WO2024050859A1 - Human breast malignant phyllodes tumor cell line sysh-mpt-03 and use thereof - Google Patents

Human breast malignant phyllodes tumor cell line sysh-mpt-03 and use thereof Download PDF

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WO2024050859A1
WO2024050859A1 PCT/CN2022/118893 CN2022118893W WO2024050859A1 WO 2024050859 A1 WO2024050859 A1 WO 2024050859A1 CN 2022118893 W CN2022118893 W CN 2022118893W WO 2024050859 A1 WO2024050859 A1 WO 2024050859A1
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cell line
human breast
malignant phyllodes
breast malignant
phyllodes tumor
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聂燕
贺诗施
雷蓉
艾力菲热伊力哈木
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中山大学孙逸仙纪念医院
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  • 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.
  • the World Health Organization divides phyllodes tumors into three categories: benign, borderline, and malignant based on histopathological characteristics. Among them, malignant phyllodes tumors have a poor prognosis, with a recurrence rate as high as 65%, and can metastasize through blood, with a metastasis rate as high as 43.1%.
  • studies have found that malignant phyllodes tumors are insensitive to radiotherapy, chemotherapy, and immunotherapy. The main clinical treatment method is surgery. Therefore, patients with malignant phyllodes tumors often die due to multiple metastases throughout the body.
  • 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 the insertion of foreign genes may have a certain impact on cell metabolism and pathways. Cell lines are cultured in vitro through conventional non-transfection methods. Due to the different characteristics of the primary cell lines, most cells have a limited number of passages in vitro. It is difficult to obtain progeny cell lines with stable biological genetic properties, and the growth rate is slow and unsuitable. Mass production and marketization.
  • the present invention provides a human breast malignant phyllodes tumor cell line and its progeny cell line that does not require immortalization, can be stably passed through indefinitely in vitro, and is particularly suitable for mass production and marketization, and further discloses the cell line. Establishment methods and related applications of its progeny cell lines.
  • the human breast malignant phyllodes tumor cell line of the present invention is derived from the right breast tumor of a 25-year-old female patient with malignant phyllodes tumor, and the cell line cultured in vitro is named the human breast malignant phyllodes tumor cell line SYSH-MPT- 03 (referred to as cell line SYSH-MPT-03), and its cell line is named human breast malignant phyllodes tumor cell line SYSH-MPT-03 (referred to as cell line SYSH-MPT-03).
  • the cell line SYSH-MPT-03 has been deposited in the China Type Culture Collection Center, and the deposit date is June 28, 2022; the deposit address is: Wuhan University, Wuhan, China, and the deposit number is CCTCC, NO.C2022189.
  • the establishment method is: take the cell line SYSH-MPT-03 in the logarithmic growth phase and culture it in vitro.
  • the cell culture conditions are: 37°C, 5% CO 2 cell culture incubator; the culture medium used is DMEM/F12 culture medium.
  • the culture medium contains epidermal cell growth factor with a final concentration of 20ng/mL, hydrocortisone at 0.5mg/mL, insulin at 10ug/ml, and penicillin-streptomycin dual antibodies accounting for 1% of the total volume of the culture medium. ; When passaging, digest the cells with trypsin and pass them at a ratio of 1:2.
  • the cell line SYSH-MPT-03 of the present invention can be used to establish a human breast malignant phyllodes tumor cell model or a human breast malignant phyllodes tumor animal model.
  • the described application scope includes: research on the pathogenesis, occurrence, development, metastasis and drug resistance mechanisms of malignant phyllodes tumors of the human breast, as well as related signaling pathways and drug targets. It can also be used in the preparation of diagnostic or detection reagents for human breast malignant phyllodes tumors, or in research on the preparation, screening and evaluation of anti-tumor drugs.
  • the primary cells of the cell line SYSH-MPT-03 of the present invention are more malignant than the general clinical malignant phyllodes tumor cells. They are derived from patients with multiple recurrences and metastases, and their invasion and migration are stronger and faster. It is more representative as a cell model.
  • the culture process Since the establishment of the cell line of the present invention does not require immortalization, 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 is of great help to study the biological characteristics and therapeutic drugs of human breast malignant phyllodes tumors.
  • Figure 1 is a photo of the cell line SYSH-MPT-03 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-03 of the present invention and the corresponding primary cell MPTPC3 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-03 of the present invention and primary cells MPTPC1 and MPTPC3.
  • 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-03 of the present invention and its primary cell MPTPC3.
  • the human breast malignant phyllodes tumor cell line SYSH-MPT-03 of the present invention is obtained by the following method:
  • Source of human breast malignant phyllodes tumor specimen The malignant phyllodes tumor specimen of the cell line SYSH-MPT-03 of the present invention is derived from the right breast tumor of a 25-year-old female patient.
  • the size of the tumor is 10cm ⁇ 10cm ⁇ 7.2cm.
  • the patient had a history of two malignant phyllodes tumors of the right breast. This patient had two recurrences and metastases to the lungs after this operation. The shortest recurrence interval was only 1 month.
  • Isolation and culture of primary cells of human breast malignant phyllodes tumor of the present invention use a sterile scalpel to cut the above-mentioned malignant phyllodes tumor tissue in the middle, take out the vigorous and active tissue, wash it with sterile PBS three times, and remove Use a sterile scalpel to chop the adipose tissue and necrotic tissue into a paste, transfer it to a 50ml centrifuge tube, add DMEM/F12 containing Worthington brand 1mg/ml type III collagenase, use tin foil to protect from light, 37 Digest 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 to obtain primary malignant phyllodes tumor cells MPTPC3.
  • Cell culture conditions are: 37°C, 5% CO 2 cell culture incubator, the medium used is Invitrogen brand DMEM/F12 medium, and the final concentration of Peprotech brand epidermal cell growth factor, 0.5 mg, is added to the medium.
  • Continuous passage Continuous passage and culture of the human breast malignant phyllodes tumor primary cell MPTPC3 of the present invention in vitro, observing changes in cell morphology and proliferation rate, and comparing it with the previously isolated primary cell MPTPC1 used to construct immortalized cell lines. Compare. MPTPC1 is the primary cell of the human breast malignant phyllodes tumor cell line HJP-0320, which is disclosed in the patent application with publication number CN111019898A. Primary cells MPTPC1 The early malignant phyllodes tumor primary cells (Early MPTPC1) at the 3rd to 5th passage are long and spindle-shaped. After passage to the 15th passage, the morphology of the late primary cells Later MPTPC1 changes and becomes hypertrophic.
  • the cell MPTPC3 is a human breast malignant phyllodes tumor cell line (line) that can be passed indefinitely, and is named the human breast malignant phyllodes tumor cell line (line) SYSH-MPT-03 (see Figure 1).
  • the human breast malignant phyllodes tumor cell line SYSH-MPT-03 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 culturing at 37 degrees for 24 hours, the cells 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-03 and its primary cells (Guangzhou Aiji Biotechnology Co., Ltd.). The results are shown in Table 1. The copy number of the 21 STR sites of the cell line SYSH-MPT-03 of the present invention completely matches that of the primary cells, indicating that the cell line SYSH-MPT-03 of the present invention is homologous to the primary cells. Contaminated by other cells.
  • the human breast malignant phyllodes tumor cell line SYSH-MPT-03 does not require immortalization and can be passaged indefinitely in vitro under conventional culture conditions. It 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 cell line SYSH-MPT-03 of the present invention can represent primary cells and be used for research on the in vitro biological functions of human breast malignant phyllodes tumors.

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Abstract

The present invention relates to the technical field of animal cell line models. Disclosed are a human breast malignant phyllodes tumor cell strain and a progeny cell line and use thereof. The cell line is named Human Breast Malignant Phyllodes Tumor Cell Line SYSH-MPT-03, which has been preserved at the China Center for Type Culture Collection with an accession number of CCTCC NO.C2022189. The cell line does not require immortalization treatment, can be passaged stably and unlimitedly in vitro, and possesses a simple culture process and low cost, making it particularly suitable for mass production in the market. The cell line can serve as a cell model to help study the biological characteristics and therapeutic drugs of human breast malignant phyllodes tumors, potentially filling the current gap in the lack of human breast malignant phyllodes tumor cell models.

Description

一种人乳腺恶性叶状肿瘤细胞系SYSH-MPT-03及其应用A human breast malignant phyllodes tumor cell line SYSH-MPT-03 and its application 技术领域Technical field
本发明涉及动物细胞系模型技术领域，尤其是一种人乳腺恶性叶状肿瘤细胞系及其建立方法和应用。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.
背景技术Background technique
人乳腺叶状肿瘤是一种较为罕见的纤维上皮性肿瘤，发病率约为全部乳腺肿瘤的1％。世界卫生组织根据组织病理特征将叶状肿瘤分为良性、交界性和恶性三类。其中恶性叶状肿瘤预后差，复发率高达65％，可通过血行转移，转移率高达43.1％。然而研究发现恶性叶状肿瘤对放疗、化疗、免疫治疗均不敏感，临床主要的治疗方式为手术治疗，因此恶性叶状肿瘤患者常因全身多处转移而死亡。细胞系通常指能在体外传代大于50代的细胞。一些细胞在外界的刺激下可获得无限增殖的能力，而大多数细胞在体外传代次数是有限的，人们可以通过转染外源永生化基因(如HPV、SV40T等病毒基因)使这些细胞获得永生化能力。例如，公布号为CN111019898A、CN114717190A的中国专利申请公开的两种人乳腺恶性叶状肿瘤细胞系均通过转染外源永生化基因获得。然而，构建永生化细胞株的成本高，且由于插入了外源基因，可能对细胞的代谢和通路产生一定的影响。通过非转染常规的方法体外培养细胞系，因原代细胞株的特性不同，大多数细胞在体外传代次数是有限，很难获得生物遗传性状稳定的子代细胞系，且生长速度慢，不适合量产市场化。Human breast phyllodes tumor is a relatively rare fibroepithelial tumor, with an incidence rate of approximately 1% of all breast tumors. The World Health Organization divides phyllodes tumors into three categories: benign, borderline, and malignant based on histopathological characteristics. Among them, malignant phyllodes tumors have a poor prognosis, with a recurrence rate as high as 65%, and can metastasize through blood, with a metastasis rate as high as 43.1%. However, studies have found that malignant phyllodes tumors are insensitive to radiotherapy, chemotherapy, and immunotherapy. The main clinical treatment method is surgery. Therefore, patients with malignant phyllodes tumors often die due to multiple metastases throughout the body. 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 the insertion of foreign genes may have a certain impact on cell metabolism and pathways. Cell lines are cultured in vitro through conventional non-transfection methods. Due to the different characteristics of the primary cell lines, most cells have a limited number of passages in vitro. It is difficult to obtain progeny cell lines with stable biological genetic properties, and the growth rate is slow and unsuitable. Mass production and marketization.
目前，由于缺乏能持续传代的人乳腺恶性叶状肿瘤细胞系，该肿瘤的研究主要基于原代细胞，然而分离肿瘤的原代细胞耗费大，周期长，使得人乳腺恶性叶状肿瘤的致病机制、发生发展机制、治疗靶点、抗肿瘤药物筛选等方面的研究均受到了限制。造成当今国内外，适宜量产、市场化的人乳腺恶性叶状肿瘤的细胞模型还处于空缺状态，因此，亟需建立一种可靠的人乳腺恶性叶状肿瘤细胞系，以便于在体内外研究人乳腺恶性叶状肿瘤的生物学功能，帮助筛选出特异标志物和有效的治疗靶点。Currently, due to the lack of human breast malignant phyllodes tumor cell lines that can be continuously passaged, research on this tumor is mainly based on primary cells. However, isolating primary tumor cells is expensive and takes a long time, which makes the pathogenesis of human breast malignant phyllodes tumor difficult. Research on mechanisms, occurrence and development mechanisms, treatment targets, and anti-tumor drug screening have been limited. As a result, there is still no cell model of human breast malignant phyllodes tumor suitable for mass production and marketization at home and abroad. Therefore, there is an urgent need to establish a reliable human breast malignant phyllodes tumor cell line to facilitate in vivo and in vitro research. The biological functions of human breast malignant phyllodes tumors help to screen out specific markers and effective therapeutic targets.
发明内容Contents of the invention
为克服以上缺陷，本发明提供了一种不需要进行永生化处理，可稳定体外无 限传代，特别适宜量产、市场化的人乳腺恶性叶状肿瘤细胞株及其子代细胞系，并进一步公开其子代细胞系的建立方法和相关应用。In order to overcome the above shortcomings, the present invention provides a human breast malignant phyllodes tumor cell line and its progeny cell line that does not require immortalization, can be stably passed through indefinitely in vitro, and is particularly suitable for mass production and marketization, and further discloses the cell line. Establishment methods and related applications of its progeny cell lines.
本发明的人乳腺恶性叶状肿瘤细胞株来源于患有恶性叶状肿瘤的25岁女病人的右乳肿物，将其体外培养的细胞系命名为人乳腺恶性叶状肿瘤细胞系SYSH-MPT-03(简称细胞系SYSH-MPT-03)，其细胞株则命名为人乳腺恶性叶状肿瘤细胞株SYSH-MPT-03(简称细胞株SYSH-MPT-03)。所述细胞系SYSH-MPT-03已保藏于中国典型培养物保藏中心，保藏日期为2022年6月28日；保藏地址为：中国，武汉，武汉大学，保藏编号为CCTCC，NO.C2022189。The human breast malignant phyllodes tumor cell line of the present invention is derived from the right breast tumor of a 25-year-old female patient with malignant phyllodes tumor, and the cell line cultured in vitro is named the human breast malignant phyllodes tumor cell line SYSH-MPT- 03 (referred to as cell line SYSH-MPT-03), and its cell line is named human breast malignant phyllodes tumor cell line SYSH-MPT-03 (referred to as cell line SYSH-MPT-03). The cell line SYSH-MPT-03 has been deposited in the China Type Culture Collection Center, and the deposit date is June 28, 2022; the deposit address is: Wuhan University, Wuhan, China, and the deposit number is CCTCC, NO.C2022189.
进一步提供本发明人乳腺恶性叶状肿瘤细胞株(或系)SYSH-MPT-03及其子代细胞系的建立方法。Further provided are methods for establishing the human breast malignant phyllodes tumor cell line (or line) SYSH-MPT-03 and its progeny cell lines of the present invention.
其建立方法为：取对数生长期的的细胞株SYSH-MPT-03在体外培养传代，细胞培养条件为：37℃，5％CO 2细胞培养箱；所用培养基为DMEM/F12培养基，该培养基中含有终浓度为20ng/mL的表皮细胞生长因子,0.5mg/mL的氢化可的松,10ug/ml的胰岛素，以及占所述培养基总体积1％的青链霉素双抗；传代时，用胰酶消化细胞，按1:2的比例传代。 The establishment method is: take the cell line SYSH-MPT-03 in the logarithmic growth phase and culture it in vitro. The cell culture conditions are: 37°C, 5% CO 2 cell culture incubator; the culture medium used is DMEM/F12 culture medium. The culture medium contains epidermal cell growth factor with a final concentration of 20ng/mL, hydrocortisone at 0.5mg/mL, insulin at 10ug/ml, and penicillin-streptomycin dual antibodies accounting for 1% of the total volume of the culture medium. ; When passaging, digest the cells with trypsin and pass them at a ratio of 1:2.
本发明的细胞系SYSH-MPT-03可用于建立人乳腺恶性叶状肿瘤细胞模型，或人乳腺恶性叶状肿瘤动物模型。所述的应用范围包括：用于人乳腺恶性叶状肿瘤的致病、发生、发展、转移和耐药机制，相关信号通路和药物靶点等方面研究。亦可应用于制备人乳腺恶性叶状肿瘤诊断或检测试剂，或应用于制备、筛选和评估抗肿瘤药物的研究中。The cell line SYSH-MPT-03 of the present invention can be used to establish a human breast malignant phyllodes tumor cell model or a human breast malignant phyllodes tumor animal model. The described application scope includes: research on the pathogenesis, occurrence, development, metastasis and drug resistance mechanisms of malignant phyllodes tumors of the human breast, as well as related signaling pathways and drug targets. It can also be used in the preparation of diagnostic or detection reagents for human breast malignant phyllodes tumors, or in research on the preparation, screening and evaluation of anti-tumor drugs.
本发明相对于现有技术具有以下优点：The present invention has the following advantages over the prior art:
1.本发明的细胞系SYSH-MPT-03的原代细胞恶性程度较一般临床的恶性叶状肿瘤细胞高，其来源于多次复发以及转移的患者,侵袭迁移性更强更快。在作为细胞模型中具有更强的代表性。1. The primary cells of the cell line SYSH-MPT-03 of the present invention are more malignant than the general clinical malignant phyllodes tumor cells. They are derived from patients with multiple recurrences and metastases, and their invasion and migration are stronger and faster. It is more representative as a cell model.
2.不需要进行永生化处理，在常规培养条件下能在体外无限传代，生长速度快，易于培养，生物遗传性状稳定，保留了其原代细胞的增殖、克隆形成、迁移、侵袭和克隆形成能力，α-SMA和FAP的表达水平与原代细胞相似，在21个STR位点的拷贝数也与原代细胞相同。因此本发明建立的细胞系能代表原代细胞，可用于人乳腺恶性叶状肿瘤体外生物学功能的研究。2. No need for immortalization treatment, it can be passaged indefinitely in vitro under conventional culture conditions, has a fast growth rate, is easy to culture, has stable biogenetic properties, and retains the proliferation, colony formation, migration, invasion and colony formation of its primary cells. The expression levels of α-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 cell line established in the present invention can represent primary cells and can be used to study the in vitro biological functions of human breast malignant phyllodes tumors.
3.因本发明细胞系的建立不需要永生化处理，所以培养工艺简单，成本低，特别适宜量产市场化，可填补目前市面上缺乏人乳腺恶性叶状肿瘤细胞系模型的空白。对研究人乳腺恶性叶状肿瘤生物学特性及治疗药物具有很大的帮助。3. Since the establishment of the cell line of the present invention does not require immortalization, 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 is of great help to study the biological characteristics and therapeutic drugs of human breast malignant phyllodes tumors.
附图说明Description of the drawings
图1是本发明细胞系SYSH-MPT-03经4％多聚甲醛固定，结晶紫染色后在光学显微镜下的照片。Figure 1 is a photo of the cell line SYSH-MPT-03 of the present invention under an optical microscope after being fixed with 4% paraformaldehyde and stained with crystal violet.
图2是人乳腺恶性叶状肿瘤细胞系HJP-0320所对应的原代细胞MPTPC1、本发明细胞系SYSH-MPT-03及对应原代细胞MPTPC3的传代状况照片(A)及CCK8检测的增殖速率图(B)。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-03 of the present invention and the corresponding primary cell MPTPC3 and the proliferation rate detected by CCK8 Figure (B).
图3是本发明细胞系SYSH-MPT-03及原代细胞MPTPC1、MPTPC3克隆形成、迁移、侵袭能力试验的结果图。Figure 3 is a diagram showing the results of the colony formation, migration and invasion ability tests of the cell line SYSH-MPT-03 of the present invention and primary cells MPTPC1 and MPTPC3.
图4是本发明细胞系SYSH-MPT-03及其原代细胞MPTPC3中恶性叶状肿瘤标志物α-SMA和FAP的mRNA(A)和蛋白质(B)表达水平试验结果图。Figure 4 is a graph showing the test results of the mRNA (A) and protein (B) expression levels of malignant phyllodes tumor markers α-SMA and FAP in the cell line SYSH-MPT-03 of the present invention and its primary cell MPTPC3.
具体实施方式Detailed ways
下面结合实施例对本发明作进一步说明。The present invention will be further described below in conjunction with examples.
实施例1Example 1
本发明的人乳腺恶性叶状肿瘤细胞系SYSH-MPT-03通过以下方法获得：The human breast malignant phyllodes tumor cell line SYSH-MPT-03 of the present invention is obtained by the following method:
1.人乳腺恶性叶状肿瘤标本来源：本发明细胞系SYSH-MPT-03的恶性叶状肿瘤标本来源于25岁女性患者的右乳肿物，肿物大小为10cm×10cm×7.2cm。该患者既往有2次右乳恶性叶状肿瘤病史，本次术后又出现了两次复发并转移至肺部，最短复发间期仅有1个月。这些临床数据说明这例恶性叶状肿瘤的恶性程度高，有较强的增殖、侵袭能力，是代表性的恶性叶状肿瘤。1. Source of human breast malignant phyllodes tumor specimen: The malignant phyllodes tumor specimen of the cell line SYSH-MPT-03 of the present invention is derived from the right breast tumor of a 25-year-old female patient. The size of the tumor is 10cm×10cm×7.2cm. The patient had a history of two malignant phyllodes tumors of the right breast. This patient had two recurrences and metastases to the lungs after this operation. The shortest recurrence interval was only 1 month. 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.
2本发明人乳腺恶性叶状肿瘤原代细胞的分离和培养：用无菌手术刀将上述恶性叶状肿瘤组织正中切开，取活力旺盛增生活跃的组织，用无菌PBS洗涤3次，去除其中的脂肪组织和坏死组织后用无菌手术刀将组织剁成糊状，转移至50ml离心管中，加入含有Worthington品牌的1mg/ml Ⅲ型胶原酶的DMEM/F12，锡箔纸避光，37度消化1小时。消化后的细胞悬液用70μm的滤网过滤，随后250g离心10min，沉淀用PBS洗涤1次后接种于培养皿，得到恶性叶状肿瘤原代细胞MPTPC3。细胞培养条件为：37℃，5％CO 2细胞培养箱，所用培养基为 Invitrogen品牌的DMEM/F12培养基，在培养基中添加终浓度为20ng/ml Peprotech品牌的表皮细胞生长因子,0.5mg/ml Sigma品牌的氢化可的松,浓度为10ug/ml Sigma品牌的胰岛素，以及占所述培养基总体积1％的Invitrogen品牌青链霉素双抗；传代时，用Gibco品牌的Tryple胰酶消化细胞，按1:2的比例传代。 2. Isolation and culture of primary cells of human breast malignant phyllodes tumor of the present invention: use a sterile scalpel to cut the above-mentioned malignant phyllodes tumor tissue in the middle, take out the vigorous and active tissue, wash it with sterile PBS three times, and remove Use a sterile scalpel to chop the adipose tissue and necrotic tissue into a paste, transfer it to a 50ml centrifuge tube, add DMEM/F12 containing Worthington brand 1mg/ml type III collagenase, use tin foil to protect from light, 37 Digest for 1 hour. The digested cell suspension was filtered through a 70 μm filter, and then centrifuged at 250 g for 10 min. The pellet was washed once with PBS and then inoculated into a culture dish to obtain primary malignant phyllodes tumor cells MPTPC3. Cell culture conditions are: 37°C, 5% CO 2 cell culture incubator, the medium used is Invitrogen brand DMEM/F12 medium, and the final concentration of Peprotech brand epidermal cell growth factor, 0.5 mg, is added to the medium. /ml Sigma brand hydrocortisone, Sigma brand insulin at a concentration of 10ug/ml, and Invitrogen brand penicillin and streptomycin double antibodies accounting for 1% of the total volume of the culture medium; when passaging, use Gibco brand Tryple trypsin Digest cells and passage them at a 1:2 ratio.
3.持续传代：在体外持续传代培养本发明人乳腺恶性叶状肿瘤原代细胞MPTPC3，观察细胞形态、增殖速度的改变，并与既往分离出的用于构建永生化细胞系的原代细胞MPTPC1进行比较。MPTPC1是人乳腺恶性叶状肿瘤细胞系HJP-0320的原代细胞，公开于公布号为CN111019898A的专利申请中。原代细胞MPTPC1第3-5代的早期恶性叶状肿瘤原代细胞(Early MPTPC1呈长梭形，当传代至第15代以后，晚期原代细胞Later MPTPC1的形态发生改变，变成肥大的不规则型，且增殖速率明显变慢，而MPTPC3传代至67代以上。该细胞仍维持原本长梭形的间质细胞形态，并保留了较快的增殖速度(结果见图2)，说明原代细胞MPTPC3为可无限传代的人乳腺恶性叶状肿瘤细胞株(系)，命名为人乳腺恶性叶状肿瘤细胞株(系)SYSH-MPT-03(见图1)。3. Continuous passage: Continuous passage and culture of the human breast malignant phyllodes tumor primary cell MPTPC3 of the present invention in vitro, observing changes in cell morphology and proliferation rate, and comparing it with the previously isolated primary cell MPTPC1 used to construct immortalized cell lines. Compare. MPTPC1 is the primary cell of the human breast malignant phyllodes tumor cell line HJP-0320, which is disclosed in the patent application with publication number CN111019898A. Primary cells MPTPC1 The early malignant phyllodes tumor primary cells (Early MPTPC1) at the 3rd to 5th passage are long and spindle-shaped. After passage to the 15th passage, the morphology of the late primary cells Later MPTPC1 changes and becomes hypertrophic. Regular type, and the proliferation rate slowed down significantly, while MPTPC3 was passaged to more than 67 generations. The cells still maintained the original long spindle-shaped interstitial cell morphology and retained a fast proliferation rate (results shown in Figure 2), indicating that the primary The cell MPTPC3 is a human breast malignant phyllodes tumor cell line (line) that can be passed indefinitely, and is named the human breast malignant phyllodes tumor cell line (line) SYSH-MPT-03 (see Figure 1).
实施例2Example 2
对实施例1得到的人乳腺恶性叶状肿瘤细胞系SYSH-MPT-03进行鉴定，具体包括以下几个方面：The human breast malignant phyllodes tumor cell line SYSH-MPT-03 obtained in Example 1 was identified, specifically including the following aspects:
1.体外功能实验：比较人乳腺恶性叶状肿瘤原代细胞MPTPC1、MPTPC3多次传代后，体外细胞增殖、克隆形成、迁移、侵袭和胶原收缩能力的改变。1. In vitro functional experiment: Compare the changes in in vitro cell proliferation, colony formation, migration, invasion and collagen contraction abilities of human breast malignant phyllodes tumor primary cells MPTPC1 and MPTPC3 after multiple passages.
细胞增殖实验：将细胞种在Corning品牌的96孔细胞培养板，每孔种1000个细胞，每组3个复孔，每隔24h吸去培养基，加入含10μL APExBio品牌的CCK8的无血清培养基，37度孵育2h后用酶标仪检测450nm处的吸光值，共检测0、24、48、72、96h五个时间点。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 μL 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.
克隆形成实验：将细胞种在Corning品牌的6孔细胞培养板，每孔种100个细胞，每组3个复孔，每3天更换培养基，10天后去培养基，PBS洗1遍，加入1mL 4％多聚甲醛固定15min，加入结晶紫染色15min，用水冲洗，晾干后计数克隆数目。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.
细胞迁移实验：Corning品牌的transwell小室上室加入200μL含20000个细胞的无血清培养基，下室加入600μL完全培养基。37度培养24h后收集小室， 4％多聚甲醛固定15min，结晶紫染色15min，用水冲洗后晾干，镜下随机5个视野计数细胞个数，取平均值。Cell migration experiment: Add 200 μL of serum-free medium containing 20,000 cells to the upper chamber of a Corning brand transwell chamber, and add 600 μL of complete medium to the lower chamber. After culturing at 37 degrees for 24 hours, the cells 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.
细胞侵袭实验：按1：8比例用无血清培养基稀释BD品牌的基质胶，预先在transwell小室上室底部铺一层基质胶，其余步骤同迁移实验。Cell invasion experiment: Dilute BD brand Matrigel with serum-free culture medium at a ratio of 1:8, and lay a layer of Matrigel at the bottom of the upper chamber of the transwell chamber in advance. The remaining steps are the same as in the migration experiment.
胶原收缩实验：将细胞与Corning品牌的酸溶性胶原蛋白Ⅰ混合，随后将胶原蛋白/细胞混合物接种到包覆牛血清白蛋白的24孔细胞培养板中，37度孵育30min后即向每孔加入1mL培养基。培养36h后，对胶原凝胶进行显像，并测量凝胶的收缩长度。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.
结果如图3，Later MPTPC1的增殖、克隆形成、迁移、侵袭和胶原收缩能力明显低于Early MPTPC1，而SYSH-MPT-03的体外生物学功能与原代细胞Early MPTPC3相同。The results are shown in Figure 3. The proliferation, colony formation, migration, invasion and collagen contraction abilities of Later MPTPC1 are significantly lower than those of Early MPTPC1, while the in vitro biological functions of SYSH-MPT-03 are the same as those of the primary cell Early MPTPC3.
2.实时荧光定量PCR、免疫荧光：比较人乳腺恶性叶状肿瘤细胞系SYSH-MPT-03与其原代细胞中人乳腺恶性叶状肿瘤标志物(α-SMA和FAP)的表达水平。2. Real-time fluorescence quantitative PCR and immunofluorescence: Compare the expression levels of human breast malignant phyllodes tumor markers (α-SMA and FAP) in the human breast malignant phyllodes tumor cell line SYSH-MPT-03 and its primary cells.
实时荧光定量PCR：消化细胞后用ESscience品牌的RNA快速提取试剂盒提取总RNA。取1μg RNA用诺唯赞品牌的
Figure PCTCN2022118893-appb-000001
II Q RT SuperMix试剂盒去除基因组DNA，用ChamQ
Figure PCTCN2022118893-appb-000002
qPCR Master Mix试剂盒逆转录为cDNA。配制以下反应体系：3.4μL cDNA，5μL ChamQ SYBR qPCR Green Master Mix，0.6μL引物混合物，1μL DEPC水。在实时荧光定量PCR仪器上进行以下程序反应：95度30sec预变性；95度10sec，60度30sec，循环反应40次；95度15sec，60度60sec，95度15sec采集融解曲线。 Real-time fluorescence quantitative PCR: After digesting the cells, use the ESscience brand RNA rapid extraction kit to extract total RNA. Take 1 μg of RNA and use Novozant brand
Figure PCTCN2022118893-appb-000001
II Q RT SuperMix kit to remove genomic DNA using ChamQ
Figure PCTCN2022118893-appb-000002
qPCR Master Mix kit for reverse transcription into cDNA. Prepare the following reaction system: 3.4 μL cDNA, 5 μL ChamQ SYBR qPCR Green Master Mix, 0.6 μL primer mix, 1 μL DEPC water. Carry out the following program reactions on the real-time fluorescence quantitative PCR instrument: pre-denaturation at 95 degrees for 30 seconds; 95 degrees for 10 seconds, 60 degrees for 30 seconds, cycle reaction 40 times; 95 degrees for 15 seconds, 60 degrees for 60 seconds, and 95 degrees for 15 seconds to collect the melting curve.
免疫荧光：用4％多聚甲醛固定细胞，索莱宝品牌的0.1％Triton X-100破膜，用山羊血清封闭30min后以1：50的比例加入R&D品牌的α-SMA和Abcam品牌的FAP的抗体，于4度孵育过夜；用PBST洗后以1：50的比例加入中杉金桥品牌的荧光二抗，室温孵育2h，PBST洗后加入DAPI染液孵育15min，PBST洗后于共聚焦显微镜采集照片。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.
结果如图4，本发明培养的细胞系SYSH-MPT-03与原代细胞中α-SMA和FAP的mRNA和蛋白质表达水平相似。The results are shown in Figure 4. The mRNA and protein expression levels of α-SMA and FAP in the cell line SYSH-MPT-03 cultured in the present invention are similar to those in primary cells.
STR鉴定：对人乳腺恶性叶状肿瘤细胞系SYSH-MPT-03及其原代细胞进行STR 鉴定(广州艾基生物技术有限公司)。结果如表1，本发明细胞系SYSH-MPT-03在21个STR位点的拷贝数与原代细胞完全匹配，说明本发明细胞系SYSH-MPT-03与原代细胞是同源的，没有被其他细胞污染。STR identification: STR identification was performed on the human breast malignant phyllodes tumor cell line SYSH-MPT-03 and its primary cells (Guangzhou Aiji Biotechnology Co., Ltd.). The results are shown in Table 1. The copy number of the 21 STR sites of the cell line SYSH-MPT-03 of the present invention completely matches that of the primary cells, indicating that the cell line SYSH-MPT-03 of the present invention is homologous to the primary cells. Contaminated by other cells.
表1Table 1
Figure PCTCN2022118893-appb-000003
Figure PCTCN2022118893-appb-000003
以上结果说明，人乳腺恶性叶状肿瘤细胞系SYSH-MPT-03不需要进行永生化处理，在常规培养条件下能在体外无限传代，生长速度快，易于培养，性状稳定，保留了其原代细胞的增殖、克隆形成、迁移、侵袭和克隆形成能力，α-SMA和FAP的表达水平与原代细胞相似，在21个STR位点的拷贝数也与原代细胞相同。因此本发明细胞系SYSH-MPT-03能代表原代细胞，用于人乳腺恶性叶状肿瘤体外生物学功能的研究。The above results show that the human breast malignant phyllodes tumor cell line SYSH-MPT-03 does not require immortalization and can be passaged indefinitely in vitro under conventional culture conditions. It 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 α-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 cell line SYSH-MPT-03 of the present invention can represent primary cells and be used for research on the in vitro biological functions of human breast malignant phyllodes tumors.

Claims (8)

  1. 一种人乳腺恶性叶状肿瘤细胞株，其特征在于：所述细胞株命名为人乳腺恶性叶状肿瘤细胞株SYSH-MPT-03，保藏于中国典型培养物保藏中心，保藏编号为CCTCC,NO.C2022189。A human breast malignant phyllodes tumor cell line, characterized in that: the cell line is named human breast malignant phyllodes tumor cell line SYSH-MPT-03, and is deposited in the China Type Culture Collection Center with the deposit number CCTCC, NO. C2022189.
  2. 如权利要求1所述的细胞株的子代细胞系。The progeny cell line of the cell line according to claim 1.
  3. 如权利要求2所述的子代细胞系的建立方法，包括以下步骤：取对数生长期的人乳腺恶性叶状肿瘤细胞株SYSH-MPT-03在体外培养传代，细胞培养条件为：37℃，5％CO 2细胞培养箱；所用培养基为DMEM/F12培养基，该培养基中含有终浓度为20ng/mL的表皮细胞生长因子,0.5mg/mL的氢化可的松,10ug/ml的胰岛素，以及占所述培养基总体积1％的青链霉素双抗；传代时，用胰酶消化细胞，按1:2的比例传代。 The method for establishing a progeny cell line as claimed in claim 2, comprising the following steps: taking the human breast malignant phyllodes tumor cell line SYSH-MPT-03 in the logarithmic growth phase and culturing it in vitro for passage, and the cell culture conditions are: 37°C , 5% CO 2 cell culture incubator; the culture medium used is DMEM/F12 culture medium, which contains epidermal cell growth factor with a final concentration of 20ng/mL, hydrocortisone 0.5mg/mL, and 10ug/ml Insulin, and penicillin and streptomycin double antibodies accounting for 1% of the total volume of the culture medium; during passage, cells were digested with trypsin and passaged at a ratio of 1:2.
  4. 如权利要求1所述的细胞株或权利要求2所述的细胞系在作为人乳腺恶性叶状肿瘤细胞模型中的应用。Use of the cell line according to claim 1 or the cell line according to claim 2 as a human breast malignant phyllodes tumor cell model.
  5. 如权利要求1所述的细胞株或权利要求2所述的细胞系在建立人乳腺恶性叶状肿瘤动物模型中的应用。Use of the cell line according to claim 1 or the cell line according to claim 2 in establishing an animal model of human breast malignant phyllodes tumor.
  6. 如权利要求4或5所述的应用，其特征在于：所述的应用范围包括在研究人乳腺恶性叶状肿瘤致病、发生、发展、转移和耐药机制，相关信号通路和药物靶点方面的应用。The application according to claim 4 or 5, characterized in that: the scope of application includes research on the pathogenesis, occurrence, development, metastasis and drug resistance mechanisms of malignant phyllodes tumors of the human breast, as well as related signaling pathways and drug targets. Applications.
  7. 如权利要求1所述的细胞株或权利要求2所述的细胞系在筛选和评估人乳腺恶性叶状肿瘤诊断或检测试剂中的应用。Use of the cell line according to claim 1 or the cell line according to claim 2 in screening and evaluating diagnostic or detection reagents for human breast malignant phyllodes tumors.
  8. 如权利要求1所述的细胞株或权利要求2所述的细胞系在筛选和评估抗人乳腺恶性叶状肿瘤药物中的应用。Use of the cell line according to claim 1 or the cell line according to claim 2 in screening and evaluating drugs against human breast malignant phyllodes tumors.
PCT/CN2022/118893 2022-09-08 2022-09-15 Human breast malignant phyllodes tumor cell line sysh-mpt-03 and use thereof WO2024050859A1 (en)

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