Method for establishing xenogeneic graft-versus-host disease model for NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice

A graft-versus-host and method-establishing technology is applied in the establishment of NOD/SCID mouse xenograft-versus-host disease models, which can solve the problems of increasing implanted cell niches, limiting the wide application of the model, and achieving the modeling process. Simple, efficient, and repeatable results

Inactive Publication Date: 2012-07-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Sublethal doses of irradiation can suppress the innate immunity of the host, while increasing the niches required for implantation of cells, so sublethal doses of irradiation are the key to the successful establishment of humanized NOD / SCID mouse GVHD models, but many domestic studies The lack of radiation-related instruments and equipment in the center limits the wide application of this model

Method used

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  • Method for establishing xenogeneic graft-versus-host disease model for NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice
  • Method for establishing xenogeneic graft-versus-host disease model for NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice
  • Method for establishing xenogeneic graft-versus-host disease model for NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1 Preparation of activated human PBMCs

[0014] 1. Add 5ml of lymphocyte separation medium into a 15ml centrifuge tube;

[0015] 2. Take 10 ml of heparin anticoagulated human peripheral venous blood and mix it with the same amount of Hank's solution, and slowly superimpose it on the layered liquid surface along the tube wall with a dropper, pay attention to keep a clear interface, and centrifuge horizontally at 600g×20 minutes;

[0016] 3. After centrifugation, the tube is divided into three layers, the upper layer is plasma and Hank's solution, the lower layer is mainly red blood cells and granulocytes, the middle layer is lymphocyte separation fluid, and there is a white cloud layer mainly composed of mononuclear cells at the interface of the upper and middle layers narrow band;

[0017] 4. Use a dropper to insert into the cloud layer, absorb mononuclear cells, put them into another 15ml centrifuge tube, add 5 times the volume of Hank's solution, centrifuge a...

Embodiment 2

[0019] Example 2 Modeling of NOD / SCID mice

[0020] 1. Pretreat NOD / SCID mice on -3 to -1 days, the pretreatment program is: intraperitoneal injection on -3 days and -2 days

[0021] Inject (or perfuse) cyclophosphamide 50mg / kg / day, intraperitoneally inject anti-mouse CD122 monoclonal antibody 100μg / mouse on day -1;

[0022] 2. On the 0th day, each mouse in the experimental group was infused (or perfused) with activated human PBMCs prepared from the tail vein 1×10 7 A total of 0.2ml, each mouse in the control group was infused (or perfused) with 0.2ml of phosphate buffered saline from the tail vein;

[0023] 3. Regularly observe the mouse weight, diarrhea, skin GVHD manifestations (hair loss, erythema, etc.), activity, etc. every day.

Embodiment 3

[0024] Example 3 Implantation detection of mouse human leukocyte quantity and content

[0025] 1. Take 1ml of heparin anticoagulated mouse blood, or 1ml of splenocyte suspension (the total number of cells is 10 6 ), add ammonium chloride solution (NH4Cl: 8.99g / L, KHCO3: 1g / L, Na4-EDTA: 0.037g / L) 3ml lysed red blood cells, mix well, act at 37°C for 10 minutes, centrifuge at 300g for 5 minutes, discard clear;

[0026] 2. Resuspend the cells in 40 μl of 1× phosphate buffer, add 20 μl of anti-CD45 FITC monoclonal antibody, mix well, and incubate at 4°C in the dark for 30 minutes;

[0027] 3. Wash twice with 2 ml of 1× phosphate buffer solution, pour out the liquid at the mouth of the tube with filter paper after each wash;

[0028] 4. Add 500 μl of 1× phosphate buffer solution to resuspend the cells, and perform flow cytometry detection on the machine; use CELL-QUEST software to obtain 10,000 cells per tube, and use human CD45 positivity as the phenotype characteristic of human ...

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Abstract

The invention provides a method for establishing a xenogeneic graft-versus-host disease model for NOD / SCID (non-obese diabetic / severe combined immunodeficient) mice, which includes: preparing active human PBMCs (peripheral blood mononuclear cells), establishing a model of NOD / SCID mice, detecting number and content of mouse-to-human white blood cells in implantation, and establishing pathologicalmanifestations of a mouse GVHD (graft-versus-host disease) target organ. Application of cyclophosphamide with anti-mouse CD122 (IL-2R beta chain) monoclonal antibody to pretreatment is used to substitute the sublethal dose irradiation process to establish the humanized GVHD model for NOD / SCID mice. The model is capable of simulating characteristics of graft-versus-host diseases after allogeneic hematopoietic stem cell transplantation in clinical chemotherapeutic pretreatment, and is high in establishing efficiency and high in repeatability. The model establishment process is simple and easy. The shortage that domestic research centers lacking relevant irradiation equipment fail to carry out relevant researches is overcome, and an experimental platform for human immune system GVHD researchon live animals can be provided.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for establishing a NOD / SCID mouse xenograft-versus-host disease model. The establishment of a disease model that simulates the characteristics of graft-versus-host disease after clinical allogeneic hematopoietic stem cell transplantation can provide a research platform for the study of the mechanism and diagnosis and treatment of graft-versus-host disease. Background technique [0002] Graft-versus-host disease (GVHD) is the main complication and cause of death after allogeneic hematopoietic stem cell transplantation, and has become a key factor hindering the success of transplantation. In vitro, animal models and clinical studies to reduce or alleviate the occurrence and degree of GVHD are particularly urgent. At present, most GVHD animal models are established by transplantation between mice with different H-2 antigen incompatibility, which are different from GVHD after hum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027G01N21/84
Inventor 黄河胡永仙顾嫣珺谭亚敏
Owner ZHEJIANG UNIV
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