Cervical cancer lymph node metastasis starting cell line and application thereof
By providing cervical cancer lymph node metastasis starting cell lines and related models, the problem of lack of cell lines and models in the prior art that effectively simulates cervical cancer lymph node metastasis is solved, and a more accurate study on cervical cancer lymph node metastasis is achieved.
Patent Information
- Application Number
- CN202510146454.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-10
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art lacks effective cell lines and related models to simulate lymph node metastasis of cervical cancer, making it difficult to conduct effective metastasis initiation cells.
A cervical cancer lymph node metastasis initiation cell line is provided, including SiHa-LNMIC and HeLa-LNMIC cell lines, and the lymph node metastasis process is simulated and studied by constructing a cervical cancer lymph node metastasis initiation model, including cell models and mouse models.
The cervical cancer lymph node metastasis initiation cell line was successfully constructed and identified, which significantly enhanced the sphere-generating ability and lymph node metastasis ability, and provided a more practical research tool to help study the mechanism of cervical cancer lymph node metastasis.
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Figure CN120060145A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of tumor cell lines, and in particular to a cervical cancer lymph node metastasis initiating cell line and its application. Background Art
[0002] Tumor metastasis originates from a tumor subset with a heterogeneous gene expression profile. This unique group of cells with the ability to form distant organ metastases is called metastasis-initiating cells (MICs). Metastasis-initiating cells are involved in multiple simultaneous or sequential steps during the metastasis process, including tumor cell proliferation, migration, and invasion of the extracellular matrix (ECM), resistance to various stresses, anchoring, and establishment of a favorable metastatic niche in distant organs. They are the key cells that initiate and regulate the cascade of the metastasis process in the early stage of metastasis. Therefore, the identification and characterization of metastasis-initiating cells are crucial for analyzing the mechanism of tumor metastasis and early detection and intervention to reduce metastasis-related mortality. However, there are few reports on the research, sorting, and identification of cervical cancer metastasis-initiating cells.
[0003] It is generally believed that metastasis initiation is the key cell driving tumor metastasis. Existing research on cervical cancer lymph node metastasis mainly uses commercially available immortalized cervical cancer cell lines for functional and mechanistic studies, and the main cell lines are derived from cervical cancer in-situ tissues, lacking cell lines and related models that are closer to simulating lymph node metastasis. Summary of the Invention
[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a cervical cancer lymph node metastasis initiating cell line that simulates lymph node metastasis and its application.
[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:
[0006] In the first aspect, the present invention provides a cervical cancer lymph node metastasis initiating cell line, characterized in that the cervical cancer lymph node metastasis initiating cell line is Homo sapiens, SiHa-LNMIC and / or Homo sapiens, HeLa-LNMIC;
[0007] The Homo sapiens, SiHa-LNMIC was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 28, 2024, and its deposit number is: GDMCC No: 65559;
[0008] The Homo sapiens, HeLa-LNMIC was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 28, 2024, and its deposit number is: GDMCC No: 65558.
[0009] In a second aspect, the present invention provides the use of the cervical cancer lymph node metastasis initiating cell line in constructing a cervical cancer lymph node metastasis initiation model.
[0010] Further, the cervical cancer lymph node metastasis initiation model includes a cervical cancer lymph node metastasis initiation mouse model and / or a cervical cancer lymph node metastasis initiation cell model.
[0011] In a third aspect, the present invention provides a product for constructing a cervical cancer lymph node metastasis initiation model, which contains the above-mentioned cervical cancer lymph node metastasis initiating cell line.
[0012] In a fourth aspect, the present invention provides a method for constructing a cervical cancer lymph node metastasis initiation model, including constructing a cervical cancer lymph node metastasis initiation cell model and / or constructing a cervical cancer lymph node metastasis initiation mouse model;
[0013] To construct a cervical cancer lymph node metastasis initiation cell model, passage culture is carried out on the above-mentioned cervical cancer lymph node metastasis initiating cell line.
[0014] To construct a cervical cancer lymph node metastasis initiation mouse model, the above-mentioned cervical cancer lymph node metastasis initiating cell line or the above-mentioned product is injected into a mouse body to form lymph nodes.
[0015] Further, to construct a cervical cancer lymph node metastasis initiation cell model, passage culture is carried out using a complete medium containing serum and antibiotics.
[0016] Further, the serum includes fetal bovine serum; the antibiotics include penicillin and / or streptomycin; the complete medium includes DMEM medium.
[0017] Further, the content of penicillin is 80 - 120 U / mL, and the content of streptomycin is 80 - 100 μg / mL.
[0018] In a specific embodiment of the present invention, experiments are carried out with the content of penicillin being 100 U / mL and the content of streptomycin being 100 μg / mL as an example.
[0019] Further, the content of the serum is 8 - 12% v / v.
[0020] In a specific embodiment of the present invention, experiments are carried out with the content of the serum being 10% v / v as an example.
[0021] Further, the culture temperature is 35 - 37°C.
[0022] In a specific embodiment of the present invention, experiments are carried out with the culture temperature being 37°C as an example.
[0023] Furthermore, a mouse model for the initiation of cervical cancer lymph node metastasis was constructed. The cervical cancer lymph node metastasis initiating cell line or the product was injected into the footpads of mice, and metastatic foci were formed in the popliteal and / or inguinal lymph nodes.
[0024] Furthermore, 1 to 5 million cervical cancer lymph node metastasis initiating cell lines were injected into each mouse.
[0025] In a specific embodiment of the present invention, an experiment was conducted by taking the injection of 2 million cervical cancer lymph node metastasis initiating cell lines as an example.
[0026] Furthermore, the mice are nude mice.
[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0028] The present invention successfully constructed and identified a cervical cancer lymph node metastasis initiating cell line. Compared with the parental cells SiHa and HeLa, the spheroid formation ability was significantly enhanced, the expression level of the stemness-related marker CD44 was increased, the lymph node metastasis ability was stronger, and the time to form lymph node metastatic foci was shorter. It can be prepared into an in vitro cell model and a mouse model, providing a favorable tool for the research related to cervical cancer lymph node metastasis. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 To isolate and construct cervical cancer lymph node metastasis initiating cells. Among them, A is the construction of a nude mouse popliteal lymph node metastasis model; B is the flow chart of continuously observing the metastasis of cervical cancer cells in the nude mouse popliteal lymph nodes at different times; C is the metastasis of cervical cancer cells in the nude mouse popliteal lymph nodes at different times, pCK represents pan-Cytokeratin, which is a cervical cancer cell marker; D is the flow chart of isolating and constructing cervical cancer lymph node metastasis initiating cells.
[0030] Figure 2 To identify cervical cancer lymph node metastasis initiating cells. Among them, A is the spheroid formation ability; B is the expression level of the stemness-related marker CD44; C is the metastasis ability and the time to form lymph node metastatic foci. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0031] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Other materials, reagents, etc. used in the embodiments can be obtained from commercial channels without special instructions.
[0032] Example 1 Isolation and Construction of Cells
[0033] I. Experimental Method
[0034] 1. Inject the human cervical cancer cell lines SiHa and HeLa with red fluorescent protein (mCherry) fluorescence (i.e., parental cells SiHa PR and HeLa PR) into the left footpads of 5-week-old female Balb / c nude mice at a quantity of 2 million cells per mouse to construct a popliteal lymph node metastasis model of nude mice ( Figure 1 A).
[0035] 2. Observe the metastasis of cervical cancer cells in the popliteal lymph nodes of nude mice at 0 week, 1 week, 2 weeks, 4 weeks, and 6 weeks ( Figure 1 B) after the injection of SiHa and HeLa respectively, so as to determine the starting time of cervical cancer metastasis when there are no obvious metastatic lesions in the pathology of the lymph nodes.
[0036] 3. As shown in Figure 1 D, separate and purify the cells:
[0037] (1) Mix 200 μL of Dispase II, 200 μL of Dnase I, and 2 mL of 0.5% (w / v) matrix collagenase, and preheat at 37°C to obtain a working solution;
[0038] (2) One week after the injection of SiHa and HeLa in step 1 of this example, sacrifice the mice by cervical dislocation after anesthesia, obtain the popliteal lymph nodes of the mice under sterile conditions, and wash them 3 times with phosphate buffer solution (PBS) containing penicillin / streptomycin double antibody with a final concentration of 3% (w / v) (300 U / mL penicillin, 300 μg / mL streptomycin) to obtain the washed lymph nodes;
[0039] (3) Mix the working solution in step (1) and the washed lymph nodes in step (2) in a gentleMACS TM tube C, and grind for 15 minutes with a fully automatic tissue processor (brand: Miltenyi Biotec, model: 130-096-427) to obtain the digested popliteal lymph node tissue liquid;
[0040] (4) Filter the popliteal lymph node tissue fluid digested in step (3) through a 70-μm cell strainer, centrifuge at 1000 rpm for 5 min to obtain a cell pellet, wash it once with PBS, and resuspend the cell pellet with DMEM (Dulbecco's Modified Eagle Medium) complete medium containing 10% (v / v) fetal bovine serum (FBS) and 1% (w / v) penicillin / streptomycin double antibody (100 U / mL penicillin, 100 μg / mL streptomycin). Flow-sort the mCherry-positive cervical cancer lymph node metastatic initiating cells SiHa-LNMIC and HeLa-LNMIC, and use DMEM cell complete medium containing 10% (v / v) FBS and 1% (w / v) penicillin / streptomycin double antibody. Incubate in a constant temperature cell incubator at 37 °C and 5% CO 2 Incubate in a constant temperature cell incubator at 37 °C and 5% CO₂. Preserve the cells with fewer passages (less than 5 passages) and store them in liquid nitrogen to obtain the cervical cancer lymph node metastatic initiating cells SiHa-LNMIC and HeLa-LNMIC with good growth status.
[0041] II. Experimental Results
[0042] As Figure 1 Shown in C, when SiHa and HeLa were injected for 1 week, there were no pathologically visible metastatic foci in the popliteal lymph nodes; at 2 weeks, micro-metastatic foci appeared; from 4 to 6 weeks, the range of metastatic foci in the lymph nodes gradually expanded. Therefore, the popliteal lymph nodes at 1 week after SiHa and HeLa injection were selected for isolation and culture to obtain cervical cancer lymph node metastatic initiating cells.
[0043] Example 2 Identification of Cells
[0044] I. Experimental Methods
[0045] 1. Sphere Formation Assay
[0046] The well-growing cervical cancer lymph node metastasis initiating cells SiHa-LNMIC and HeLa-LNMIC and their corresponding parental cells SiHa PR and HeLa PR in Example 1 were each washed twice with PBS. Then, 1 mL of 0.25% (w / v) trypsin was added and the cells were digested at room temperature for 3 min. An equal volume of DMEM medium containing 10% (v / v) FBS was added to neutralize the trypsin. The cells were centrifuged at 1000 rpm for 5 min, the cells were collected, the medium was removed, and the cells were washed twice with PBS. The cells were counted and seeded into an ultra-low attachment six-well plate at a density of 1000 cells per well. DMEM / F12 medium (Dulbeccos Modified Eagle Medium / Nutrient Mixture F-12) containing human leukocyte antigen B27 (1×B27), 20 ng / mL basic fibroblast growth factor (bFGF), and 20 ng / mL epidermal growth factor (EGF) was used, and the cells were cultured in a constant temperature cell incubator at 37°C and 5% CO 2 for 10 days, and the cell spheroid formation was observed and the spheroid formation rate was calculated.
[0047] 2. Expression of cell surface receptor protein CD44
[0048] The well-growing cervical cancer lymph node metastasis initiating cells SiHa-LNMIC and HeLa-LNMIC and their corresponding parental cells SiHa PR and HeLa PR in Example 1 were digested and centrifuged for collection according to the method in step 1 respectively. The medium was removed, and the cells were washed twice with PBS. The cells were counted and evenly seeded into a 24-well plate pre-coated with cell slides at a density of 10000 cells per well. 500 μL of DMEM complete cell medium containing 10% (v / v) FBS and 1% (w / v) penicillin / streptomycin double antibody was added, and the cells were cultured in a constant temperature cell incubator at 37°C and 5% CO 2 for 48 h to obtain the cultured cells.
[0049] The cultured cells were washed twice with PBS, fixed with 4% (w / v) paraformaldehyde at room temperature for 15 min, then washed three times with PBS, 5 min each time. After blocking with donkey serum for 30 min, CD44 antibody was added and incubated overnight at 4°C to obtain the cells after CD44 antibody incubation; the cells after CD44 antibody incubation were washed three times with PBS, 5 min each time, corresponding immunofluorescent secondary antibody was added and incubated at room temperature for 1 h, then the cells were washed three times with PBS, 5 min each time, and a mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) was added for mounting. The CD44 expression level of the cells was observed and calculated.
[0050] 3. Animal experiments: The well-conditioned cervical cancer lymph node metastasis initiating cells SiHa-LNMIC and HeLa-LNMIC and their corresponding parental cells SiHa PR and HeLa PR in Example 1 were digested and centrifuged for collection according to the method in Step 1 respectively. The cells were resuspended with PBS to obtain cell suspensions, and the cell suspensions were injected into the left footpads of 5-week-old female Balb / c nude mice at a dose of 2 million cells per mouse to construct a nude mouse popliteal lymph node metastasis model. At the predetermined time points (the 2nd, 3rd, and 4th weeks after injection), the mice were sacrificed by cervical dislocation after anesthesia, and their popliteal lymph nodes were taken for paraffin embedding, sectioning, and staining to determine the lymph node metastasis situation.
[0051] II. Experimental results
[0052] 1. As Figure 2 shown in A, the sphere-forming ability of the cervical cancer lymph node metastasis initiating cells SiHa-LNMIC and HeLa-LNMIC was significantly enhanced compared with their corresponding parental cells SiHa PR and HeLa PR.
[0053] 2. As Figure 2 shown in B, the expression level of the stemness-related marker CD44 in the cervical cancer lymph node metastasis initiating cells SiHa-LNMIC and HeLa-LNMIC was increased compared with their corresponding parental cells SiHa PR and HeLa PR.
[0054] 3. As Figure 2 shown in C, in the nude mouse popliteal lymph node metastasis model, compared with the parental cells SiHa PR and HeLa PR, the cervical cancer lymph node metastasis initiating cells SiHa-LNMIC and HeLa-LNMIC in Example 1 had stronger lymph node metastasis ability and shorter time to form lymph node metastasis foci.
[0055] The cervical cancer lymph node metastasis initiating cells SiHa-LNMIC and HeLa-LNMIC in Example 1 were identified as human cells. The obtained SiHa-LNMIC cells were named Homo sapiens SiHa LNMIC and were deposited at the Guangdong Provincial Microbial Culture Collection Center on November 28, 2024, with the deposit number: GDMCC No: 65559, and the deposit address is the 5th floor of the Experimental Building, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province. The obtained HeLa-LNMIC cells were named Homo sapiens HeLa-LNMIC and were deposited at the Guangdong Provincial Microbial Culture Collection Center on November 28, 2024, with the deposit number: GDMCC No: 65558, and the deposit address is the 5th floor of the Experimental Building, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province. Example 3 constructs a cervical cancer lymph node metastasis initiating model
[0056] 1. Establish a cervical cancer lymph node metastasis initiating cell model
[0057] (1) Wash the well - identified cervical cancer lymph node metastasis initiating cells SiHa - LNMIC or HeLa - LNMIC in Example 2 twice with PBS. Add 1 mL of trypsin with a concentration of 0.25% (w / v) and digest at room temperature for 3 min. Add an equal volume of DMEM medium containing 10% (v / v) fetal bovine serum to neutralize the trypsin. Centrifuge at 1000 rpm for 5 min, discard the supernatant, and collect the cells.
[0058] (2) Culture the cervical cancer lymph node metastasis initiating cells SiHa - LNMIC and HeLa - LNMIC cells respectively with DMEM complete cell medium containing 10% (v / v) FBS and 1% (w / v) penicillin / streptomycin double - antibody. Conduct sub - culture in a constant - temperature cell incubator at 37 °C and 5% CO 2 to establish an in - vitro cell model of cervical cancer lymph node metastasis for various conventional cell experiments. According to the cell experiments in Example 2, the in - vitro cell model of cervical cancer lymph node metastasis is successfully constructed.
[0059] 2. Establish a mouse model of cervical cancer lymph node metastasis
[0060] (1) Wash the well - identified cervical cancer lymph node metastasis initiating cells SiHa - LNMIC or HeLa - LNMIC in Example 2 twice with PBS. Add 1 mL of trypsin and digest at room temperature for 3 min. Add an equal volume of DMEM medium containing 10% (v / v) FBS to neutralize the trypsin. Centrifuge at 1000 rpm for 5 min, discard the supernatant, and collect the cells.
[0061] (2) Wash the cells in step (1) twice with PBS and resuspend the cells to obtain a cell suspension. Inject the cell suspension into the left footpads of 5 - week - old female Balb / c nude mice at a quantity of 2 million cells per mouse. After 1 week, a mouse model of cervical cancer lymph node metastasis is obtained. According to the animal experiments in Example 2, the mouse model is successfully constructed.
[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.
Claims
1. A cervical cancer lymph node metastasis initiating cell line, characterized in that: The cervical cancer lymph node metastasis initiating cell line is SiHa-LNMIC of the genus Homo sapiens and / or HeLa-LNMIC of the genus Homo sapiens; The Homo sapiens SiHa-LNMIC was deposited in Guangdong Microbial Culture Collection Center on November 28, 2024, and its deposit number is: GDMCC No: 65559; The Homo sapiens HeLa-LNMIC was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 28, 2024, and its deposit number is: GDMCC No: 65558.
2. Use of the cervical cancer lymph node metastasis initiating cell line according to claim 1 in constructing a cervical cancer lymph node metastasis initiating model.
3. A product for constructing a cervical cancer lymph node metastasis initiation model, characterized in that: The product contains the cervical cancer lymph node metastasis initiating cell line according to claim 1.
4. A method for constructing a cervical cancer lymph node metastasis initiation model, characterized in that: Including constructing a cervical cancer lymph node metastasis initiating cell model and / or constructing a cervical cancer lymph node metastasis initiating mouse model; Constructing a cervical cancer lymph node metastasis initiating cell model, and subculturing the cervical cancer lymph node metastasis initiating cell line described in claim 1; A mouse model of cervical cancer lymph node metastasis initiation is constructed, and the cervical cancer lymph node metastasis initiation cell line described in claim 1 or the product described in claim 3 is injected into the mouse to form lymph nodes.
5. The method according to claim 4, characterized in that A cervical cancer lymph node metastasis initiating cell model was constructed and subcultured in complete medium containing serum and antibiotics.
6. The method according to claim 5, characterized in that The content of the serum is 8-12% v / v.
7. The method according to claim 5, characterized in that The culture temperature is 35-37°C.
8. The method according to claim 4, characterized in that A mouse model of cervical cancer lymph node metastasis initiation is constructed, and the cervical cancer lymph node metastasis initiation cell line described in claim 1 or the product described in claim 3 is injected into the mouse foot pad to form metastatic foci in the popliteal fossa and / or inguinal lymph nodes.
9. The method according to claim 4, characterized in that Each mouse was injected with 1 to 5 million cervical cancer lymph node metastasis initiating cell lines.
10. The method according to any one of claims 4, 8 and 9, characterized in that: The mice are nude mice.