Cervical cancer lymph node high metastasis model and preparation method thereof
By isolating and preserving U14-LNM3 cell lines and constructing a high metastasis model for cervical cancer lymph nodes, the problem of obtaining effective cell lines and lacking clinically relevant models in the existing technology is solved, and an economical and reliable research tool is provided, which enhances the clinical relevance of cervical cancer lymph node metastasis research.
Patent Information
- Application Number
- CN202510146392.0
- 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 is difficult to obtain effective cell lines related to cervical cancer lymph node metastasis, and there is a lack of a clinically prone cervical cancer lymph node metastasis model for research.
By isolating and preserving a high metastasis cell line of cervical cancer lymph nodes called U14-LNM3, and providing its preparation method, including subculture and mouse model construction, a high metastasis model of cervical cancer lymph nodes was established.
It provides an economical, reliable and repeatable high metastasis model of cervical cancer lymph nodes, helping researchers explore the mechanisms and potential therapeutic targets of cervical cancer lymph node metastasis, and enhances the clinical relevance of the study.
Smart Images
Figure CN120060144A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cervical cancer cells, and particularly relates to a cervical cancer lymph node high-metastasis model and a preparation method thereof. Background Art
[0002] Lymph node metastasis is the main metastasis mode and cause of death in cervical cancer patients. Therefore, exploring the specific mechanism of lymph node metastasis and mining potential therapeutic targets are particularly important for the treatment of cervical cancer patients. At present, various tumor cell lines mainly originate from the in-situ lesions of tumor patients rather than the tumor cells after metastasis to lymph nodes, making it relatively difficult to compare the gene expression and various biological behaviors between cervical cancer cells with strong metastatic ability and those without lymph node metastasis. Therefore, obtaining cervical cancer cells with strong lymph node metastasis ability and exploring related research on the driving factors of cervical cancer lymph node metastasis have become important problems that need to be solved urgently.
[0003] Existing research on cervical cancer lymph node metastasis mainly uses methods such as in vitro transfection to pre-edit the genes of tumor cells derived from in-situ lesions, and then injects them into the footpads of mice to observe the changes in their metastatic ability. There is a lack of a more clinically relevant spontaneous cervical cancer lymph node metastasis model for lymph node metastasis-related research. 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 high-metastasis model with more clinical relevance for spontaneous cervical cancer lymph node metastasis and a preparation method thereof.
[0005] To achieve the above purpose, the technical solutions adopted by the present invention are as follows:
[0006] In the first aspect, the present invention provides a cervical cancer lymph node high-metastasis cell line, and the cervical cancer lymph node high-metastasis cell line is U14-LNM3 of the genus Talpa and the species Mus musculus. The U14-LNM3 of the genus Talpa and the species Mus musculus was deposited in the Guangdong Provincial Microbial Culture Collection Center on November 28, 2024, and its deposit number is: GDMCC No: 65557.
[0007] In the second aspect, the present invention provides the application of the cervical cancer lymph node high-metastasis cell line in the preparation of a cervical cancer lymph node high-metastasis model.
[0008] Furthermore, the cervical cancer lymph node high-metastasis model includes a cervical cancer lymph node high-metastasis in vitro cell model and / or a cervical cancer lymph node high-metastasis mouse model.
[0009] In the third aspect, the present invention provides a reagent for preparing a cervical cancer lymph node high-metastasis model, and the reagent contains the cervical cancer lymph node high-metastasis cell line described above.
[0010] Fourthly, the present invention provides a method for preparing an in vitro cell model of highly metastatic cervical cancer lymph nodes, and subculturing the highly metastatic cell line of cervical cancer lymph nodes.
[0011] Further, subculturing is carried out using a complete medium containing serum and antibiotics.
[0012] As a preferred embodiment, the serum includes fetal bovine serum, the antibiotics include at least one of penicillin, streptomycin, and puromycin, and the complete medium includes DMEM medium.
[0013] In a specific embodiment of the present invention, the content of fetal bovine serum is 8-12% v / v, preferably 10% v / v.
[0014] In a specific embodiment of the present invention, the content of penicillin is 80-120 U / mL, preferably 100 U / mL; the content of streptomycin is 80-120 μg / mL, preferably 100 μg / mL.
[0015] As a preferred embodiment, the culture temperature is 35-37 °C, preferably 37 °C.
[0016] Fifthly, the present invention provides a method for preparing a highly metastatic mouse model of cervical cancer lymph nodes, injecting the highly metastatic cell line of cervical cancer lymph nodes or the reagent into a mouse body to form lymph nodes.
[0017] Further, 1-2 million highly metastatic cell lines of cervical cancer lymph nodes are injected into each mouse body, preferably 1.5 million.
[0018] In a specific embodiment of the present invention, the highly metastatic cell line of cervical cancer lymph nodes or the reagent can be injected into the footpad of a mouse to form lymph nodes in the popliteal fossa.
[0019] Further, the mouse is a C57BL / 6 mouse.
[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0021] The present invention has isolated a highly metastatic cell line U14-LNM3 of cervical cancer lymph nodes with strong lymph node metastasis ability, which is more similar to the spontaneous cervical cancer lymph node metastasis in clinical practice, and is conducive to providing an economical, reliable, and highly reproducible animal model and in vitro cell model for basic research related to cervical cancer lymph node metastasis, and providing multiple cell lines for verification in related research. Description of the Drawings
[0022] Figure 1To construct the process and situation of cervical cancer lymph node highly metastatic cells. Among them, A is the flow chart for constructing cervical cancer lymph node highly metastatic cells; B is the metastatic ability of cervical cancer lymph node highly metastatic cells.
[0023] Figure 2 The Transwell experiment was used to compare the migration and invasion abilities between parental cells and lymph node metastatic cells. Specific implementation manners
[0024] 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.
[0025] Example 1 Isolation of cervical cancer lymph node highly metastatic cell line U14-LNM3
[0026] As Figure 1 shown in A, the mouse cervical cancer cell line U14-GFP with GFP (green fluorescent protein) fluorescence and Puromycin resistance was cultured in vitro, and mycoplasma was regularly removed and relevant detections were carried out.
[0027] U14-GFP cells in good growth state were injected into the left footpad of 5-week-old female C57BL / 6 mice at a dose of 1.5 million cells per mouse to construct a popliteal lymph node highly metastatic model, and the primary cancer and lymph node metastatic lesions of the mice were monitored under a stereomicroscope. Four weeks after inoculating U14-GFP cells, the mice were sacrificed by cervical dislocation under anesthesia. The popliteal lymph nodes of the mice were obtained under sterile conditions and washed 3 times with PBS (phosphate buffer) 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 popliteal lymph nodes.
[0028] Prepare the working solution: Mix 2 mL of 0.5% (w / v) matrix collagenase, 200 μL of Dnase I (deoxyribonuclease I) and 200 μL of Dispase II (dispase II), and preheat at 37 °C; Add the washed popliteal lymph nodes and the working solution to gentleMACS TMIn the C tube, grind for 15 min with a fully automatic tissue processor (brand: Miltenyi Biotec, model: 130-096-427) to obtain the digested tissue liquid. Filter the digested tissue liquid through a 70 μm cell sieve, centrifuge at 1000 rpm for 5 min to obtain cell pellets, wash the cell pellets once with PBS, resuspend the cell pellets with DMEM medium containing 10% (v / v) fetal bovine serum. After sorting with a flow cytometer, use DMEM (Dulbecco's Modified Eagle Medium) complete cell medium supplemented with Puromycin at a final concentration of 2 μg / mL, 10% (v / v) FBS (fetal bovine serum), and 1% (w / v) penicillin / streptomycin double antibody (100 U / mL penicillin, 100 μg / mL streptomycin), and culture in a 37 °C, 5% CO 2 cell incubator to obtain purified cells U14-LNM.
[0029] Inject U14-LNM into the mouse footpads again according to the method of this example to enable secondary cell metastasis. After re-isolation and culture, U14-LNM2 is obtained; inject U14-LNM2 into the mouse footpads again according to the method of this example to enable tertiary cell metastasis, thereby obtaining highly metastatic cervical cancer lymph node cells U14-LNM3.
[0030] Example 2 Identification of highly metastatic cervical cancer lymph node cells U14-LNM3
[0031] I. Experimental method
[0032] Wash the parental cells U14-GFP with good growth status in Example 1, highly lymph node metastatic cells U14-LNM, U14-LNM2, and U14-LNM3 with different lymph node metastasis times twice with PBS, digest the cells with 1 mL of 0.25% (w / v) trypsin for 3 min, add DMEM medium containing 10% (v / v) fetal bovine serum equal in volume to the trypsin to neutralize the trypsin, centrifuge at 1000 rpm for 5 min, wash the cells twice with PBS to obtain digested cells, and inject them into the footpads of female C57BL / 6 mice at a dose of 1.5 million per mouse.
[0033] Regularly observe the tumor formation in the mouse footpads and the size of the corresponding popliteal lymph nodes. At the predetermined time point, sacrifice the mice by cervical dislocation after anesthesia, take their lymph nodes, fix, embed, section, and stain them to compare the lymph node metastasis of the parental cells U14-GFP, highly lymph node metastatic cells U14-LNM, U14-LNM2, and U14-LNM3 with different lymph node metastasis times.
[0034] 2. Compare the differences in migration and invasion abilities between cervical cancer lymph node metastatic cells and parental cells
[0035] According to the method of step 1 of this embodiment, the parental cells U14-GFP with good growth status in Example 1, the highly lymph node metastatic cells U14-LNM, U14-LNM2, and U14-LNM3 with different lymph node metastasis times were digested respectively to obtain digested cells.
[0036] Resuspend the digested cells with serum-free DMEM medium and inoculate them onto the upper layer of a transwell chamber (a permeable cell culture chamber) at a density of 50,000 cells / 250 μL. Add 500 μL of complete DMEM cell medium containing 10% (v / v) fetal bovine serum and 1% (w / v) penicillin / streptomycin double antibody to the lower layer of the chamber. Incubate in a 37 °C, 5% CO 2 cell incubator for 24 h. After 24 h, take out the transwell chamber, wash it twice with PBS, gently wipe the cells on the upper chamber with a clean cotton swab, fix the transwell chamber in 4% (w / v) paraformaldehyde at room temperature for 15 min, stain with crystal violet for 15 min, wash the cells 5 - 6 times with PBS, and observe and photograph under a microscope.
[0037] II. Experimental Results
[0038] As Figure 1 shown in Figure 2 B, U14-LNM3 has a stronger lymph node metastasis ability compared to the parental cell lines U14-GFP, U14-LNM, and U14-LNM2. As
[0039] shown, U14-LNM3 has stronger migration and invasion abilities compared to the parental cell lines U14-GFP, U14-LNM, and U14-LNM2.
[0040] Example 3 Preparation of a highly lymph node metastatic model of cervical cancer
[0041] 1. Highly lymph node metastatic mouse model of cervical cancer
[0042] (1) Digest U14-LNM3 cells with 0.25% (w / v) trypsin for 3 min, add DMEM medium containing 10% (v / v) fetal bovine serum with the same volume as trypsin to neutralize trypsin, centrifuge at 1000 rpm for 5 min, and discard the supernatant to obtain cells.
[0043] (2) Wash the cells in step (1) twice with PBS, resuspend the cells to obtain a cell suspension, and inject the cell suspension into the left footpad of 5-week-old female C57BL / 6 nude mice at a quantity of 1.5 million cells per mouse. After 4 weeks, a mouse model with high cervical cancer lymph node metastasis is obtained. According to the animal experiment in Example 2, the mouse model is successfully constructed. According to the cell experiment in Example 2, the mouse model with high cervical cancer lymph node metastasis is successfully constructed.
[0044] 2. In vitro cell model of high cervical cancer lymph node metastasis
[0045] (1) Digest U14-LNM3 cells with 0.25% (w / v) trypsin for 3 min, add DMEM medium containing 10% (v / v) fetal bovine serum with the same volume as trypsin to neutralize trypsin, centrifuge at 1000 rpm for 5 min, and discard the supernatant to obtain cells.
[0046] (2) Culture U14-LNM3 cells with DMEM complete cell medium containing 10% (v / v) FBS (fetal bovine serum) and 1% (w / v) penicillin / streptomycin double antibody, and perform subculture in a 37 °C, 5% CO 2 incubator for constant temperature culture, and use it as an in vitro cell model of high cervical cancer lymph node metastasis for various conventional cell experiments.
[0047] 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 high metastasis cell line, characterized in that: The cervical cancer lymph node highly metastatic cell line is U14-LNM3 of the genus Moss and the species Mus musculus. The genus Moss and the species Mus musculus U14-LNM3 was preserved in the Guangdong Provincial Microbiological Culture Collection Center on November 28, 2024, and its preservation number is: GDMCCNo: 65557.
2. Use of the cervical cancer lymph node high metastasis cell line according to claim 1 in preparing a cervical cancer lymph node high metastasis model.
3. The use according to claim 2, characterized in that: The cervical cancer lymph node high metastasis model includes a cervical cancer lymph node high metastasis in vitro cell model and / or a cervical cancer lymph node high metastasis mouse model.
4. A reagent for preparing a cervical cancer lymph node high metastasis model, characterized in that: The reagent contains the cervical cancer lymph node highly metastatic cell line according to claim 1.
5. A method for preparing an in vitro cell model of cervical cancer with high lymph node metastasis, characterized in that: The cervical cancer lymph node highly metastatic cell line according to claim 1 is subcultured.
6. The method according to claim 5, characterized in that Subculture was performed using complete medium containing serum and antibiotics.
7. A method for preparing a mouse model of cervical cancer with high lymph node metastasis, characterized in that: The cervical cancer lymph node highly metastatic cell line according to claim 1 or the reagent according to claim 4 is injected into mice to form lymph nodes.
8. The method according to claim 7, characterized in that One to two million cervical cancer lymph node highly metastatic cell lines were injected into each mouse.
9. The method according to claim 7, characterized in that: The cervical cancer lymph node highly metastatic cell line according to claim 1 or the reagent according to claim 4 is injected into the foot pad of mice to form lymph nodes in the popliteal fossa.
10. The method according to claim 9, characterized in that The mice are C57BL / 6 mice.