A kind of construction humanized mouse tumor model and its preparation method and application

A mouse tumor model, humanized technology, applied in the field of biomedicine, can solve the problems of the application of humanized mouse solid tumor models that have not been reported yet, achieve scientific research benefits and economic benefits, and make up for the singleness and model incompleteness , the effect of maintaining accessibility

Active Publication Date: 2021-12-03
THE EIGHTH AFFILIATED HOSPITAL SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the application of humanized murine solid tumor models of thymus and hematopoietic stem cell transplantation in tumor immunotherapy

Method used

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  • A kind of construction humanized mouse tumor model and its preparation method and application
  • A kind of construction humanized mouse tumor model and its preparation method and application
  • A kind of construction humanized mouse tumor model and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Construction of humanized mice with immune system

[0036] Immunodeficient mice aged 6-10 weeks were selected, and the mice were irradiated with sublethal doses (1.00-2.00Gy, 1Gy / min) of X-rays. After the mice were anesthetized, about 1m 3 Embryo-derived human thymus tissue (Advanced Bioscience Resources, USA) was implanted under the renal capsule, and after the mice woke up, 2-5×10 5 Syngeneic CD34+ human fetal liver hematopoietic stem cells (Advanced Bioscience Resources, USA) were fed for 8-10 weeks.

[0037] 2. Detection of human immune cells in peripheral blood

[0038] Take the venous blood of the mouse 8 weeks after operation in step 1, collect it in 5mM EDTA / PBS buffer, centrifuge to remove the supernatant, lyse the red blood cells with ACK buffer, use CD45, CD3, CD19, CD4, CD8, CD14, CD11c antibody was used for staining, and after incubation in the dark for 45 minutes, 500 microliters of flow loading buffer was used to resuspend the sample for flow analysi...

Embodiment 2

[0046] 1. Construction of humanized mouse tumor model and immunodeficiency mouse tumor model

[0047] Collect 5×10 5 H460 tumor cells were made into 100 microliters of single-cell PBS suspension, added 50 microliters of Matrigel, and transplanted into the mice in Example 1 and subcutaneously in immunodeficient mice to construct a humanized mouse tumor model and immunodeficiency mice. As for the mouse tumor model (as a control), the formation of tumors in the two model mice was observed. Three weeks after transplantation of tumor cells, the mice were sacrificed, and the tumors were photographed and analyzed.

[0048] 2. Detection of immune microenvironment in humanized mouse tumor model and immunodeficiency mouse tumor model

[0049] Add 500 microliters of TRIZOL reagent homogenate to the tumor tissue collected in the above step 1, place it on ice until it is completely lysed, centrifuge to remove the lysed part and cell debris, add 1 / 5 chloroform after taking the supernatant,...

Embodiment 3

[0059] Example 3 Construction of CD19-CAR-T cells

[0060] 1. Construction of tumor cells expressing antigen CD19

[0061] Get linearized 2.5ug human CD19 gene (its nucleotide sequence is shown in SEQ ID NO: 1) vector mixed with 5ulLipo2000 liposomes, transfected to H460 cells cultured in 6-well plate, 48 hours after transfection, The tumor cells were screened with 4ug / ml puromycin for one week, and the obtained monoclonal cells were identified by flow cytometry.

[0062] 2. Construction of CD19-CAR plasmid and T cells

[0063] Preparation method of CD19-CAR plasmid: The DNA sequence of CD19-CAR gene was biosynthesized by Guangzhou Aiji, the gene was amplified by polymerase chain reaction, and then the gene was connected to pLenti-EF1a-GFP by Gibson Assembly DNase ligation method In the lentiviral vector, replace the GFP gene sequence in the vector.

[0064] Take 20ug CD19-CAR lentiviral plasmid (the artificial chimeric protein sequence encoded by the CAR gene is shown in S...

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Abstract

The invention provides a humanized mouse tumor model, which is characterized in that the humanized mouse tumor model is obtained by transplanting human thymus tissue and CD34+ hematopoietic stem cells homologous to the thymus tissue. The present invention constructs a humanized mouse tumor model transplanted from thymus and hematopoietic stem cells for the first time. The humanized mouse tumor model has confirmed that it has a complete immunosuppressive microenvironment, and is suitable for the overall evaluation of solid tumor immunotherapy, and can be used to evaluate the feasibility and effectiveness of immunotherapy. The invention makes up for the singleness and incompleteness of the existing tumor immunotherapy evaluation methods, provides reference value for clinical research, and complements the key role of the human immune system in immunotherapy compared with traditional models.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a humanized mouse tumor model and its preparation method and application. Background technique [0002] Tumor refers to the new organism formed by the proliferation of local tissue cells under the action of various tumorigenic factors, especially in malignant tumors, it grows rapidly, has strong invasion ability, is prone to metastasis, and is easy to relapse after treatment, which can make patients The quality of life and life health have declined significantly. Although there are many medical methods for tumor treatment, such as surgery, chemotherapy, radiotherapy, etc., there is no effective method that can completely cure the tumor. In recent years, tumor immunotherapy has made great progress. Among them, artificial chimeric antigen receptor T cells (CAR-T cells) technology and immune checkpoint inhibitors are the main means. Tumor immunotherapy is in the bas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01K67/027
CPCA01K67/0271A01K67/0275A01K2217/30A01K2227/105A01K2267/03
Inventor 徐洋陈渠付雪梅
Owner THE EIGHTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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