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CN-122004172-A - Construction method and application of multiple myeloma extramedullary lesion mouse model

CN122004172ACN 122004172 ACN122004172 ACN 122004172ACN-122004172-A

Abstract

The invention belongs to the technical field of bioengineering, and relates to a construction method and application of a multiple myeloma extramedullary lesion mouse model. The preparation method comprises the steps of constructing a Vk MYC myeloma mouse by intravenous injection of Vk12653 tumor cell suspension in the tail of a C57BL/6J mouse, feeding the Vk MYC myeloma mouse to obtain tumor infiltrated spleen cells, knocking out fibrinogen alpha chains from the whole body of the C57BL/6J mouse, transplanting the spleen cells, and continuing to culture to obtain a multiple myeloma extramedullary disease small mouse model. The animal transplanting model provided by the invention can be used for effectively screening the medicines for the extramedullary transfer of the multiple myeloma, can test multiple medicines and combined medicines, can be used for more comprehensively and deeply researching the in-vivo mechanism of the extramedullary transfer of the multiple myeloma, and provides basic support for developing new specific medicines.

Inventors

  • LIU ZHIQIANG
  • XIE YING
  • LI WENJING
  • WANG JINGJING
  • GUO JING

Assignees

  • 山东第一医科大学附属肿瘤医院(山东省肿瘤防治研究院、山东省肿瘤医院)

Dates

Publication Date
20260512
Application Date
20260414

Claims (6)

  1. 1. A method for constructing a multiple myeloma extramedullary lesion mouse model, which is characterized by comprising the following steps: (1) Vk×myc myeloma mice were constructed by intravenous injection of Vk12653 tumor cell suspension at the mouse tail of C57BL/6J mice; (2) After raising vk×myc myeloma mice, dissecting spleen, and obtaining tumor-infiltrated spleen cells by grinding; (3) Knocking out fibrinogen alpha chain from a whole body of a C57BL/6J mouse, performing gene editing to obtain Fga-KO mouse, and transplanting the spleen cells into a bone marrow cavity in a Fga-KO mouse, wherein the sequence of the fibrinogen alpha chain is shown as SEQ ID NO. 1, and the knocking-out target sequence is shown as any sequence of SEQ ID NO. 2 or SEQ ID NO. 3; (4) And continuing to culture until the mice have multiple organ tumor metastasis phenotype, thereby obtaining the multiple myeloma extramedullary disease small mouse model.
  2. 2. The method of claim 1, wherein the concentration of Vk12653 tumor cell suspension in step (1) is 1-2 x 10 7 /mL and the injection amount is 95-105 μl.
  3. 3. The method according to claim 1, wherein the feeding time of vk×myc myeloma mice in step (2) is 3 to 5 weeks, and the feeding condition is confirmed by observing splenomegaly and detecting the expression level of M protein in peripheral blood and the proportion of B220 - /CD138 + cells.
  4. 4. The method according to claim 1, wherein the spleen cell transplantation method in the step (3) is a single-sided bone marrow cavity injection method, the concentration of the spleen cells is 1-2×10 8 /mL, and the injection amount is 9-11 μl.
  5. 5. The method of claim 1, wherein the multiple myeloma extramedullary lesion mice in step (4) exhibit multiple organ tumor metastasis phenotypes including splenomegaly, expression of M protein in bone marrow blood and peripheral blood serum, tumor infiltration of liver and spleen, tumor metastasis in brain, and elevated cell proportions of B220 - /CD138 + in peripheral blood, spleen, liver and bone marrow cells.
  6. 6. Use of the mouse model prepared by the method of claims 1-5 for screening drugs for extramedullary lesions.

Description

Construction method and application of multiple myeloma extramedullary lesion mouse model Technical Field The invention belongs to the technical field of bioengineering, and relates to a construction method and application of a multiple myeloma extramedullary lesion mouse model. Background Multiple Myeloma (MM) is a hematological malignancy characterized by malignant proliferation of clonal plasma cells in the bone marrow. Although the use of novel drugs such as proteasome inhibitors, immunomodulators and monoclonal antibodies significantly improve patient survival, most patients eventually relapse and progress to incurable advanced disease. Among them, the extramedullary lesion (EMD) is an aggressive expression form of MM, and indicates that myeloma cells break through the bone marrow barrier, infiltrate into extramedullary tissues such as spleen, liver, lymph node, central nervous system, etc., or form soft tissue plasmacytoma. The occurrence of EMD is closely related to the poor prognosis, for which the traditional treatment regimen has limited efficacy, and therefore, it is urgent to understand the pathogenesis of EMD and develop effective treatment strategies. Preclinical studies are highly dependent on animal models that can accurately mimic the pathological features of human disease. Currently, the conventional MM mouse models mainly comprise (1) an immunodeficiency mouse transplantation model, such as transplanting human MM cell lines or tumor cells derived from patients into immunodeficiency mice such as NOD/SCID, NSG and the like, which are main tools for researching the growth of human MM cells, but are mainly limited in that tumors are usually limited to bone marrow cavity growth, difficult to spontaneously form extensive extramedullary metastasis, and lack of a complete host immune system, and cannot be used for researching immune-mediated anti-tumor responses or immunotherapy. (2) Transgenic mice, such as vk×myc transgenic mice, can spontaneously produce monoclonal gammaglobulinosis with unknown meaning and progress to MM, and part of mice can have extramedullary lesions in later stages. However, the disease has randomness, long incubation period and different disease degrees, which is unfavorable for large-scale and synchronous drug screening experiments. (3) In situ transplantation model murine MM cell lines such as 5TGM1, vk MYC derived cells were transplanted into immunocompetent host mice (C57 BL/KaLwRij, C57BL/6J, respectively). Such models retain the immune system and can be used to study tumor interactions with the microenvironment, but their extramedullary metastatic capacity is often limited and the site and incidence of metastasis is unstable. The existing MM mouse model has obvious defects in the aspect of simulating EMD, namely, stable and extensive extramedullary metastasis is difficult to spontaneously form, or the support of an immune system is lacked, or the modeling period is too long and uncontrollable factors are more. Therefore, the development of a mouse model capable of rapidly, stably and repeatedly inducing the multi-bone marrow rumen multi-organ extramedullary metastasis in an immune sound host has great significance for revealing the pathological mechanism of EMD and accelerating the development of anti-metastatic drugs. Disclosure of Invention The invention provides a construction method and application of a multiple myeloma extramedullary disease mouse model aiming at the technical problem of simulating EMD of a traditional MM mouse model. In order to achieve the above purpose, the invention is realized by adopting the following technical scheme: A construction method of a multiple myeloma extramedullary lesion mouse model comprises the following steps: (1) Vk×myc myeloma mice were constructed by intravenous injection of Vk12653 tumor cell suspension at the mouse tail of C57BL/6J mice; (2) After raising vk×myc myeloma mice, dissecting spleen, and obtaining tumor-infiltrated spleen cells by grinding; (3) Knocking out fibrinogen alpha chain from a C57BL/6J mouse whole body, performing gene editing to obtain Fga-KO mouse, and transplanting the spleen cells into Fga-KO mouse, wherein the fibrinogen alpha chain sequence is shown as SEQ ID NO. 1, and the knocking-out target sequence is shown as any sequence of SEQ ID NO. 2 or SEQ ID NO. 3; (4) And continuing to culture until the mice have multiple organ tumor metastasis phenotype, thereby obtaining the multiple myeloma extramedullary disease small mouse model. Preferably, the concentration of the Vk12653 tumor cell suspension in the step (1) is 1-2×10 7/mL, and the injection amount is 95-105. Mu.L. The specific steps include thawing vk×myc (Vk 12653) cells frozen in liquid nitrogen in water bath, recovering cells, washing with 1×pbs, centrifuging at normal temperature, adding 1×pbs, counting cells, preparing cell suspension with 1×pbs, preparing 6-week-old mice with the same sex of C57BL/6J, injecting with sterile syring