Construction method and application of colorectal cancer liver metastasis cell strain

Abstract

The invention relates to a construction method and application of a colorectal cancer liver metastasis cell strain, and the construction method specifically comprises the following steps: transfecting a tdToma / Luciferase gene to CT26. WT to obtain a CT26-TL cell strain; the colorectal cancer cell strain capable of directionally and stably transferring the liver is obtained by taking a CT26-TL cell strain as a starting material and carrying out three rounds of spleen injection screening and five rounds of cecum injection screening, the colorectal cancer cell strain is named as a mouse colorectal cancer cell clone strain CT26-TL-CL5, and the preservation number is CCTCC NO: C2024307. According to the CT26-TL-CL5 cell strain, the technical difficulty of constructing a liver metastasis model starting from in situ in basic research is solved, the problem of observing colorectal cancer liver metastasis in a living body or an isolated organ is solved, and the modeling time is effectively shortened. The CT26-TL-CL5 cell strain can provide a good colorectal cancer liver metastasis animal model for researching molecules and mechanisms for promoting colorectal cancer liver metastasis, and provides cells and related animal models for industrially screening drugs related to colorectal cancer metastasis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent application No. CN202411268651.9 filed on September 11, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine technology, and in particular to a method for constructing and applying a colorectal cancer liver metastasis cell line, specifically to an application of the colorectal cancer liver metastasis cell line in studying components that promote or inhibit colorectal cancer liver metastasis, which is suitable for basic research on intestinal cancer metastasis and the development of drugs related to intestinal cancer metastasis. Background Art

[0004] The current methods for constructing animal models of colorectal cancer liver metastasis include: (1) Zhang L et al. constructed an orthotopic liver metastasis animal model by injecting colorectal cancer cells CT26 into the cecal serosa of mice, with a metastasis rate of only 40% (Zhang L, Bu P. Generation of an orthotopic mouse model to study colorectal cancer metastasis [J]. STAR protocols, 2021, 2 (4)); (2) Zhang Y et al. implanted CT26 cells into the subcutaneous tissue of mice, and killed the tumor-bearing mice 2 weeks later, and digested them into single cells with digestive enzymes, which were called CT26-F1 cell lines; implanted CT26-F1 cell lines into the cecum, and cut off the tumor tissue of liver metastasis 4 weeks later, digested them into single cells, and then implanted them into the cecum, and repeated this cycle 3 times to obtain the highly metastatic cell line CT26-FL3, which can achieve a metastasis rate of up to 80%-90% after in situ transplantation into the cecum (directly injecting CT26 cell lines into the subserous layer of the cecum of Balb / cByJ mice, the liver metastasis rate is only 8%). (Zhang Y, Davis C, Ryan J, et al. Development and characterization of a reliable mouse model of colorectal cancer metastasis to the liver [J]. Clinical & experimental all metastasis, 2013, 30: 903-918.). (3) Chinese patent CN110760477A discloses a method for constructing a mouse liver highly metastatic colorectal cancer cell line, the steps of which include: labeling mouse colorectal cancer cells with antibiotics and bioluminescence, injecting the cell suspension into the mouse subcutaneously for in vivo adaptation; after 4 weeks or when the tumor volume exceeds 2 cm 3 The mice were then killed, the subcutaneous tumor tissue was removed, the mouse intestinal cancer cells were screened for antibiotic resistance in vitro, and cells that were positive for biomarkers were selected; the obtained cells were expanded and cultured in vitro, and a cell suspension was made, and new live mice were treated with the cecal wall implantation method; after 4-6 weeks or when the in situ tumor in the mouse body grew to 1cm 3 The mice were then killed and the metastatic tissue of their livers was removed. Mouse intestinal cancer cells were screened and cultured for antibiotic resistance in vitro, and cells that were positive for biomarkers were selected. The screening was repeated 1-3 times, and cell lines were obtained 4-5 weeks later or when more than 80% of the mice developed liver metastasis after cecal injection.

[0005] However, the above methods have the following shortcomings: (1) The CRC metastasis reported in existing articles is difficult to reproduce. BABL / c mice were purchased from several domestic experimental animal sales agencies (including Shanghai Slake Experimental Animal Co., Ltd., Shanghai Xipul-Bike Experimental Animal Co., Ltd. and Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) and injected into the mouse serosa with CT26.WT, which successfully established cecal in situ tumors, but no liver metastasis occurred. (2) The high metastatic cell line screening scheme reported in the literature is less efficient. The CRC cecal model was constructed by using CT26 cells obtained from subcutaneous tumors, which successfully established cecal in situ tumors, but no liver metastasis occurred. (3) The CT26.WT cell line cannot be used for in vivo or ex vivo organ tumor imaging. (4) The modeling cycle is generally long. Summary of the invention

[0006] In order to overcome at least one problem existing in the prior art, the present invention transfects CT26.WT with tdTomato / Luciferase gene to obtain CT26-TL cell line; and starting with CT26-TL cell line, optimizes the screening process to obtain colorectal cancer liver metastasis cell line CT26-TL-CL5 (a colorectal cancer cell line with stable and high metastasis to the liver). The CT26-TL-CL5 cell line obtained by the above screening method can significantly shorten the animal modeling time to 18 days, and the liver metastasis rate is not less than 80%.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention is to provide a method for constructing a colorectal cancer liver metastasis cell line, which comprises the steps of:

[0009] Step S1, obtaining a CT26-TL cell line by lentiviral transfection, wherein the CT26-TL cell line is obtained by transfecting tdTomato / Luciferase gene into CT26.WT cells;

[0010] Step S2, injecting the CT26-TL cell line obtained in step S1 into the spleen of mice to construct a spleen-injected colorectal cancer liver metastasis model; after the liver metastasis is formed, collecting the liver metastasis cells, digesting and expanding them, and then continuing to perform 2 rounds of screening via spleen injection;

[0011] Step S3, injecting the CT26-TL cell line obtained in step S2 into the cecum to construct a colorectal cancer liver metastasis model injected in situ into the cecum; after the liver metastasis is formed, collecting the liver metastasis cells, digesting and expanding them, and then continuing to perform 4 rounds of screening via cecal serosa injection to finally obtain a colorectal cancer liver metastasis cell line, named CT26-TL-CL5 cell line.

[0012] Furthermore, in step S1, the preparation step of the CT26-TL cell line includes:

[0013] Step S1-1, preparing a transfection mixture, wherein the transfection mixture comprises p-PEI, a viral expression plasmid pCDH-tdTomato-Luciferase, a viral packaging plasmid pMD2.G, and a viral packaging plasmid psPAX2 in a predetermined ratio;

[0014] Step S1-2, taking 293FT cells and adding them to the transfection mixture obtained in step S1-1 (preferably the cell density is 70%, 4 mL of transfection mixture), incubating for a predetermined time (preferably 37° C., 5% CO2 for 6 hours), adding a complete culture medium of 293FT cells, culturing for a predetermined time (preferably culturing for 24 hours), discarding the culture medium of 293FT cells, adding fresh culture medium (preferably 10 mL), and amplifying the virus (preferably 32° C., 5% CO2 for virus amplification);

[0015] Step S1-3, culturing CT26.WT cells in a complete culture medium of CT26.WT cells to a predetermined density (preferably culturing CT26.WT cells after 48 hours, and the cell density reaches 50-60% after culturing for 24 hours);

[0016] Step S1-4, after the virus is amplified for a predetermined time (preferably 72 hours), the supernatant of the 293FT cells with amplified virus is collected, filtered (preferably using a 0.45 μm filter), centrifuged (preferably at 4°C, 20,000 rpm, centrifuged for 100 min), the supernatant is discarded, the CT26.WT cell complete medium (preferably 2 mL) obtained in step S1-3 is added to resuspend the virus, and then an infection enhancer (preferably HB, with a final concentration of 8 μg / mL) is added, cultured (preferably at 37°C, 5% CO2 for 24 hours, then replaced with new complete medium, 37°C, 5% CO2 for 24 hours), and passaged;

[0017] Step S1-5: Perform flow cytometry sorting on the cells obtained in step S1-4 by tdTomato fluorescent labeling to obtain CT26-TL positive cell lines expressing tdTomato and Luciferase.

[0018] Further, the step S1-1 specifically includes the steps of: 1) preparing a p-PEI mixed solution: adding 30 μL p-PEI to 2 mL serum-free culture medium, mixing, and standing at room temperature for 5 min; 2) preparing a plasmid mixed solution containing 3 μg of viral expression plasmid pCDH-tdTomato-Luciferase, 1.87 μg of viral packaging plasmid pMD2.G, and 3.43 μg of viral packaging plasmid psPAX2, mixing, adding to the centrifuge tube of the p-PEI mixed solution, mixing again, and standing at room temperature for 20 min. The above-mentioned serum-free culture medium is serum-free DMEM HG culture medium.

[0019] Furthermore, in step S1-2, the complete culture medium of 293FT cells contains DMEM HG, 10% FBS and 100 U / ml penicillin-streptomycin; the fresh culture medium replaced subsequently is DMEM HG culture medium.

[0020] Furthermore, in step S1-3, the components of the CT26.WT cell culture medium include RPMI-1640, 10% fetal bovine serum, 1×GlutaMAX TM and 100U / ml penicillin-streptomycin; the new complete culture medium replaced subsequently is the complete culture medium for CT26.WT cells.

[0021] It is understandable that the above-mentioned step of screening positive cells by flow cytometry can be performed by conventional steps.

[0022] Furthermore, in step S2, in the screening for spleen injection, CT26-TL cells were resuspended in PBS solution to obtain a cell resuspension. In the first round of screening, the injected cell concentration was 10 7 / mL, in the second round of screening, the injection cell concentration was 3×10 6 / mL, in the third round of screening, the injection cell concentration was 10 6 / mL, and the injection volume in each round was 100 μL of cell resuspension.

[0023] Furthermore, in step S3, in the screening of cecal serosa injection, CT26-TL cells were diluted to 1×10 6 / 15μL, add an equal amount of matrix gel to the cell suspension to obtain a cell suspension, and the injection volume for each round is 30μL of cell suspension.

[0024] Furthermore, in the above-mentioned construction method, the method for obtaining tumor cells from liver metastasis by digestion includes the steps of: cutting the metastasis tissue into pieces, digesting the tissue, and forming the tissue into a single cell suspension; adding the cell digestion solution to a buffer solution, centrifuging, and discarding the supernatant; adding a complete cell culture medium to resuspend the cell pellet; adding the resuspended cells to a cell culture dish for culturing overnight; the next day, discarding the culture medium, adding fresh complete cell culture medium, and waiting for the cells at the bottom of the culture dish to grow to a coverage of 60%-80%, the cells are passaged and amplified.

[0025] It is understandable that the reagents, culture conditions, etc. used in the above method of obtaining tumor cells from liver metastases by digestion can refer to conventional methods in the art.

[0026] Furthermore, the CT26-TL cell line is used for in vivo imaging or in vitro organ imaging after modeling.

[0027] Furthermore, the liver metastasis rate of the CT26-TL-CL5 cell line is not less than 80%.

[0028] The second aspect of the present invention is to provide a colorectal cancer liver metastasis cell line, which is named mouse colon cancer cell clone CT26-TL-CL5 (Mus musculus), with a preservation number of CCTCC NO: C2024307, a preservation date of September 5, 2024, and a preservation unit of China Center for Type Culture Collection (abbreviated as CCTCC), and the preservation unit address is Wuhan University, Wuhan, China.

[0029] Furthermore, the CT26-TL-CL5 cell line is obtained by any construction method described in the first aspect.

[0030] Furthermore, the preservation and resuscitation method of the CT26-TL-CL5 cell line is as follows: 1) resuspending the cells in a complete culture medium containing 10% DMSO, placing them at -80°C for 2 days, and then transferring them to a liquid nitrogen tank for storage; 2) during resuscitation, taking the cells out of the liquid nitrogen tank, quickly placing them in 37°C warm water until they are completely thawed, adding the thawed cells to the complete culture medium, discarding the supernatant after centrifugation, adding the complete culture medium to resuspend, inoculate, and culture.

[0031] The third aspect of the present invention is to provide a colorectal cancer liver metastasis cell line obtained by any construction method described in the first aspect, or the use of the colorectal cancer liver metastasis cell line as described in any one of the second aspects in constructing a colorectal cancer liver metastasis animal model.

[0032] Furthermore, the colorectal cancer liver metastasis animal model includes a colorectal cancer in situ-liver metastasis model and a spleen injection colorectal cancer liver metastasis model.

[0033] The fourth aspect of the present invention is to provide a method for constructing a colorectal cancer liver metastasis mouse model, which comprises injecting the colorectal cancer liver metastasis cell line obtained by any of the construction methods described in the first aspect or the colorectal cancer liver metastasis cell line described in any of the second aspect into the cecal serosa or spleen of mice to construct a colorectal cancer liver metastasis mouse model.

[0034] Furthermore, in the above construction method, the CT26-TL-CL5 cell line was diluted to 1×10 5 / 15μL, add an equal amount of matrix gel to the cell suspension to obtain a cell suspension for cecal in situ injection, and the cecal serosa injection volume is 30μL of cell suspension.

[0035] Furthermore, in the above construction method, the cells were resuspended in PBS solution to obtain a cell concentration of 10 6 / mL of cell resuspension for spleen injection, and the spleen injection volume is 100μL of cell resuspension.

[0036] Furthermore, a colorectal cancer liver metastasis mouse model was successfully established 18 days after injection of the CT26-TL-CL5 cell line, and the liver metastasis rate was no less than 80%.

[0037] The fifth aspect of the present invention is to provide an application of a colorectal cancer liver metastasis mouse model obtained by any of the construction methods described in the fourth aspect, which is selected from at least one of the following applications: use as an animal model in screening components that promote or inhibit colorectal cancer liver metastasis or in screening for colorectal cancer metastasis-related preventive or therapeutic drugs, and use as an animal model in studying the mechanism of colorectal cancer liver metastasis.

[0038] It is understandable that the above-mentioned components or drugs include antibiotics, traditional Chinese medicine ingredients, antagonistic bacteria or bacteriophages against liver metastasis pathogens, etc.

[0039] Furthermore, the component or medicine contains Enterococcus gallinarumphage, and its deposit number is CCTCC NO: M20241928.

[0040] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0041] The present invention obtains a CT26-TL-CL5 cell line with a high liver metastasis rate by optimizing the screening method for colorectal cancer cell lines with stable liver metastasis. The above screening method provides a reference for a more efficient screening method for colorectal cancer high metastasis cell lines that are directed to the liver. The above CT26-TL-CL5 cell line solves the technical difficulties of constructing a liver metastasis model starting from the in situ in basic research, and solves the problem of observing colorectal cancer liver metastasis in living or ex vivo organs. The use of the CT26-TL-CL5 cell line to construct a colorectal cancer liver metastasis animal model can achieve a liver metastasis rate of not less than 80% in about 18 days. The above CT26-TL-CL5 cell line can provide a good colorectal cancer liver metastasis animal model for the study of molecules and mechanisms that promote colorectal cancer liver metastasis, and provides cells and related animal models for industrial screening of colorectal cancer metastasis-related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0043] Figure 1 This is a schematic diagram of screening a liver-directed metastatic cell line CT26-TL-CL5 of the present invention;

[0044] Figure 2 1 is a schematic diagram of the results of screening tdTomato-positive CT26-TL cells by flow cytometry in one embodiment of the present invention; wherein the left figure is a negative control, and the box in the right figure is a tdTomato-positive cell population;

[0045] Figure 3 This is a schematic diagram of the results of liver metastasis rate of the cecum in situ model constructed with CT26-TL-CL5 cell line in one embodiment of the present invention; wherein, part A shows that 4 out of 5 livers have obvious metastatic foci; part B shows H&E staining of liver metastasis to verify the occurrence of liver metastasis;

[0046] Figure 4 This is a schematic diagram of the results of constructing a cecum in situ model mouse with CT26-TL-CL5 in one embodiment of the present invention, showing that other organs of the mouse have no tumor bioluminescent signals;

[0047] Figure 5 It is a schematic diagram of the results of constructing an in situ tumor and liver metastasis in the cecum in situ model using the CT26-TL-CL5 cell line in one embodiment of the present invention; wherein, Part A shows the formation of in situ tumor and liver metastasis in the model mouse, indicating that the model was successfully constructed; Part B shows the bioluminescent signal of the in situ tumor in the model mouse in vivo imaging; and Part C shows the bioluminescent signal imaging of the liver tumor in vitro.

[0048] Figure 6 This is a schematic diagram of the results of using a colorectal cancer liver metastasis model constructed by spleen injection of CT26-TL-CL5 cell line to verify the effect of quail and gall Enterococcus phage Phi_Eg_SY1 on colorectal cancer liver metastasis in one embodiment of the present invention; wherein, part a is the in vitro tumor signal imaging of the liver at the end point of the experiment of the colorectal cancer liver metastasis model intervened by spleen injection of Enterococcus phage Phi_Eg_SY1; part b is the tumor signal statistics in part a; *p<0.05.

[0049] The above-mentioned colorectal cancer liver metastasis cell line CT26-TL-CL5 has been preserved and named as mouse colon cancer cell clone CT26-TL-CL5 (Mus musculus). The preservation number is CCTCC NO: C2024307. The preservation date is September 5, 2024. The preservation unit is China Center for Type Culture Collection (CCTCC for short), and the preservation unit address is Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0050] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials and reagents without the source in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0051] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0052] In certain embodiments of the present invention, Figure 1The screening schematic diagram shows a method for constructing a metastatic cell line for colorectal cancer liver metastasis, comprising the following steps: (1) obtaining a CT26-TL cell line stably expressing tdTomato and Luciferase by lentiviral transfection, which can be used for in vivo imaging or in vitro organ imaging after modeling; (2) optimizing the in vivo screening method: first, injecting the mouse spleen with the CT26-TL cell line to construct a spleen-injected colorectal cancer liver metastasis model; after the formation of liver metastases, taking the metastatic cells and continuing to perform 2 rounds of screening via spleen injection; injecting the obtained cell line into the cecal serosa, and at the end of the experiment, collecting the liver metastases, digesting and expanding them, and then performing a total of 4 rounds of screening until a high metastatic cell line CT26-TL-CL5 (preservation number: CCTCC NO: C2024307) with a liver metastasis rate of not less than 80% is obtained.

[0053] In the following examples, the method for obtaining tumor cells by digestion from liver metastases includes the following steps: (1) at the end of the experiment, the mouse is euthanized, the abdominal cavity is opened, and the metastatic lesions in the liver are removed under a dissecting microscope; (2) the metastatic lesions are placed in a sterile 1.5 mL EP tube, the metastatic lesions are rinsed 3 times with sterile PBS, and the tissue is cut into pieces of about 1 mm 3(3) Add appropriate amount of collagenase (type II) (Yisheng, 40508ES60) to a final concentration of 1 mg / mL and incubate at 37°C for 1 h; (4) Use a 1 mL pipette tip to repeatedly pipette and aspirate to form a single cell suspension; Transfer the cell digestion solution into a 15 mL centrifuge tube and add 2 mL HBSS buffer, 1000rpm, centrifuge for 5min, discard the supernatant; repeat washing with HBSS twice; (5) add 2mL of complete culture medium and resuspend the cell pellet; the complete culture medium used for the different cell lines obtained by screening is the same as the complete culture medium required for CT26.WT culture; (6) add the resuspended cells to a 3.5cm pore cell culture dish and culture overnight; (7) the next day, discard the culture medium, gently wash the surface of the adherent cells with PBS, repeat twice, and add 2mL of fresh complete culture medium; (8) when the cells at the bottom of the culture dish grow to 60%-80% coverage, subculture and expand the cells; (9) discard the supernatant, wash once with PBS, aspirate PBS, and add 200μL Digest with 0.25% trypsin for 2 min, add 1 mL of complete culture medium to terminate digestion, transfer the cell suspension into a centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant; (10) Add 1 mL of complete culture medium to resuspend, centrifuge at 1000 rpm for 5 min, and discard the supernatant; (11) Take a 10 cm cell culture dish, add about 10 mL of complete culture medium, and then inoculate all the cell suspension obtained in the previous step into it to expand the cells; after 2 days of culture, digest and subculture in the same way, with a subculture ratio of 1:3. The complete culture medium used for screening the different CT26-TL cell lines obtained above is the same as the complete culture medium required for CT26.WT culture, which contains RPMI-1640 (Gibco, 11875093), 10% fetal bovine serum (Cegrogen, A0500-3010), 1×GlutaMAXTM (Gibco, 35050061), and 100U / ml penicillin-streptomycin (Yamei, CB010).

[0054] In the following embodiments, the Enterococcus gallinarum phage Phi_Eg_SY1 used has been deposited, and its classification name is Enterococcus gallinarum phage, its deposit number is CCTCC NO: M20241928, the deposit date is September 6, 2024, the depository is China Center for Type Culture Collection (abbreviated as CCTCC), and the depository address is Wuhan University, Wuhan, China.

[0055] The following embodiments are only used to illustrate the technical solutions of the present invention.

[0056] Example 1 - Construction of CT26-TL cell line by lentiviral transfection

[0057] This example is a method and steps for transfecting CT26.WT (ATCC CRL-2638) with tdTomato / Luciferase gene, including:

[0058] (1) Packaging and amplification of lentivirus;

[0059] 1) Prepare transfection mixture;

[0060] p-PEI mixed solution: Add 30 μL p-PEI (Polysciences, 23966) to 2 mL serum-free DMEM HG (Gibco, 11965092) medium, mix well, and let stand at room temperature for 5 min. Prepare a plasmid mixture containing 3 μg of viral expression plasmid pCDH-tdTomato-Luciferase, 1.87 μg of viral packaging plasmid pMD2.G (addgene, 12259), and 3.43 μg of viral packaging plasmid psPAX2 (addgene, 12260), mix well, and add to the centrifuge tube of p-PEI mixed solution, mix again, and let stand at room temperature for 20 min.

[0061] 2) Take out the 293FT cells that were passaged 24 hours ago and have a density of 70%, discard the culture medium, slowly add 4 mL of the transfection mixture prepared in the previous step, and incubate at 37°C, 5% CO2 for 6 hours.

[0062] 3) After 6 hours, the complete medium of 293FT cells was slowly added, which contained DMEM HG (Gibco, 11965092), 10% FBS (Cegrogen, A0500-3010) and 100 U / ml penicillin-streptomycin (Yamo, CB010).

[0063] 4) After 24 hours, the culture medium of 293FT cells was discarded, and 10 mL of DMEM HG culture medium was added at 32° C. and 5% CO 2 to amplify the virus.

[0064] 5) After 48 hours, CT26.WT cells were transferred into 6 cm cell culture dishes so that the density reached 50-60% after 24 hours. The complete culture medium of CT26.WT cells contained RPMI-1640 (Gibco, 11875093), 10% fetal bovine serum (Cegrogen, A0500-3010), 1×GlutaMAXTM (Gibco, 35050061), and 100 U / ml penicillin-streptomycin (Yamei, CB010).

[0065] 6) After 72 hours of virus amplification, the supernatant of the 293FT cells in which the virus was amplified was collected, filtered through a 0.45 μm filter into a virus centrifuge tube, and centrifuged at 4° C., 20,000 rpm, for 100 min, and the supernatant was discarded.

[0066] 7) Add 2 mL of CT26.WT complete cell culture medium to resuspend the virus, then add infection enhancer HB (final concentration 8 ug / mL) and culture at 37°C, 5% CO2 for 24 h.

[0067] 8) After 24 hours, replace with new CT26.WT complete culture medium and culture at 37°C, 5% CO2 for 24 hours. Then subculture and screen for positive cells.

[0068] (2) Screening of positive cells;

[0069] Flow cytometry was performed using tdTomato (excitation wavelength 554 nm, emission wavelength 581 nm) fluorescent labeling, and the sorted positive cells were subsequently cultured.

[0070] 1) After 24 hours, add 2 mL of freshly prepared RPMI-1640 medium and culture at 37°C, 5% CO2 for 24 hours.

[0071] 2) Discard the supernatant, wash once with PBS, remove the PBS, add 200 μL 0.25% trypsin (Gibco, 27250-018) and digest for 2 min, add 1 mL CT26.WT cell culture medium to terminate digestion, transfer the cell suspension into a centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0072] 3) Add 10 mL PBS to resuspend and centrifuge at 1000 rpm for 5 min.

[0073] 4) Add 1 mL of RPMI-1640 medium, resuspend the cells, and add the cells to the flow cytometry tube. At the same time, prepare a flow cytometry recovery tube and add 300 μL of medium to the flow cytometry recovery tube.

[0074] 5) When sorting on the machine, the control group is CT26.WT cells that are not transfected with fluorescent plasmids. Use a sorting flow cytometer, select the PE channel, and sort to obtain CT26-TL positive cell lines expressing tdTomato and Luciferase (such as Figure 2 shown).

[0075] Example 2-Screening of colorectal cancer liver metastasis cell line CT26-TL-CL5

[0076] This embodiment is a screening optimization scheme for highly metastatic cell lines of colorectal cancer liver metastasis, which specifically includes the following steps:

[0077] (1) The CT26-TL cells obtained in Example 1 were digested and filtered through a 70 μm filter. The obtained single cells were washed twice with PBS. After the last centrifugation, they were resuspended in an appropriate volume of PBS to adjust the cell concentration to 10 7 / mL.

[0078] (2) The mice that had been previously depilated were anesthetized by induction and maintenance of anesthesia with 1-3% isoflurane, and the epidermis and peritoneum were cut open by a scalpel for about 0.5 cm at the lower edge of the left rib cage of the mouse.

[0079] (3) Gently pick out the mouse spleen with a sterile cotton swab and place it on a sterile gauze moistened with saline to prepare for cell injection. Mix the cell suspension thoroughly and draw up 100 μL of the cell suspension with a 29G insulin syringe. Insert the needle into the spleen about 1 cm at a straight angle and slowly inject the cell suspension.

[0080] (4) Gently press with a cotton swab until the blister produced by the injection disappears, then remove the insulin needle. Continue pressing the needle hole for 2-3 minutes.

[0081] (5) Use No. 4 suture to ligate the blood vessels at both ends of the spleen and remove the spleen.

[0082] (6) Suture the wound of the mouse, and then place the mouse on a 37°C heating pad to keep warm and wait for it to recover. Place the recovered mouse in a sterile cage, mark the date of surgery, and return it to a ventilated cage for breeding.

[0083] (7) The experimental endpoint was 4 weeks after modeling. The mice were euthanized, the liver metastases were digested, and the obtained cells were expanded and cultured. The cells were digested and the cell concentration was adjusted to 3×10 6 / mL.

[0084] (8) The second round of spleen injection modeling was performed using the same operation method.

[0085] (9) At the end of the experiment, the liver metastases were collected and the obtained cells were expanded and cultured. The cells were digested and the cell concentration was adjusted to 10 6 / mL.

[0086] (10) Perform the third round of spleen injection using the same procedure.

[0087] (11) At the end of the experiment, liver metastases were collected and the obtained cells were expanded and cultured.

[0088] (12) The digested cells were resuspended in RPMI-1640 medium, centrifuged again, and the supernatant was discarded.

[0089] (13) Resuspend the cells in a small amount of RPMI-1640 medium, aspirate 10 μL of the cell suspension, dilute it appropriately, and count the live cells.

[0090] (14) The cells were diluted to an appropriate concentration using RPMI-1640 medium for subsequent construction of a mouse model of cecal orthotopic CRC liver metastasis. The cell concentration was adjusted to 1×10 6 / 15μL.

[0091] (15) Add an equal amount of Matrigel (Yisheng, 40183ES10) to the cell suspension diluted to an appropriate concentration, mix well, and place on ice. Pipette 30 μL of the cell suspension into a 30G insulin syringe and place on ice for later use.

[0092] (16) Anesthesia was induced and maintained in mice with pre-depilated abdomen using 1-3% isoflurane. The epidermis and peritoneum were cut open about 1 cm along the linea alba about 1.5 cm above the mouse anus.

[0093] (17) The cecum was gently picked out using a cotton swab moistened with saline and placed on a sterile gauze moistened with saline in preparation for cell injection.

[0094] (18) Gently press one side of the cecum with a cotton swab moistened with saline, and place the prepared insulin needle tip with the cell suspension facing upwards. Carefully pierce the cecum subserosa and inject the cell suspension into the subserosa. After successful injection, a transparent bubble-like structure will bulge on the surface of the serosa. Allow the needle tip to remain in the subserosa for about 20 seconds, remove the needle, and immediately glue the needle hole with a small amount of medical glue.

[0095] (19) The wound of the mouse was sutured and the mouse was placed on a 37°C heating pad to keep warm. After the mouse recovered, it was placed in a sterile cage, marked with the date of surgery, and returned to a ventilated cage for breeding.

[0096] (20) The experimental endpoint was 3 weeks after modeling. The mice were euthanized, the liver metastases of the mice were digested, and the obtained cells were named CT26-TL-CL1 for expansion and culture. The cells were digested and the cell concentration was adjusted to 1×10 6 / 15μL.

[0097] (21) The same operation method was used to perform cecal modeling in the 2nd to 5th rounds.

[0098] (22) At the end of the experiment, the mice were euthanized, the liver metastases were harvested and digested, and the obtained cells, named CT26-TL-CL5, were expanded and cultured.

[0099] The colorectal cancer liver metastasis cell line CT26-TL-CL5 obtained by the above screening has been preserved, and its preservation number is CCTCC NO: C2024307. The freezing and recovery of the above cell lines also follow the "slow freezing and quick dissolution principle": a portion of the amplified cells are resuspended in a complete culture medium containing 10% DMSO, transferred to a cryopreservation tube, and placed in a gradual freezing box. After being placed at -80 degrees for 2 days, they are transferred to a liquid nitrogen tank for storage; when recovering, the cells are taken out of the liquid nitrogen tank and quickly placed in 37°C warm water until completely thawed. The thawed cells are transferred to a 15mL centrifuge tube, 4 times the volume of complete culture medium is added, and the supernatant is discarded after centrifugation (1000rpm, 5min), 1mL of complete culture medium is added to resuspend, and inoculated into a 10mL cell culture dish. Place in a cell culture incubator (37°C, containing 5% CO2) for culture.

[0100] Example 3 - Construction of a mouse model of colorectal cancer liver metastasis using cell line CT26-TL-CL5

[0101] In this example, the cell line CT26-TL-CL5 obtained in Example 2 was used to construct a mouse model of colorectal cancer liver metastasis. The cecum model was established in the same manner as in Example 2, and the cells were digested to reduce the cell concentration to 1×10 5 / 15μL, verify the proportion of liver metastasis in the cecum orthotopic model mice constructed by CT26-TL-CL5 cell line, and confirm whether there is metastasis to other organs. The experimental results include:

[0102] (1) Figure 3 As shown in the figure, the proportion of liver metastasis in mice with the cecum orthotopic model constructed by the CT26-TL-CL5 cell line was 80%, and 4 of the 5 livers had visible metastatic foci, which were the areas encircled by the yellow solid circle ( Figure 3 H&E tissue staining also revealed metastatic lesions in 4 of the 5 cases, namely, the darkly stained areas with hematoxylin ( Figure 3 Part B of Figure 4 As shown in the figure, there are no metastatic foci in the heart, spleen, lung, kidney and brain of the cecum orthotopic model mice constructed by the CT26-TL-CL5 cell line. The above results indicate that only liver metastasis occurs in this model, and no metastasis occurs in other common organs (from left to right: heart, spleen, lung, kidney, brain).

[0103] (2) Figure 5 As shown, the mice were euthanized 18 days after modeling, and after dissection, it was found that there was orthotopic tumor growth in the cecum and obvious liver metastasis in the liver ( Figure 5 At the end of the experiment, D-luciferin was injected into the orbital venous plexus of mice to detect the tumor cell signals in vivo. The results showed that the in situ tumor signals ( Figure 5The mice were euthanized, the livers were removed, and tumor signal imaging was performed on the isolated livers. It was found that 4 out of 5 mice had tumor cell signals in their livers ( Figure 5 The above results show that the colorectal cancer liver metastasis model was successfully constructed, and the model can be constructed in as short as 18 days, which greatly shortens the modeling time.

[0104] Example 4 - Application of Colorectal Cancer Liver Metastasis Mouse Model

[0105] This example uses a mouse model of colorectal cancer liver metastasis constructed by spleen injection of CT26-TL-CL5 cell line to verify the function of Enterococcus gallinarum phage Phi_Eg_SY1 on colorectal cancer liver metastasis, which specifically includes the following steps:

[0106] (1) Five 6-week-old BALB / c female mice (from Shanghai Xipu-Bikai Laboratory Animal Co., Ltd.) were used and injected intraperitoneally with 100 μL of 10 8 PFU Phi_Eg_SY1 phage particles in normal saline, and the control group was injected with an equal amount of normal saline at the same time.

[0107] (2) After three injections of phage, the mouse spleens of the experimental and control groups were injected with the mouse colorectal cancer liver metastasis cell line CT26-TL-CL5 the next day to construct a colorectal cancer liver metastasis mouse model. The cell concentration was adjusted to 10 6 / mL, spleen modeling was performed in the same manner as in Example 2;

[0108] (3) Three weeks after the establishment of the colorectal cancer liver metastasis mouse model, the mice were injected with D-luciferin sodium salt, and the livers were removed after euthanasia to image the tumor bioluminescence signal in vitro ( Figure 6 Part a of the study), and quantitative and comparative analysis of the bioluminescent signal intensity of liver tumors ( Figure 6 Part b) was used to evaluate the effect of phage Phi_Eg_SY1 intervention on liver metastasis of colorectal cancer in mice. The above experimental results prove that phage Phi_Eg_SY1 significantly inhibits liver metastasis of colorectal cancer and reduces the degree of liver metastasis, thereby achieving the prevention or auxiliary intervention of liver metastasis.

[0109] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for constructing a colorectal cancer liver metastasis cell line, characterized in that: Includes steps: Step S1, obtaining a CT26-TL cell line by lentiviral transfection, wherein the CT26-TL cell line is obtained by transfecting tdTomato / Luciferase gene into CT26.WT cells; Step S2, injecting the CT26-TL cell line obtained in step S1 into the spleen of mice to construct a spleen-injected colorectal cancer liver metastasis model; after the liver metastasis is formed, collecting the liver metastasis cells, digesting and expanding them, and then continuing to perform 2 rounds of screening via spleen injection; Step S3, injecting the CT26-TL cell line obtained in step S2 into the cecum to construct a colorectal cancer liver metastasis model injected in situ into the cecum; after the liver metastasis is formed, collecting the liver metastasis cells, digesting and expanding them, and then continuing to perform 4 rounds of screening via cecal serosa injection to finally obtain a colorectal cancer liver metastasis cell line, named CT26-TL-CL5 cell line.

2. The construction method according to claim 1, characterized in that: In step S1, the preparation steps of the CT26-TL cell line include: Step S1-1, preparing a transfection mixture, wherein the transfection mixture comprises p-PEI, a viral expression plasmid pCDH-tdTomato-Luciferase, a viral packaging plasmid pMD2.G, and a viral packaging plasmid psPAX2 in a predetermined ratio; Step S1-2, adding 293FT cells to the transfection mixture obtained in step S1-1, incubating for a predetermined time, adding a complete culture medium of 293FT cells, culturing for a predetermined time, discarding the culture medium of 293FT cells, adding fresh culture medium, and amplifying the virus; Step S1-3, culturing CT26.WT cells in CT26.WT cell complete culture medium to a predetermined density; Step S1-4: After the virus is amplified for a predetermined time, the supernatant of the 293FT cells with amplified virus is collected, filtered, centrifuged, and the supernatant is discarded. The complete medium of CT26.WT cells obtained in step S1-3 is added to resuspend the virus, and then an infection enhancer is added for culture and passage; Step S1-5: Perform flow cytometry sorting on the cells obtained in step S1-4 by tdTomato fluorescent labeling to obtain CT26-TL positive cell lines expressing tdTomato and Luciferase.

3. The construction method according to claim 1, characterized in that: In step S2, in the screening of spleen injection, CT26-TL cells were resuspended in PBS solution to obtain a cell resuspension. In the first round of screening, the injection cell concentration was 10 7 / mL, in the second round of screening, the injection cell concentration was 3×10 6 / mL, in the third round of screening, the injection cell concentration was 10 6 / mL, and the injection volume in each round was 100 μL of cell resuspension; In step S3, in the screening of cecal serosa injection, CT26-TL cells were diluted to 1×10 6 / 15μL, add an equal amount of matrix gel to the cell suspension to obtain a cell suspension, and the injection volume for each round is 30μL of cell suspension.

4. The construction method according to claim 1, characterized in that: The method for obtaining tumor cells by digestion in liver metastasis includes the following steps: cutting up metastasis tissue, digesting the tissue, and forming the tissue into a single cell suspension; adding a buffer solution to the cell digestion solution, centrifuging, and discarding the supernatant; adding a complete cell culture medium, and resuspending the cell precipitate; adding the resuspended cells to a cell culture dish and culturing overnight; the next day, discarding the culture medium, adding fresh complete cell culture medium, and when the cells at the bottom of the culture dish grow to a coverage of 60%-80%, the cells are passaged and amplified.

5. The construction method according to claim 1, characterized in that: The CT26-TL cell line is used for in vivo imaging or in vitro organ imaging after modeling, and the liver metastasis rate of the CT26-TL-CL5 cell line is not less than 80%.

6. A colorectal cancer liver metastasis cell line, characterized in that: The colorectal cancer liver metastasis cell line is a mouse colon cancer cell clone CT26-TL-CL5, and its preservation number is CCTCC NO: C2024307.

7. Use of a colorectal cancer liver metastasis cell line obtained by the construction method described in any one of claims 1 to 5, or a colorectal cancer liver metastasis cell line described in claim 6 in constructing a colorectal cancer liver metastasis animal model.

8. A method for constructing a mouse model of colorectal cancer liver metastasis, characterized in that: The colorectal cancer liver metastasis cell line obtained by the construction method described in any one of claims 1 to 5 or the colorectal cancer liver metastasis cell line described in claim 6 is injected into the cecal serosa or spleen of mice to construct a colorectal cancer liver metastasis mouse model.

9. The construction method according to claim 8, characterized in that: CT26-TL-CL5 cells were diluted to 1×10 5 / 15μL, add an equal amount of matrix gel to the cell suspension to obtain a cell suspension for in situ injection in the cecum, and the cecal serosa injection volume is 30μL of cell suspension; or, Resuspend the cells in PBS to obtain a cell concentration of 10 6 / mL of cell resuspension for spleen injection, and the spleen injection volume is 100μL of cell resuspension.

10. An application of a mouse model of colorectal cancer liver metastasis obtained by the construction method according to claim 8 or 9, characterized in that: The application is selected from at least one of the following applications: use as an animal model in screening components that promote or inhibit colorectal cancer liver metastasis or in screening colorectal cancer metastasis-related preventive or therapeutic drugs, and use as an animal model in studying the mechanism of colorectal cancer liver metastasis.

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