Mouse intrahepatic bile duct cancer cell line mICC-K and application thereof

Abstract

The invention belongs to the technical field of microbial animal cell lines, and particularly relates to a mouse intrahepatic bile duct cancer cell line mICC-K and application thereof. The mouse intrahepatic bile duct cancer cell line mICC-K is preserved in the China Center for Type Culture Collection on January 23, 2025, and the preservation number of the mouse intrahepatic bile duct cancer cell line mICC-K is CCTCC NO: C202538. The mouse intrahepatic cholangiocarcinoma spontaneous tumor is induced through AKT, YAP and SB plasmid systems by utilizing a high-pressure hydrodynamic method caudal vein plasmid injection method, so that the mouse intrahepatic cholangiocarcinoma cell line is obtained, and meanwhile, a KRASG12D mutation site is introduced, so that the blank of lack of a stable mouse in-vivo and in-vitro model in the current domestic intrahepatic cholangiocarcinoma research is filled, and the mouse intrahepatic cholangiocarcinoma cell line is developed. In addition, a stable model is provided for drug target research aiming at KRAS mutation.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial animal cell lines, and particularly relates to a mouse intrahepatic cholangiocarcinoma cell line mICC-K and an application thereof. Background Art

[0002] Intrahepatic cholangiocarcinoma is a type of liver tumor. Currently, research on intrahepatic cholangiocarcinoma is limited by the lack of a complete animal model. Human intrahepatic cholangiocarcinoma models constructed by existing human cholangiocarcinoma cell lines, PDX (human tumor xenografts), organoids, etc. can only be implanted in mice with severe immune deficiencies. These models can be used to study pathogenic genes of intrahepatic cholangiocarcinoma or screen drugs that simply kill tumor cells, but cannot be used to study tumor immune-related mechanisms and drugs. Existing immune-related research on intrahepatic cholangiocarcinoma is through humanized immune mice. This model can partially simulate the effect of the human immune system on humanized intrahepatic cholangiocarcinoma, but the model is expensive, has large heterogeneity, is difficult to simulate the in situ tumor environment, and has a short treatment window. Therefore, it cannot be widely used in the study of intrahepatic cholangiocarcinoma. The model constructed by mouse intrahepatic cholangiocarcinoma cells can truly simulate the interaction between mouse immune cells and intrahepatic cholangiocarcinomas, which has significant advantages in studying the tumorigenesis and development mechanism of intrahepatic cholangiocarcinoma, especially the immune interaction in the tumor environment.

[0003] At present, foreign scientist Holger Willenbring (J Clin Invest. 2012; 122 (8): 2911-2915) reported that a spontaneous intrahepatic cholangiocarcinoma model was constructed in FVB / N mice by using tail vein high pressure hydrodynamic injection of NICD / AKT plasmid. However, the existing technology still has many disadvantages: (1) Direct construction by tail vein high pressure hydrodynamic method will cause diffuse growth of intrahepatic tumors in mice and serious damage to mouse liver function within 5 weeks. Therefore, the experimental window period for drug research is short, experimental research is difficult, and it is difficult to use for drugs that require long-term observation for efficacy; (2) Individual heterogeneity of spontaneous tumor models is large; (3) Most models are induced in FVB / N mice, which are expensive and require large research investment; (4) Almost all models come from foreign laboratories and are difficult to obtain for domestic research in China. Therefore, it is necessary to construct a domestically-owned, stable, short-modeling and simple intrahepatic cholangiocarcinoma cell line to solve the above problems. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a mouse intrahepatic cholangiocarcinoma cell line mICC-K and applications thereof.

[0005] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0006] In a first aspect, the present invention provides a mouse intrahepatic cholangiocarcinoma cell line, comprising mouse intrahepatic cholangiocarcinoma cell mICC-K Mus musculus, and the mouse intrahepatic cholangiocarcinoma cell line mICC-K was deposited in the China Center for Type Culture Collection on January 23, 2025, with a deposit number of CCTCC NO: C202538.

[0007] The present invention adopts the high-pressure hydrodynamic method to inject plasmids into the tail vein, and successfully induces the formation of spontaneous tumors of intrahepatic cholangiocarcinoma in mice through the AKT, YAP, and SB plasmid systems, thereby obtaining a stable mouse intrahepatic cholangiocarcinoma cell line. At the same time, the KRAS G12D mutation site is introduced into the cell line, thereby filling the gap of the lack of stable mouse in vitro and in vivo models in the study of intrahepatic cholangiocarcinoma in China. In addition, the cell line provides a reliable model for studying drug targets related to KRAS mutations. The cell line of the present invention is stable in passage and rapid in proliferation, and can be stably passaged through conventional DMEM or 1640 culture medium. Due to its good growth characteristics, the cell line can not only be used for the construction of intrahepatic cholangiocarcinoma models of mice in vitro and in vivo, but also can be widely used in basic research and drug screening of intrahepatic cholangiocarcinoma, providing a stable and efficient experimental platform for related research. Through the cell line obtained by the present invention, researchers can more accurately study the pathogenesis of intrahepatic cholangiocarcinoma, the role of KRAS mutations, and new therapeutic targets, thereby promoting the progress of treatment research on intrahepatic cholangiocarcinoma.

[0008] Preferably, the mouse intrahepatic cholangiocarcinoma cell line also includes a progeny cell line of the mouse intrahepatic cholangiocarcinoma cell line mICC-K.

[0009] Preferably, the method for constructing the mouse intrahepatic cholangiocarcinoma cell line comprises the following steps:

[0010] YAP plasmid S127A , plasmid AKT, plasmid KRAS G12D and plasmid SB are dissolved in physiological saline, injected into the tail vein of mice by high-pressure hydrodynamics, and then the mouse tumor tissue is separated, the tumor tissue is cut into pieces and transplanted into the mouse liver tissue for culture, the tumor tissue in the mouse liver is obtained and tumor cells are extracted, and the tumor cells are subcultured to obtain the mouse intrahepatic cholangiocarcinoma cell line.

[0011] Preferably, the plasmid YAP S127A , plasmid AKT, plasmid KRAS G12D The mass ratio of p361.4 to plasmid SB is 3:2:1:1.

[0012] The experimental investigation found that the AKT and YAP S127A , SB and KRAS G12DThe dosage of the plasmid has an important influence on the tumor formation rate of spontaneous tumors of intrahepatic cholangiocarcinoma in mice, as well as the stability and growth characteristics of the cell line. The construction of the cell line using the above-defined amount ratio of the present invention can effectively improve the tumor formation rate and ensure the stability and repeatability of tumor formation. In addition, the obtained intrahepatic cholangiocarcinoma cell line mICC-K has stable passage and rapid growth. The cell line is more in line with the biological characteristics of intrahepatic cholangiocarcinoma and can provide a reliable model for subsequent drug screening and mechanism research.

[0013] Preferably, the mouse is a C57BL / 6 mouse.

[0014] In a second aspect, the present invention provides a method for constructing the mouse intrahepatic cholangiocarcinoma cell line, comprising the following steps:

[0015] YAP plasmid S127A , plasmid AKT, plasmid KRAS G12D and plasmid SB are dissolved in physiological saline, injected into the tail vein of mice by high-pressure hydrodynamics, and then the mouse tumor tissue is separated, the tumor tissue is cut into pieces and transplanted into the mouse liver tissue for culture, the tumor tissue in the mouse liver is obtained and tumor cells are extracted, and the tumor cells are subcultured to obtain the mouse intrahepatic cholangiocarcinoma cell line.

[0016] Preferably, the plasmid YAP S127A , plasmid AKT, plasmid KRAS G12D The mass ratio of p361.4 to plasmid SB is 3:2:1:1.

[0017] Preferably, the mouse is a C57BL / 6 mouse.

[0018] In a third aspect, the present invention provides an application of the mouse intrahepatic cholangiocarcinoma cell line, wherein the application includes any one of the following:

[0019] (1) Used to study the mechanism of occurrence, development and metastasis of intrahepatic cholangiocarcinoma;

[0020] (2) Establish an animal model of intrahepatic cholangiocarcinoma;

[0021] (3) Screening or evaluating drugs for the treatment of intrahepatic cholangiocarcinoma;

[0022] (4) Construction of drug-resistant cell models;

[0023] (5) Screening biomarkers or identifying molecular targets related to intrahepatic cholangiocarcinoma.

[0024] In a fourth aspect, the present invention provides a method for constructing a mouse intrahepatic cholangiocarcinoma model, the construction method comprising any one of the following (a)-(b):

[0025] (a) injecting the mouse intrahepatic cholangiocarcinoma cell line subcutaneously into mice and culturing for 1-2 weeks until a tumor is felt under the mouse skin;

[0026] (b) The mouse intrahepatic cholangiocarcinoma cell line is injected subcutaneously into mice, and the formed tumor mass is used for orthotopic liver transplantation to obtain an orthotopic tumor model.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention uses the high-pressure hydrodynamic method to inject plasmids into the tail vein to induce spontaneous tumors of intrahepatic cholangiocarcinoma in mice through the AKT, YAP, and SB plasmid systems, thereby obtaining a mouse intrahepatic cholangiocarcinoma cell line and introducing KRAS G12D Mutation sites can fill the gap in the current domestic research on intrahepatic cholangiocarcinoma, which lacks a stable in vivo and in vitro mouse model. It also provides a stable model for the study of drug targets targeting KRAS mutations.

[0029] (2) The cell line of the present invention has stable passage and rapid growth, can be stably passaged using conventional DMEM or 1640 culture medium, and can be directly used for the construction of intrahepatic cholangiocarcinoma models in mice in vitro and in vivo;

[0030] (3) The cell line of the present invention is convenient and quick to construct an in vivo tumor model. The tumor model can be directly constructed by subcutaneous injection of the cell line into mice, or the formed tumor mass can be used to make an orthotopic liver tumor model. Compared with the spontaneous tumor model by tail vein injection of plasmids, the modeling cycle is greatly shortened. At the same time, the modeling is stable and consistent.

[0031] (4) The cell line of the present invention is the same as the previously commercialized human intrahepatic cholangiocarcinoma cell line. It can be cultured in vitro under conventional cell culture conditions and can be used to study the molecular mechanism of the in vitro development of mouse intrahepatic cholangiocarcinoma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the cell morphology of the mouse intrahepatic cholangiocarcinoma cell line when the tumor reached the 40th to 50th generation;

[0033] Figure 2 CK7, CK19, and KRAS in the extracted mouse intrahepatic cholangiocarcinoma cells G12D Immunofluorescence results;

[0034] Figure 3 It is the Western Blot image of the cell lines after being treated with different concentrations of MRTX1133 drugs;

[0035] Figure 4 This is a picture of subcutaneous tumor formation in nude mice;

[0036] Figure 5This is a picture of orthotopic tumor formation in the liver of C57BL / 6 mice;

[0037] Figure 6 This is the immunohistochemical staining of CK7 and CK19 in nude mouse subcutaneous tumor;

[0038] Figure 7 This is an immunohistochemical staining of CK7 and CK19 in the liver orthotopic tumor of C57BL / 6 mice. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] Unless otherwise specified, the reagents used in the examples are conventional reagents in the art and can be purchased through commercial channels. The experimental operations not specifically described in the examples are conventional operations in the art or can be understood or known by those skilled in the art based on the existing technology or common knowledge they master.

[0041] Plasmid YAP described in Example S127A Plasmid AKT and plasmid SB were purchased from Addgene (YAP S127A , Catalog No.: 46049; Plasmid AKT, Catalog No.: 31789; Plasmid SB, Catalog No.: 20207) Plasmid KRAS G12D The donation comes from Professor Gao Qiang's research group at Zhongshan Hospital Affiliated to Fudan University.

[0042] Example 1

[0043] This embodiment provides a mouse intrahepatic cholangiocarcinoma cell line mICC-K, and the construction method thereof comprises the following steps:

[0044] (1) Plasmid YAP S127A (30 μg per mouse) + plasmid AKT (20 μg per mouse) + plasmid KRAS G12D (10 μg per mouse) + plasmid SB (10 μg per mouse) dissolved in 2 mL of saline and injected into the tail vein of C57BL / 6 mice within 6-8 seconds;

[0045] (2) After 4-5 weeks, the mice were anesthetized and killed by cervical dislocation, and the tumor-infected livers of the mice were obtained in a sterile environment. The tumor tissues were cut into pieces of about 0.5 mm in a sterile environment and transplanted into the liver tissues of C57BL / 6 mice;

[0046] (3) After 3-4 weeks, the mice were euthanized and the tumor tissue in the mouse liver was obtained under a sterile environment. The tumor tissue was washed, minced, digested, and filtered through a 70 μm mesh. The large tumor tissue was centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded. Two times the volume of red blood cell lysis buffer was added and lysed at room temperature for 3 minutes. The cells were centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded. The cells were resuspended in 10% Australian fetal bovine serum + double antibody and F12 medium and cultured at 37°C with 5% carbon dioxide.

[0047] (4) The culture medium (10% Australian fetal bovine serum + double antibody, F12 medium) was changed every 3-4 days for 2 months. After 2 months, the serum was replaced with 10% fetal bovine serum + double antibody, 1640 or DMEM medium, and the culture was continued for 2 months until the tumor cells could be stably passaged, thus obtaining mouse intrahepatic cholangiocarcinoma cell mICC-K Mus musculus. The cell morphology of the tumor at the 40th to 50th passage during the establishment of the mouse intrahepatic cholangiocarcinoma cell line is shown in the figure. Figure 1 As shown, it was deposited in the China Center for Type Culture Collection on January 23, 2025, with the deposit number CCTCC NO: C202538, and the deposit address is Wuhan University, Wuhan, China.

[0048] CK7 and CK19 are common markers of intrahepatic cholangiocarcinoma. The expression of CK7 and CK19 in the extracted mouse intrahepatic cholangiocarcinoma cells was detected by immunofluorescence staining. Figure 2 The results showed that both CK7 and CK19 in the cells showed strong positive reactions, indicating that the cells had the characteristics of intrahepatic bile duct cells; KRAS G12D The mutation is an important mutation site specially introduced in this study, which is highly specific and can be detected by immunofluorescence detection of KRAS G12D The expression of KRAS G12D Highly expressed in cells ( Figure 2 ), indicating that the cell line successfully carried the mutation, and this mutation was closely related to the occurrence and progression of intrahepatic cholangiocarcinoma; therefore, the results of immunofluorescence detection showed that CK7, CK19, and KRAS in the extracted mouse intrahepatic cholangiocarcinoma cells G12D The high expression of can effectively support that the mouse intrahepatic cholangiocarcinoma cell line has the cell marker characteristics of intrahepatic cholangiocarcinoma. In addition, this study also used Western Blot to further analyze the KRAS after the cell line was treated with different concentrations of MRTX1133 (a KRAS G12D specific inhibitor). G12D The expression of proteins related to mutation and its signaling pathway, Figure 3 The results showed that MRTX1133 can effectively inhibit KRAS G12D The expression of downstream signaling pathway proteins (such as ERK) of KRAS was significantly decreased, indicating that MRTX1133 can effectively target KRASG12D , verifying KRAS G12D The key role of KRAS in tumor formation and also provide a stable model for drug target research against KRAS mutations. Then the intrahepatic cholangiocarcinoma cell line extracted above was divided into 5×10 6 The cells were injected subcutaneously into nude mice or C57BL / 6 mice. Tumors could be felt under the skin of the mice 1-2 weeks later. The subcutaneous tumor formation in nude mice is shown in the figure below. Figure 4 In addition, pathological staining can further verify that the subcutaneous tumor of nude mice has the characteristics of mouse intrahepatic cholangiocarcinoma ( Figure 6 ). At the same time, the generated tumor mass was cut into pieces and transplanted into the liver of C57BL / 6 mice, which also formed tumors in 2 weeks. The figure of in situ tumor formation in the liver of C57BL / 6 mice is shown in Figure 2. Figure 5 As shown, the immunohistochemical staining of CK7 and CK19 in mouse liver orthotopic tumor is shown in Figure 7 As shown, the results can fully prove that the liver tumor in situ also has the characteristics of mouse intrahepatic cholangiocarcinoma. Therefore, the mouse intrahepatic cholangiocarcinoma cell line mICC-K of the present invention can be stably passaged, and the model can be used for oncology-related experiments of mouse intrahepatic cholangiocarcinoma in vivo and in vitro.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A mouse intrahepatic cholangiocarcinoma cell line, characterized in that The cell line is the mouse intrahepatic cholangiocarcinoma cell line mICC-K, which was deposited in the China Center for Type Culture Collection on January 23, 2025, with the deposit number CCTCC NO: C202538.

2. The mouse intrahepatic cholangiocarcinoma cell line according to claim 1, characterized in that The method for constructing the mouse intrahepatic cholangiocarcinoma cell line comprises the following steps: YAP plasmid S127A , plasmid AKT, plasmid KRAS G12D and plasmid SB are dissolved in physiological saline, injected into the tail vein of mice by high-pressure hydrodynamics, and then the mouse tumor tissue is separated, the tumor tissue is cut into pieces and transplanted into the mouse liver tissue for culture, the tumor tissue in the mouse liver is obtained and tumor cells are extracted, and the tumor cells are subcultured to obtain the mouse intrahepatic cholangiocarcinoma cell line.

3. The mouse intrahepatic cholangiocarcinoma cell line according to claim 2, characterized in that The YAP plasmid S127A , plasmid AKT, plasmid KRAS G12D The mass ratio of p361.4 to plasmid SB is 3:2:1:

1.

4. The mouse intrahepatic cholangiocarcinoma cell line according to claim 2, characterized in that The mice are C57BL / 6 mice.

5. The method for constructing a mouse intrahepatic cholangiocarcinoma cell line according to any one of claims 1 to 4, characterized in that: The following steps are involved: YAP plasmid S127A , plasmid AKT, plasmid KRAS G12D and plasmid SB are dissolved in physiological saline, injected into the tail vein of mice by high-pressure hydrodynamics, and then the mouse tumor tissue is separated, the tumor tissue is cut into pieces and transplanted into the mouse liver tissue for culture, the tumor tissue in the mouse liver is obtained and tumor cells are extracted, and the tumor cells are subcultured to obtain the mouse intrahepatic cholangiocarcinoma cell line.

6. The construction method according to claim 5, characterized in that: The YAP plasmid S127A , plasmid AKT, plasmid KRAS G12D The mass ratio of p361.4 to plasmid SB is 3:2:1:

1.

7. The use of the mouse intrahepatic cholangiocarcinoma cell line according to any one of claims 1 to 4, characterized in that: The application includes any of the following: (1) Used to study the mechanism of occurrence, development and metastasis of intrahepatic cholangiocarcinoma; (2) Establish an animal model of intrahepatic cholangiocarcinoma; (3) Screening or evaluating drugs for the treatment of intrahepatic cholangiocarcinoma; (4) Construction of drug-resistant cell models; (5) Screening biomarkers or identifying molecular targets related to intrahepatic cholangiocarcinoma.

8. A method for constructing a mouse intrahepatic cholangiocarcinoma model, characterized in that: The construction method comprises any one of the following (a)-(b): (a) injecting the mouse intrahepatic cholangiocarcinoma cell line according to any one of claims 1 to 5 subcutaneously into mice and culturing for 1 to 2 weeks until a tumor is felt under the mouse skin; (b) injecting the mouse intrahepatic cholangiocarcinoma cell line according to any one of claims 1 to 5 subcutaneously into mice, and performing orthotopic liver transplantation using the formed tumor mass to obtain an orthotopic tumor model.

Images