WO2024050858A1 - Lignée cellulaire tumorale phyllode maligne du sein humain sysh-mpt-04 et son utilisation - Google Patents
Lignée cellulaire tumorale phyllode maligne du sein humain sysh-mpt-04 et son utilisation Download PDFInfo
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Definitions
- the present invention relates to the technical field of animal cell line models, in particular to a human breast malignant phyllodes tumor cell line and its establishment method and application.
- Human breast phyllodes tumor is a relatively rare fibroepithelial tumor, with an incidence rate of approximately 1% of all breast tumors.
- Malignant phyllodes tumors have a poor prognosis, with a recurrence rate as high as 65%, and can metastasize through the blood, with a metastasis rate as high as 43.1%.
- Studies have found that malignant phyllodes tumors are insensitive to radiotherapy, chemotherapy, and immunotherapy. Therefore, patients with malignant phyllodes tumors often die due to multiple metastases throughout the body.
- the medical research on the biology of human breast phyllodes malignant tumors and its anti-tumor drugs is still in the preliminary stage.
- human breast phyllodes malignant tumors are relatively rare, and second, it is difficult to obtain stable products on the market.
- a sufficient number of cell models Research on human breast malignant phyllodes tumors is mainly based on primary tumor cells. However, isolating primary tumor cells is expensive and takes a long time, which makes the pathogenesis, occurrence and development mechanism, therapeutic targets, and antibiotics of human breast malignant phyllodes tumors unclear. Research in areas such as tumor drug screening has been limited.
- Cell lines usually refer to cells that can be passaged in vitro for more than 50 generations. Some cells can acquire the ability to proliferate indefinitely under external stimulation, while most cells have a limited number of passages in vitro. People can make these cells immortal by transfecting exogenous immortalization genes (such as HPV, SV40T and other viral genes) ization ability. For example, two human breast malignant phyllodes tumor cell lines disclosed in Chinese patent applications with publication numbers CN111019898A and CN114717190A were both obtained by transfecting exogenous immortalization genes. However, the cost of constructing immortalized cell lines is high, and due to the insertion of foreign genes, which has a certain impact on the metabolism and pathways of cells, it is difficult to obtain stable progeny cell lines.
- exogenous immortalization genes such as HPV, SV40T and other viral genes
- cell lines are cultured in vitro. Due to the different characteristics of the primary cell lines, most cells have a limited number of passages in vitro. It is also difficult to obtain progeny cell lines with stable biological genetic properties. The growth rate is slow and is not suitable for Mass production and marketization. Therefore, it is very necessary to find a representative cell strain (line) that is suitable for stable passage and has a simple culture process.
- the purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art, provide a human malignant phyllodes tumor cell line SYSH-MPT-04 and its progeny cell lines, and further disclose the establishment method of its cell line and related application.
- This cell line (line) is derived from primary cells extracted from Chinese human breast malignant phyllodes tumor tissue.
- This cell line and cell line do not require immortalization and can be stably passed down indefinitely in vitro. They are particularly suitable for mass production and market culture.
- the cell line of the present invention is named human breast malignant phyllodes tumor cell line SYSH-MPT-04 (referred to as cell line SYSH-MPT-04), and its cell line is named human breast malignant phyllodes tumor cell line SYSH-MPT-04 (referred to as cell line strain SYSH-MPT-04), this cell line is deposited in the China Type Culture Collection Center, the deposit date is June 28, 2022; the deposit address is: Wuhan University, Wuhan, China, and the deposit number is CCTCC, NO.C2022190.
- the method for establishing the progeny cell line of the cell line of the present invention includes the following steps: taking the cell line SYSH-MPT-04 in the logarithmic growth phase and culturing it in vitro for passage.
- the cell culture conditions are: 37°C, 5% CO 2 cell culture box; the culture medium used is DMEM/F12 culture medium, which contains epidermal cell growth factor at a final concentration of 20ng/mL, hydrocortisone at 0.5mg/mL, insulin at 10ug/mL, and 10% of the culture medium. 1% of the total volume of penicillin-streptomycin double antibody; when passage, use trypsin to digest the cells and passage them in a ratio of 1:2.
- This cell line SYSH-MPT-04 as a human breast malignant phyllodes tumor cell model or animal model.
- the application scope includes studying the pathogenesis, occurrence, development, metastasis and metastasis of human breast malignant phyllodes tumors.
- the cell strain (line) SYSH-MPT-04 of the present invention has the following advantages:
- the cell line of the present invention does not require immortalization, can be passed down indefinitely in vitro under conventional culture conditions, has a fast growth rate, is easy to culture, has stable biogenetic properties, and retains the proliferation, clone formation, and migration of its primary cells. , invasion and colony formation ability, the expression levels of ⁇ -SMA and FAP are similar to those of primary cells, and the copy numbers of 21 STR loci are also the same as those of primary cells. Therefore, the cell line SYSH-MPT-04 can represent primary cells and be used to study the in vitro biological functions of human breast malignant phyllodes tumors.
- the culture process is simple and low-cost, and is particularly suitable for mass production and marketization. It can fill the gap in the current lack of human breast malignant phyllodes tumor cell line models on the market. It has a great promotion effect on the biological characteristics and therapeutic drug research of human breast malignant phyllodes tumors.
- Figure 1 is a photo of the cell line SYSH-MPT-04 of the present invention under an optical microscope after being fixed with 4% paraformaldehyde and stained with crystal violet.
- Figure 2 is a picture (A) of the passage status of the primary cell MPTPC1 corresponding to the human breast malignant phyllodes tumor cell line HJP-0320, the cell line SYSH-MPT-04 of the present invention and the corresponding primary cell MPTPC4 and the proliferation rate detected by CCK8 Figure (B).
- Figure 3 is a diagram showing the results of the colony formation, migration and invasion ability tests of the cell line SYSH-MPT-04 of the present invention and primary cells MPTPC1 and MPTPC4.
- Figure 4 is a graph showing the test results of the mRNA (A) and protein (B) expression levels of malignant phyllodes tumor markers ⁇ -SMA and FAP in the cell line SYSH-MPT-04 of the present invention and its primary cell MPTPC4.
- the human breast malignant phyllodes tumor cell line SYSH-MPT-04 of the present invention is obtained by the following method:
- Source of tumor specimens The human breast malignant phyllodes tumor specimen from which MPTPC1 was isolated was derived from the right breast tumor of a 47-year-old female patient.
- MPTPC1 is the primary cell of the human malignant phyllodes tumor cell line HJP-0320, which was published in the publication No.
- the patent application for CN111019898A is pending.
- the malignant phyllodes tumor specimen of SYSH-MPT-04 was isolated from the right breast mass of a 51-year-old female patient. The size of the mass was 9cm ⁇ 8.2cm ⁇ 6.1cm. The patient had two recurrences after this operation, with an interval of about 6 months. Eleven months after the last recurrence of the tumor, the patient developed cough and found multiple metastases in both lungs. These clinical data indicate that this malignant phyllodes tumor is highly malignant and has strong proliferation and invasion capabilities. It is a representative malignant phyllodes tumor of the human breast.
- Isolation and culture of primary tumor cells Use a sterile scalpel to cut the above tumor tissue in the middle, take out the vigorous and proliferating tissue, wash it 3 times with sterile PBS, remove the fat tissue and necrotic tissue, and then use Chop the tissue into a paste with a sterile scalpel, transfer it to a 50 mL centrifuge tube, add DMEM/F12 containing type III collagenase digestion (1 mg/mL, Worthington), protect from light with tin foil, and digest at 37 degrees for 1 hour. The digested cell suspension was filtered through a 70 ⁇ m filter, and then centrifuged at 250 g for 10 min.
- the pellet was washed once with PBS and then inoculated into a culture dish.
- Cell culture conditions are: 37°C, 5% CO2 cell culture incubator, the medium used is Invitrogen brand DMEM/F12 medium; the final concentration is 20ng/mL, Peprotech brand epidermal cell growth factor; 0.5mg/mL. , Sigma brand hydrocortisone; 10ug/mL, Sigma brand insulin; and accounting for 1% of the total volume of the medium, Invitrogen brand penicillin-streptomycin double antibody; when passaged, use Gibco brand Tryple trypsin digestion Cells were passaged at a ratio of 1:2.
- Continuous passage Continuously passage and culture the above-mentioned human breast malignant phyllodes tumor primary cells in vitro, and observe the changes in cell morphology and proliferation rate.
- the morphology of the early malignant phyllodes tumor primary cells (Early MPTPC1) at the 3rd to 5th passage was long and spindle-shaped. After passage to the 15th passage, the morphology of the late primary cells Later MPTPC1 changed and became hypertrophic and irregular. , and the proliferation rate slows down significantly.
- the cells still maintain their original long spindle-shaped stromal cell morphology and retain a rapid proliferation rate (see Figure 2), indicating that the cells are malignant phyllodes tumors of the human breast that can be passed indefinitely.
- the cell line was named human breast malignant phyllodes tumor cell line SYSH-MPT-04, and its morphology is shown in Figure 1.
- the human breast malignant phyllodes tumor cell line SYSH-MPT-04 obtained in Example 1 was identified, specifically including the following aspects:
- Cell proliferation experiment Seed cells in a Corning brand 96-well cell culture plate, with 1,000 cells in each well, and 3 duplicate wells in each group. Aspirate the culture medium every 24 hours, and add 10 ⁇ L of APExBio brand CCK8 for serum-free culture. Base, after incubation at 37 degrees for 2 hours, use a microplate reader to detect the absorbance value at 450 nm. A total of five time points of 0, 24, 48, 72, and 96 hours were detected.
- Clone formation experiment Plant the cells in a Corning brand 6-well cell culture plate. Plant 100 cells in each well, with 3 duplicate wells in each group. Change the medium every 3 days. Remove the medium after 10 days, wash once with PBS, and add Fix 1 mL of 4% paraformaldehyde for 15 minutes, add crystal violet to stain for 15 minutes, rinse with water, dry and count the number of clones.
- Cell migration experiment Add 200 ⁇ L of serum-free medium containing 20,000 cells to the upper chamber of a Corning brand transwell chamber, and add 600 ⁇ L of complete medium to the lower chamber. After 24 hours of incubation at 37 degrees, the chambers were collected, fixed with 4% paraformaldehyde for 15 minutes, stained with crystal violet for 15 minutes, rinsed with water and dried, and the number of cells was counted in 5 random fields of view under the microscope, and the average value was taken.
- Collagen shrinkage experiment Mix the cells with Corning brand acid-soluble collagen I, and then inoculate the collagen/cell mixture into a 24-well cell culture plate coated with bovine serum albumin. Incubate at 37 degrees for 30 minutes and then add it to each well. 1mL culture medium. After 36 h of culture, the collagen gel was visualized and the shrinkage length of the gel was measured.
- Real-time fluorescence quantitative PCR After digesting the cells, use the ESscience brand RNA rapid extraction kit to extract total RNA. Take 1 ⁇ g of RNA and use Novozant brand II Q RT SuperMix kit to remove genomic DNA using ChamQ qPCR Master Mix kit for reverse transcription into cDNA. Prepare the following reaction system: 3.4 ⁇ L cDNA, 5 ⁇ L ChamQ SYBR qPCR Green Master Mix, 0.6 ⁇ L primer mix, 1 ⁇ L DEPC water.
- Immunofluorescence Fix the cells with 4% paraformaldehyde, rupture the membrane with Soleba brand 0.1% Triton The antibody was incubated overnight at 4 degrees; after washing with PBST, add Zhongshan Jinqiao brand fluorescent secondary antibody at a ratio of 1:50, and incubate at room temperature for 2 hours. After washing with PBST, add DAPI dye and incubate for 15 minutes. After washing with PBST, collect under a confocal microscope. photo.
- STR identification was performed on the human breast malignant phyllodes tumor cell line SYSH-MPT-04 and its primary cells. The results are shown in Table 1. The copy number of the human breast malignant phyllodes tumor cell line SYSH-MPT-04 at 21 STR sites completely matches that of the original cells, indicating that the human breast malignant phyllodes tumor cell line SYSH-MPT-04 is identical to the original cells. The generation cells are homologous and not contaminated by other cells.
- the human breast malignant phyllodes tumor cell line SYSH-MPT-04 does not require immortalization, can be passed indefinitely in vitro under conventional culture conditions, has a fast growth rate, is easy to culture, has stable traits, and retains its original characteristics.
- the cell proliferation, colony formation, migration, invasion and colony formation abilities, the expression levels of ⁇ -SMA and FAP were similar to those of primary cells, and the copy number of 21 STR loci was also the same as that of primary cells. Therefore, the human breast malignant phyllodes tumor cell line SYSH-MPT-04 can represent primary cells and be used to study the in vitro biological functions of human breast malignant phyllodes tumors.
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Abstract
Souche cellulaire de tumeur phyllode maligne du sein humain, sa lignée cellulaire de génération filiative et son utilisation. La lignée cellulaire est appelée lignée cellulaire de tumeur phyllode maligne du sein humain SYSH-MPT-04, et a été déposée sous le numéro CCTCC NO. C2022190 auprès du China Center for Type Culture Collection. La lignée cellulaire n'a pas besoin de traitement d'immortalisation et peut être passée de manière stable et infinie in vitro, et le procédé de culture de la lignée cellulaire est simple et peu coûteux. La lignée cellulaire est particulièrement appropriée pour être produite en masse et commercialisée. La lignée cellulaire peut être utilisée comme modèle cellulaire pour promouvoir la recherche sur les caractéristiques biologiques et les médicaments pour traiter les tumeurs phyllodes malignes du sein humain.
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JP2007190013A (ja) * | 2005-12-19 | 2007-08-02 | Univ Nagoya | ヒト悪性卵巣胚細胞腫瘍の細胞株の樹立方法、ヒト悪性卵巣胚腫瘍細胞株、及びその利用 |
WO2007107038A1 (fr) * | 2006-03-20 | 2007-09-27 | Hua Liu | Construction d'un modèle tumoral in vitro et application |
CN111019898A (zh) * | 2019-02-14 | 2020-04-17 | 中山大学孙逸仙纪念医院 | 人恶性叶状肿瘤细胞系hjp-0320及其应用 |
CN113293133A (zh) * | 2021-03-05 | 2021-08-24 | 中山大学孙逸仙纪念医院 | 一种人乳腺恶性叶状肿瘤细胞株及其应用 |
CN114717190A (zh) * | 2022-04-20 | 2022-07-08 | 中山大学孙逸仙纪念医院 | 一种人乳腺恶性叶状肿瘤细胞系bpt0713及其应用 |
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JP2007190013A (ja) * | 2005-12-19 | 2007-08-02 | Univ Nagoya | ヒト悪性卵巣胚細胞腫瘍の細胞株の樹立方法、ヒト悪性卵巣胚腫瘍細胞株、及びその利用 |
WO2007107038A1 (fr) * | 2006-03-20 | 2007-09-27 | Hua Liu | Construction d'un modèle tumoral in vitro et application |
CN111019898A (zh) * | 2019-02-14 | 2020-04-17 | 中山大学孙逸仙纪念医院 | 人恶性叶状肿瘤细胞系hjp-0320及其应用 |
CN113293133A (zh) * | 2021-03-05 | 2021-08-24 | 中山大学孙逸仙纪念医院 | 一种人乳腺恶性叶状肿瘤细胞株及其应用 |
CN114717190A (zh) * | 2022-04-20 | 2022-07-08 | 中山大学孙逸仙纪念医院 | 一种人乳腺恶性叶状肿瘤细胞系bpt0713及其应用 |
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Title |
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HE SHISHI, XIAO XIAOYUN, LEI RONG, CHEN JIEWEN, HUANG HONGYAN, YILIHAMU AILIFEIRE, GUO MINGYAN, TAN CUI, LI XUN, ZHUANG ZILIN, ER : "Establishment of Breast Phyllodes Tumor Cell Lines Preserving the Features of Phyllodes Tumors", BIO INTEGRATION, vol. 4, no. 1, 1 January 2023 (2023-01-01), pages 7 - 17, XP093147166, ISSN: 2712-0074, DOI: 10.15212/bioi-2022-0025 * |
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