WO2024050858A1 - Malignant human breast phyllodes tumor cell line sysh-mpt-04 and use thereof - Google Patents

Malignant human breast phyllodes tumor cell line sysh-mpt-04 and use thereof Download PDF

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WO2024050858A1
WO2024050858A1 PCT/CN2022/118892 CN2022118892W WO2024050858A1 WO 2024050858 A1 WO2024050858 A1 WO 2024050858A1 CN 2022118892 W CN2022118892 W CN 2022118892W WO 2024050858 A1 WO2024050858 A1 WO 2024050858A1
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cell line
human breast
sysh
mpt
cell
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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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    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

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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.
  • 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

A malignant human breast phyllodes tumor cell strain, a filial generation cell line thereof, and use thereof. The cell line is named malignant human breast phyllodes tumor cell line SYSH-MPT-04, and is deposited under CCTCC NO. C2022190 in the China Center for Type Culture Collection. The cell line needs no immortalization treatment and can be stably and infinitely passaged in vitro, and the process for culturing the cell line is simple and cheap. The cell line is particularly suitable for being mass-produced and marketed. The cell line can be used as a cell model to promote research on the biological characteristics of and drugs for treating malignant human breast phyllodes tumors.

Description

一种人乳腺恶性叶状肿瘤细胞系SYSH-MPT-04及其应用A human breast malignant phyllodes tumor cell line SYSH-MPT-04 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％。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％。研究发现恶性叶状肿瘤对放疗、化疗、免疫治疗均不敏感，因此恶性叶状肿瘤患者常因全身多处转移而死亡。目前医药界对人乳腺叶状恶性肿瘤的生物学和其抗肿瘤药物的相关研究还处于初步阶段，原因主要有两方面:一是人乳腺叶状恶性肿瘤较为罕见，二是市面上难以获得稳定数量足够的细胞模型。人乳腺恶性叶状肿瘤的研究主要基于该肿瘤原代细胞，然而分离肿瘤原代细胞的耗费大，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。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. At present, the medical research on the biology of human breast phyllodes malignant tumors and its anti-tumor drugs is still in the preliminary stage. There are two main reasons: first, 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.
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，对细胞的代谢和通路产生一定的影响,较难获得稳定的子代细胞系。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.
通过非转染的常规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有限，亦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。因此，寻求一种适合稳定传代，培养工艺简单具有代表性的细胞株(系)是非常必要的。Through conventional non-transfection methods, 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.
发明内容Contents of the invention
基于上述问题，本发明的目的在于克服上述现有技术的不足，提供一种人恶性叶状肿瘤细胞株SYSH-MPT-04及其子代细胞系，并进一步公开其细胞系的建立 方法和相关应用。该细胞株(系)来源于中国人乳腺恶性叶状肿瘤组织提取的原代细胞，该细胞株和细胞系不需要进行永生化处理，可稳定体外无限传代，特别适合量产市场化培养。Based on the above problems, 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.
本发明的细胞系命名为人乳腺恶性叶状肿瘤细胞系SYSH-MPT-04(简称细胞系SYSH-MPT-04)，其细胞株命名为人乳腺恶性叶状肿瘤细胞株SYSH-MPT-04(简称细胞株SYSH-MPT-04)，该细胞系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.C2022190。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.
本发明的细胞株的子代细胞系的建立方法，包括以下步骤：取对数生长期的细胞株SYSH-MPT-04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 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.
本发明细胞株及其子代细胞系的应用：Applications of the cell lines of the present invention and their progeny cell lines:
1.本细胞系SYSH-MPT-04在作为人乳腺恶性叶状肿瘤细胞模型或动物模型中的应用，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。1. The application of 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. Applications in drug resistance mechanisms, related signaling pathways and drug targets.
2.本细胞系SYSH-MPT-04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。2. Application of this cell line SYSH-MPT-04 in screening and evaluating diagnostic or detection reagents for human breast malignant phyllodes tumors.
3.本细胞系SYSH-MPT-04在筛选和评估治疗人乳腺恶性叶状肿瘤药物中的应用。3. The application of this cell line SYSH-MPT-04 in screening and evaluating drugs for the treatment of malignant phyllodes tumors of the human breast.
本发明的细胞株(系)SYSH-MPT-04具有以下优点：The cell strain (line) SYSH-MPT-04 of the present invention has the following advantages:
1.本发明的细胞系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。因此细胞系SYSH-MPT-04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。1. 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 α-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.
2.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。对人乳腺恶性叶状肿瘤生物学特性及治疗 药物研究方面具有很大的促进作用。2. 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.
附图说明Description of the drawings
图1是本发明细胞系SYSH-MPT-04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-04 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-04及对应原代细胞MPTPC4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-04 of the present invention and the corresponding primary cell MPTPC4 and the proliferation rate detected by CCK8 Figure (B).
图3是本发明细胞系SYSH-MPT-04及原代细胞MPTPC1、MPTPC4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-04 of the present invention and primary cells MPTPC1 and MPTPC4.
图4是本发明细胞系SYSH-MPT-04及其原代细胞MPTPC4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-04 of the present invention and its primary cell MPTPC4.
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-04通过以下方法获得：The human breast malignant phyllodes tumor cell line SYSH-MPT-04 of the present invention is obtained by the following method:
1.肿瘤标本来源：分离MPTPC1的人乳腺恶性叶状肿瘤标本来源于47岁女性患者的右乳肿物，MPTPC1是人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。1. 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.
分离SYSH-MPT-04的恶性叶状肿瘤标本来源于51岁女性患者的右乳肿物，肿物大小为9cm×8.2cm×6.1cm。该患者本次术后又出现了2次复发，复发间期约6个月，在最后一次复发切除肿瘤后11个月，患者出现咳嗽并发现双肺多发转移灶。这些临床数据说明该例恶性叶状肿瘤恶性程度高，有较强的增殖、侵袭能力，是代表性的人乳腺恶性叶状肿瘤。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.
2.肿瘤原代细胞的分离和培养：用无菌手术刀将上述肿瘤组织正中切开，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Ⅲ型胶原酶消化(1mg/mL，Worthington)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。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；其中添加终浓度为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；10ug/mL,Sigma品牌的胰岛素；以及占所述培养基总体积1％,Invitrogen品牌的青链霉素双抗；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 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 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. 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.
3.持续传代：在体外持续传代培养上述人乳腺恶性叶状肿瘤原代细胞，观察细胞形态、增殖速度的改变。第3-5代的早期恶性叶状肿瘤原代细胞(Early MPTPC1)形态呈长梭形，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MPTPC4传至62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(见图2)，说明该细胞为可无限传代的人乳腺恶性叶状肿瘤细胞系，命名为人乳腺恶性叶状肿瘤细胞系SYSH-MPT-04，形态如图1所示。3. 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. However, after MPTPC4 has been passed on for more than 62 generations, 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.
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-04进行鉴定，具体包括以下几个方面：The human breast malignant phyllodes tumor cell line SYSH-MPT-04 obtained in Example 1 was identified, specifically including the following aspects:
1.体外功能实验：比较早期、晚期人乳腺恶性叶状肿瘤原代细胞MPTPC1、人乳腺恶性叶状肿瘤细胞系SYSH-MPT-04与其原代细胞的体外细胞增殖、克隆形成、迁移、侵袭和胶原收缩能力。1. In vitro functional experiments: Compare the in vitro cell proliferation, colony formation, migration, invasion and Collagen shrinkage ability.
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 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.
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，而人乳腺恶性叶状肿瘤细胞系SYSH-MPT-04的体外生物学功能与原代细胞相同。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 human breast malignant phyllodes tumor cell line SYSH-MPT-04 are the same as those of primary cells. .
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-04与其原代细胞中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 malignant phyllodes tumor markers (α-SMA and FAP) in the human breast malignant phyllodes tumor cell line SYSH-MPT-04 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 PCTCN2022118892-appb-000001
II Q RT SuperMix试剂盒去除基因组DNA，用ChamQ
Figure PCTCN2022118892-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 PCTCN2022118892-appb-000001
II Q RT SuperMix kit to remove genomic DNA using ChamQ
Figure PCTCN2022118892-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，人乳腺恶性叶状肿瘤细胞系SYSH-MPT-04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 human breast malignant phyllodes tumor cell line SYSH-MPT-04 are similar to those in primary cells.
3.STR鉴定：对人乳腺恶性叶状肿瘤细胞系SYSH-MPT-04及其原代细胞进行STR鉴定。结果如表1，人乳腺恶性叶状肿瘤细胞系SYSH-MPT-04在21个STR位点的拷贝数与原代细胞完全匹配，说明人乳腺恶性叶状肿瘤细胞系SYSH-MPT-04与原代细胞是同源的，没有被其他细胞污染。3. STR identification: 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.
表1Table 1
Figure PCTCN2022118892-appb-000003
Figure PCTCN2022118892-appb-000003
以上结果说明，人乳腺恶性叶状肿瘤细胞系SYSH-MPT-04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。因此人乳腺恶性叶状肿瘤细胞系SYSH-MPT-04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-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 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 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.

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-04，保藏于中国典型培养物保藏中心，保藏编号为CCTCC,NO.C2022190。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-04, and is deposited in the China Type Culture Collection Center with the deposit number CCTCC, NO. C2022190.
  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-04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-04 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 medium used is DMEM/F12 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/CN2022/118892 2022-09-08 2022-09-15 Malignant human breast phyllodes tumor cell line sysh-mpt-04 and use thereof WO2024050858A1 (en)

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