40, Aibimeng, Zhenjiang).

[0052] Example 1: Establishment of human TETs cell line

[0053] Under sterile surgical conditions, human thymoma tissue specimens were immediately immersed in cryopreservation tubes containing tissue protective cryopreservation solution after being removed and transported to the laboratory at low temperature.

[0054] Take out the above tissues in a sterile clean bench, put them in a culture dish, use a dropper to absorb 5 mL of PBS buffer, and repeatedly rinse the above tissues at least 2 times to discard the blood clots and necrotic tissues visible to the naked eye.

[0055] Use sterile surgical scissors to cut it into small tissue blocks of 1 to 3 mm in size, place these tissue blocks in a 15 mL sterile centrifuge tube, add 5 mL of PBS buffer to mix, centrifuge at 800 r / min at 4°C for 3 minutes, and discard the supernatant; repeat the washing twice, discard the supernatant, and collect the precipitated tissue blocks and cells.

[0056] Add 10 mL of cell culture medium to the centrifuge tube containing the precipitated tissue blocks and cells, gently blow and mix to obtain a suspension, transfer the mixed suspension to a 10 cm culture dish, and culture in a 37°C, 5% CO2 incubator. Replace with new cell culture medium after 24 hours, and then replace the cell culture medium every 48 to 72 hours, and observe cell growth regularly. During this period, the suspended lymphocytes and necrotic tissue blocks in the culture flask will be gradually removed by changing the fluid. The growth of live cells of the thymoma primary cells was observed under an optical microscope (Olympus BX43 biological microscope from Japan) with a magnification of 40 times. The optical morphology picture is as follows Figure 1A As shown. Figure 1A It can be seen that the primary thymoma cells initially crawled out from around the tissue mass, grew adherently, and grew in oval and cobblestone-shaped sheets.

[0057] 7 to 10 days after the first replacement of the cell culture medium, it was observed that the cells growing on the wall around the tissue merged into a sheet (the area occupied about 90% of the bottom of the dish). At this time, the tissue block was removed, and 0.25% trypsin (w / v)-EDTA was added for digestion for 1 to 2 minutes. When the cell morphology became round, the cell gap was obvious, and some cells just began to detach from the bottle wall, 10 mL of cell culture medium was added to terminate the digestion, and the cells were carefully blown down with the cell culture medium, and the mixed liquid was transferred to a 15 mL centrifuge tube, centrifuged at 1000 rpm / min at room temperature for 5 minutes to resuspend, and the supernatant was removed by centrifugation to obtain a tumor cell precipitate, which was subcultured to a T25 culture bottle at a ratio of 1:2 or 1:3. At this time, the bottle contained the first generation of thymoma primary cells, which were placed in a 37°C, 5% CO2 incubator for culture, and fresh cell culture medium was replaced after 2 to 3 days for continued culture. The growth of the first generation of thymoma primary cells was observed under an optical microscope (Olympus BX43 biological microscope in Japan), and the magnification was 40 times. Optical morphology pictures Figure 1B As shown. Figure 1B It can be seen that after one generation of thymoma primary cells, the cell morphology began to change, and after digestion, the cell volume increased.

[0058] When the first generation of thymoma primary cells in the T25 bottle grows to 80-90% density, remove the cell culture medium in the original T25 culture bottle, add 3-4ml PBS to the T25 bottle and wash 1-2 times; discard PBS, digest with 0.25% trypsin (w / v)-EDTA and add culture medium to 5-6mL, perform digestion and subculture, repeat the above digestion and subculture process to obtain the second generation of thymoma primary cells. Observe the growth of the living cells of the second generation of thymoma cells under an optical microscope (Olympus BX43 biological microscope in Japan) with a magnification of 40 times. The optical morphology picture is as follows Figure 1C As shown. Figure 1CIt can be seen that the gaps between the second-generation primary cells of thymoma become larger, the growth and proliferation become slower, the cells extend filopodia, and their morphology resembles fibroblasts.

[0059] The above digestion and subculture process was repeated to obtain the third generation thymoma primary cells. The viable cell growth of the third generation thymoma primary cells was observed under an optical microscope (Olympus BX43 biological microscope, Japan) at a magnification of 40 times. Figure 1D As shown by Figure 1D It can be seen that the cell morphology of the third generation thymoma primary cells changed: the cell volume gradually increased, tentacles grew around it, the tentacles became longer and appeared to be in a wire-like state, the state of the cells obviously deteriorated, and there was no tendency to proliferate.

[0060] The third generation thymoma primary cells were resuspended in cell culture medium and the number of cells per well was about 2×10 4 Inoculate a 24-well plate, culture it in a 37°C, 5% CO2 incubator, and incubate it overnight; after the cells adhere to the wall, the cell density rises to about 20-30%, and replace it with a new cell culture medium containing SV40 overexpression lentivirus (Order Number: CS5003948, Abimeng, Zhenjiang) for transfection. The amount of SV40 overexpression lentivirus is 20uL (MOI=40). 24 hours after adding the virus, replace it with a new cell culture medium and continue culturing. Next, replace the fresh cell culture medium every 2-3 days for culture, and observe the cell morphology. After about 10 days, changes in cell morphology can be found in the wells that have been successfully transfected. The growth of cells successfully transfected with the lentivirus SV40 was observed under an optical microscope (Olympus BX43 biological microscope, Japan), with a magnification of 40 times. The optical morphology picture is as follows Figure 2 As shown. Figure 2 It can be seen that the morphology of successfully transfected cells changed from an irregular elongated state to an oval shape, similar to the morphology of primary cells that had just crawled out of the tissue block, and the proliferation rate was accelerated. When the oval cells in the well plate were filled to 90% density, they basically all recovered to the morphology consistent with the primary thymoma cells.

[0061] Using cell culture medium, the cells that were successfully transfected in the wells were plated on 96-well plates for monoclonal screening and culture. Monoclonal cells with excellent growth were selected for expansion culture, and the expanded cells were the P1 generation (the first generation after immortalization); they were subcultured and amplified at 1:2 or 1:3, and each subculture was frozen and amplified to at least 50 generations. Each subculture takes about 3 to 4 days. It was observed that all generations of cells grew adherently, had good morphology, and were consistent before and after subculture. The P30 cell line was preserved and named human thymoma cell Hthy2301 Homo sapiens (also abbreviated as human thymoma cell Hthy2301 or Hthy2301 cell), and was deposited in the China Center for Type Culture Collection (CCTCC) on March 14, 2024, with a preservation number of CCTCC NO: C202478.

[0062] The above-mentioned monoclonal cells, P1 cells, P30 cells and P50 cells with good growth potential were placed under an optical microscope (Japan Olympus BX43 biological microscope) to observe the growth of living cells. The magnification was 40 times. The optical morphological pictures were as follows: Figure 3A to Figure 3D As shown in the figure, the morphology of the selected monoclonal cells with good growth and the P1, P30 and P50 cells after passage is clear and intact, arranged in an epithelial-like pattern, and the cells grow attached to the wall. The cells of each generation show better and similar cell morphology, and are similar to each other. Figure 1A The thymoma primary cells in the samples showed consistent morphology.

[0063] Transmission electron microscopy was used to observe cell morphology in more detail: When Hthy2301 cells grew to a density of 80% to 90%, the cells were washed 2 to 3 times with pre-cooled PBS, collected with a cell scraper and transferred to a 1.5 mL EP tube, centrifuged, the supernatant discarded, and fixed with 2.5% glutaraldehyde solution, and placed at 4°C for overnight fixation. The next day, the fixative was discarded, and the cells were washed 2 to 3 times with pre-cooled PBS. The cells were fixed for 1 hour with pre-cooled 1% osmium acid fixative (Hangzhou Hao Ke Biological Company), gently washed twice with PBS, and dehydrated with graded ethanol solutions (30%, 50%, 70%, 80% and 100%) in sequence, each for about 15 minutes, and encapsulated with epoxy resin, dried and sliced. The cells were stained with lead citrate and uranyl acetate, and the cell structure was observed using a transmission electron microscope. The results are as follows Figure 4A As shown in the figure, it can be seen that: transmission electron microscopy shows that Hthy2301 cells are epithelial-like in morphology, with large nuclei and irregular outlines, and the cytoplasm contains abundant mitochondria, ribosomes, endoplasmic reticulum, Golgi bodies and other cell sub-organs, indicating that the cell structure is complete and the cell vitality is good. Figure 4BAs shown, it can be seen that the mitochondria of the cells are abundant and numerous, presenting various forms, mainly linear (short rod-shaped) and granular, indicating that the tumor cells have active metabolism and grow rapidly.

[0064] Example 2: Biological characteristics and applications of cell lines

[0065] (I) STR analysis of genetic material and homology identification of cell lines

[0066] Short tandem repeat (STR), also known as microsatellite DNA, refers to a type of DNA sequence on chromosomes that is formed by tandem repetition of several base pairs (2 to 6 base pairs) as core units (the number of repetitions is 10 to 60 times, and the gene fragment is less than 400 base pairs); the number of times each core unit is repeated will vary from individual to individual, thus forming alleles with different fragment lengths. Therefore, the number of repetitions of a set of STR sequences is almost unique in different individuals, which is an individual's genetic identity feature and the main method for cell biology to identify cell identity and origin.

[0067] Detection method: Freshly cultured Hthy2301 cells were taken and DNA was extracted using Microread Genomic DNA Kit. 20 STR loci and sex identification loci were amplified using MicroreaderTM21ID System. PCR products were detected using ABI 3730xl genetic analyzer. The test results were analyzed using GeneMapper3.2 software (Applied Biosystems) (see Table 1). The results were compared with the American Type Culture Collection (ATCC) and the German Microbiological Collection (DSMZ) databases, and no identical STR test results were found. That is, the 2455 cell line STR databases from ATCC, DSMZ, JCRB and RIKEN databases did not find a site matching the cell, indicating that this cell line is a new cell line; no multi-allelic loci were found, indicating that this cell line is a single cell without contamination from other cells. All of these can prove the uniqueness of this cell and that there is no cross-contamination with other cells during the culture process.

[0068] The number of repetitions of a set of STR sequences is almost unique in different individuals. It is an individual's genetic identity feature and the main method for cell biology to identify cell identity and origin. According to the ATCC Standards Committee Identification Standard (ANSI / ATCC ASN-0002-2011), a match of ≥80% is considered to be from the same source.

[0069] According to the experimental results, the STR genotype match between the Hthy2301 cell line and the patient's blood is 100, indicating that the Hthy2301 cell line is derived from the patient's human thymic tumor tissue, retains the characteristics of type B3 thymoma well, and has high clinical guidance value.

[0070] The STR analysis of the genetic material of the cell lines and their derived blood is shown in Table 1 .

[0071] Table 1: Genotyping results of STR loci of cell lines and their derived blood

[0072]

[0073] (II) Immunohistochemical Characterization Analysis of Cell Lines

[0074] Hthy2301 cells in the logarithmic growth phase were digested and centrifuged to obtain cell pellets. The cell pellets were fixed in 4% formaldehyde solution, embedded in paraffin, made into 3 μm sections, and fixed on polylysine-coated slides. After the cell sections were dewaxed and dehydrated, heat-induced antigen retrieval was performed at 98°C. After cooling to room temperature, the slides were covered with 3% hydrogen peroxide to inhibit endogenous peroxidase activity, blocked with 5% goat serum at room temperature for 30 minutes, and the sections were incubated with diluted primary antibodies (SV40T, CK, CK8, CK18, CK19) at 4°C overnight. The second day, the secondary antibody was added and incubated at room temperature for 30 minutes. After washing with PBS three times, the color was developed with 3,3′-diaminobenzidine hydrochloride DAB reagent color development box. The cells were placed in distilled water to stop color development, and the nuclei were counterstained with hematoxylin for 3 minutes. After rinsing with tap water until the water was colorless, the cells were dehydrated with ethanol in a gradient manner. After being transparentized with xylene twice, the cells were naturally dried and sealed with neutral gum. The expression of SV40T in the cells was observed under an optical microscope and photographed. The results are shown in the figure. Figure 5A The expression of SV40T, CK, CK8, CK18, and CK19 in cells was observed under an optical microscope and photographed. The results are shown in Figure 6A to Figure 6D shown.

[0075] Depend on Figure 5A and Figures 6A to 6D It can be seen that the human thymoma cell line expresses positive SV40T, positive CK, positive CK8, positive CK18 and positive CK19. Among them, SV40T is mainly expressed in the nucleus; CK, CK8, CK18 and CK19 are mainly expressed in the cytoplasm.

[0076] For comparison, the same operation method and steps were used to perform immunohistochemical analysis on thymoma tissues. The expression of SV40T was observed under an optical microscope and photographed. The results are shown in Figure 2. Figure 5BThe expression of SV40T, CK, CK8, CK18, and CK19 was observed under an optical microscope and photographed. The results are shown in Figure 7A to Figure 7D As shown. Figure 5B and Figures 7A to 7D It can be seen that the thymoma tissue is positive for SV40T, CK, CK8, CK18 and CK19. Among them, SV40T is mainly expressed in the nucleus; CK, CK8, CK18 and CK19 are mainly expressed in the cytoplasm.

[0077] It can be seen that the immunohistochemical detection of the human thymoma cell line is consistent with that of the tumor tissue derived from the patient.

[0078] (III) Biological behavior analysis of cell lines

[0079] (3.1) Cell proliferation assay to determine cell growth curve

[0080] The growth curve of P30 cells was measured. The P30 cells with good growth status were digested with trypsin, resuspended after centrifugation, and the cells were counted. They were seeded in a 96-well plate at a density of 3000 cells per well. 100 μL of cell culture medium was added to each well, and 100 μl of PBS buffer (pH=7.4) was added to the control well. The corresponding 96-well plates were taken out after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days, and after the culture medium in the wells was sucked out, 100 μl of fresh cell culture medium and 10 μl of CCK8 (Apexbio, No.K101819133EF5E) were added to each well and incubated in a 37°C incubator away from light. Ensure that the incubation time of each well is consistent every day, measure the absorbance value at 450 nm with an enzyme reader, continuously measure the cell growth and draw a growth curve, and use GraphPad Prism software to calculate the doubling time according to the regression equation of the curve. The cell growth curve is as shown in Figure 8 As shown, it can be seen that the population doubling time of Hthy2301 cells is about 23.9 hours.

[0081] (3.2) Scratch assay to determine cell migration ability

[0082] P30 cells in the logarithmic growth phase were taken and 5×10 5P30 cells were inoculated into 6-well plates at a density of cells / well, 2 mL of cell culture medium was added to each well, and the plates were cultured at 37°C in a cell culture incubator containing 5% CO2. When the cells grew to a density of 80-90%, the 6-well plates were taken out, and a 1000 μL pipette tip was used to scratch the plates perpendicularly from one end of the well to the other. The scratched cells were washed with PBS, and complete DMEM culture medium containing 1% (v / v) FBS was added for serum starvation. The plates were then cultured at 37°C in a cell culture incubator containing 5% CO2. The healing of the cell scratches was observed under an inverted microscope at 0h and 24h, and photographed. The results are shown in the figure. Fig.9A and Fig. 9B As shown, it can be seen that human thymoma cells Hthy2301 showed a strong metastatic ability. 24 hours after the scratch, the new growing cells collectively migrated and grew toward the middle area and covered the scratch area.

[0083] (3.3) Transwell assay to determine cell migration ability

[0084] Prepare Matrigel ( 365234) was placed in a 4°C refrigerator to thaw and turn into liquid. 100 μL of medium containing Matrigel (containing 10 μL Matrigel and 90 μL DMEM complete medium) was added to the pre-cooled Transwell chamber and incubated at 37°C for 1 to 2 hours until a "white layer" solid appeared. Human thymoma cells Hthy2301 in the logarithmic growth phase were selected, digested with trypsin, centrifuged, and the supernatant was discarded. The cells were resuspended in DMEM complete medium and the cell density was adjusted to 5×10 5 / mL, add 100μL of cell suspension to each well. At the same time, add 500μL of DMEM complete medium containing 10% FBS to the lower layer of the chamber, and incubate in an incubator at 37℃ with 5% CO2. After 24h, take out the chamber, wipe off the uninvaded cells in the upper layer with a cotton swab, rinse the cells twice with ultrapure water, fix the cells in the lower layer of the chamber with 4% paraformaldehyde for 15min, and then stain with 0.5% crystal violet for 15min. After washing with ultrapure water, observe and take pictures under a microscope and count the cells that migrated to the lower layer. The invasion and migration results are as follows: Fig. 10A and Fig. 10B As shown, it can be seen that human thymoma cells Hthy2301 cells can smoothly pass through the basement membrane and show strong invasive ability.

[0085] Comparative Example 1

[0086] Under sterile surgical conditions, human thymoma tissue specimens were immediately immersed in cryopreservation tubes containing tissue protective cryopreservation solution after being removed and transported to the laboratory at low temperature.

[0087] Take out the above tissues in a sterile clean bench, put them in a culture dish, use a dropper to absorb 5 mL of PBS buffer, and repeatedly rinse the above tissues at least 2 times to discard the blood clots and necrotic tissues visible to the naked eye.

[0088] Use sterile surgical scissors to cut it into small tissue blocks of 1 to 3 mm in size, place these tissue blocks in a 15 mL sterile centrifuge tube, add 5 mL of PBS buffer to mix, centrifuge at 800 r / min at 4°C for 3 minutes, and discard the supernatant; repeat the washing twice, discard the supernatant, and collect the precipitated tissue blocks and cells.

[0089] Add 10 mL of cell culture medium to the centrifuge tube containing the above-precipitated tissue cells, gently blow and mix to obtain a suspension, transfer the mixed suspension to a 10 cm culture dish, and culture in a 37°C, 5% CO2 incubator. Replace the new cell culture medium after 24 hours, and then replace the cell culture medium every 48 to 72 hours, and observe the cell growth regularly. During this period, the suspended lymphocytes and necrotic tissue blocks in the culture flask will be gradually removed by changing the medium. 7 to 10 days after the first replacement of the cell culture medium, the tissue blocks were removed and digested with 0.25% trypsin (w / v)-EDTA for 1 to 2 minutes. When the cell morphology became round, the cell gaps were obvious, and some cells just began to detach from the flask wall, 10 mL of cell culture medium was added to terminate the digestion, and the cells were mixed by pipetting. The cells were carefully pipetted down with the cell culture medium, and the mixed liquid was transferred to a 15 mL centrifuge tube. The mixture was centrifuged at 1000 rpm / min at room temperature for 5 min to be resuspended, and the supernatant was removed by centrifugation to obtain a tumor cell pellet. The tumor cell pellet was obtained and passaged to a T25 culture flask at a ratio of 1:2 or 1:3. At this time, the flask contained the first generation of thymoma primary cells, which were cultured in a 37°C, 5% CO2 incubator. After 2 to 3 days, fresh cell culture medium was replaced for continued culture. When the first generation of thymoma primary cells in the T25 bottle grows to 80-90% density, remove the culture medium in the original T25 culture bottle, add 3-4 ml PBS to the T25 bottle and wash 1-2 times; discard PBS, digest with 0.25% trypsin (w / v)-EDTA and add culture medium to 5-6 mL, perform digestion and subculture, repeat the above digestion and subculture process to obtain thymoma primary cells of the second to fifth generations. Until the fifth generation of thymoma primary cells are obtained, the cells no longer proliferate and cannot be used for subsequent basic cytological experiments.

[0090] The growth of the living cells of the 1st to 5th generations of the primary thymoma cells was observed under an optical microscope (Japan Olympus BX43 biological microscope), and it was found that: the state of the cells deteriorated with the increase of cell generations, and the three-dimensional sense of the aged cells gradually disappeared, and they began to become flat. The intervals between cells decreased or mixed together, and the cells were no longer spindle-shaped, but various irregular shapes. The cell adhesion weakened, and cells began to float in the culture medium; the number of dividing cells decreased significantly and the cell proliferation rate also decreased greatly; the cells began to grow horn differentiation. The optical morphology of the living cells of the 5th generation of the primary thymoma cells is shown in the figure below. Fig.11 shown.

[0091] Comparative Example 2

[0092] The same as Example 1, except that the content of SV40 lentivirus was 10uL, and the morphology of the obtained cells was as follows Fig.12 As shown, it can be seen that the morphology of cells into which the lentivirus has not been successfully introduced remains unchanged, and like aged primary cells, the cell volume is reduced, the cytoplasm is concentrated, and the cell membrane is bubbling and shrinking.

[0093] In summary, in the present invention, primary cells derived from thymoma tissue are subcultured, and after infection with SV40 lentivirus, an immortalized human TETs cell line with continuous subculture and good proliferation ability is obtained, and the monoclonal selection is followed by continuous subculture for more than 50 generations. The Chinese thymoma cell line established by the present invention is of great significance for studying the pathogenesis, invasion, metastasis, and molecular mechanism of chemotherapy resistance of Chinese thymoma, as well as developing new anti-thymoma therapeutic drugs; at the same time, the method for establishing the Chinese thymoma cell line Hthy2301 disclosed in the present invention also has great practical application value.

[0094] It can be seen that the purpose of the present invention has been completed and effectively achieved. The method and principle of the present invention have been demonstrated and explained in the embodiments, and the implementation method can be modified arbitrarily without departing from the principles. Therefore, the present invention includes all modified implementation methods based on the spirit and scope of the claims. It is understandable that for those skilled in the art, equivalent replacement or change of the technical solution and inventive concept of the present invention should belong to the protection scope of the claims attached to the present invention.

Claims

1. A human thymoma cell line, characterized in that The cell line was named human thymoma cell Hthy2301 Homo sapiens and was deposited in China Center for Type Culture Collection on March 14, 2024 with the deposit number CCTCC NO: C202478.

2. A progeny cell line of the human thymoma cell line according to claim 1.

3. The human thymoma cell line according to claim 1 or the progeny cell line according to claim 2, characterized in that: SV40T expression was positive.

4. The human thymoma cell line according to claim 1 or the progeny cell line according to claim 2, characterized in that: CK expression was positive.

5. The human thymoma cell line according to claim 1 or the progeny cell line according to claim 2, characterized in that: CK8 expression was positive.

6. The human thymoma cell line according to claim 1 or the progeny cell line according to claim 2, characterized in that: CK18 expression was positive.

7. The human thymoma cell line according to claim 1 or the progeny cell line according to claim 2, characterized in that: CK19 expression was positive.

8. The method for constructing a human thymoma cell line or a progeny cell line according to any one of claims 1 to 7, comprising the following steps: Obtain thymoma specimens from patients with thymoma type B3, perform enzymatic digestion and primary culture, and obtain thymoma primary cells; The primary thymoma cells are transfected with SV40 to obtain immortalized cells, which are then cloned and purified as single cell suspensions and cultured for at least 50 generations.

9. Use of the human thymoma cell line or progeny cell line according to any one of claims 1 to 7 as a cell model of a human thymoma cell line.

10. Use of the human thymoma cell line or progeny cell line according to any one of claims 1 to 7 in studying the drug resistance mechanism of human thymoma, screening drugs for treating human thymoma, and evaluating the efficacy of drugs for human thymoma.

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