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Constructed humanized mouse tumor model and preparation method and application thereof

A mouse tumor model and 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, maintain accessibility, and make up for singleness and the effect of model incompleteness

Active Publication Date: 2020-03-31
中山大学附属第八医院 +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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  • Constructed humanized mouse tumor model and preparation method and application thereof
  • Constructed humanized mouse tumor model and preparation method and application thereof
  • Constructed humanized mouse tumor model and preparation method and application thereof

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 2, 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. The humanized mouse tumor model is characterized by being obtained by transplanting human thymus tissues and CD34+ hematopoietic stem cells homologous with the thymus tissues. The humanized mouse tumor model transplanted by thymus and hematopoietic stem cells is constructed for the first time. The humanized mouse tumor model is determined to have a complete immunosuppression microenvironment, is suitable for overall evaluation of solid tumor immunotherapy, and can be applied to evaluation of feasibility and effectiveness of immunotherapy curative effects. The humanized mouse tumor model makes up for singleness and model incompleteness of existing tumor immunotherapy evaluation methods, and provides reference value for clinical research.Compared with traditional models, the humanized mouse tumor model supplements key effects of a human immune system in immunotherapy.

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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IPC IPC(8): A01K67/027
CPCA01K67/0271A01K67/0275A01K2217/30A01K2227/105A01K2267/03
Inventor 徐洋陈渠付雪梅
Owner 中山大学附属第八医院
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