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A kind of establishment method of C5AR gene knockout mouse cgvhd model

A gene knockout mouse, a technique for establishing a method, which is applied in the field of establishing a mouse cGVHD model

Active Publication Date: 2019-11-08
GUANGDONG GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no research has explored the pathogenic mechanism of the C5a-C5aR complement pathway in the cGVHD disease process.

Method used

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  • A kind of establishment method of C5AR gene knockout mouse cgvhd model
  • A kind of establishment method of C5AR gene knockout mouse cgvhd model
  • A kind of establishment method of C5AR gene knockout mouse cgvhd model

Examples

Experimental program
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Effect test

Embodiment 1

[0021] This example is an example of establishing a chronic graft-versus-host disease model after allogeneic bone marrow transplantation in C5aR knockout mice of the present invention, and the operation is as follows:

[0022] 1. Rat donor preparation

[0023] Select B10.D2 (Hc1 H2d H2-T18c) males of Specific pathogen Free (SPF) grade and raise them to 16 weeks old as donor mice.

[0024] 2. Preparation for receiving rats

[0025] SPF grade C5aR- / - (C.129S4(B6)-C5ar1tm1Cge / J), female, 16 weeks old, served as recipient mice. The rats were randomly divided into 4 groups, 5 in each group; among them,

[0026] A. Normal control group: synchronous feeding, no intervention;

[0027] B. Radiation control group: TBI+RPMI 1640, that is, the mice in the group were treated with total body irradiation (TBI), and injected with PRMI1640 culture solution through the tail vein;

[0028] C. Transplant control group: TBI+4×10^7 BMC, that is, the mice in the group were treated with total bod...

Embodiment 2

[0041] This example is an example of detection of allogeneic bone marrow implantation in mice.

[0042] Take the recipient mice +60 days after transplantation for detection, and proceed according to the following steps:

[0043] 1. Colchicine treatment: 0.1% colchicine (2-4 μg / g) was intraperitoneally injected into mice 3 to 4 hours before the experiment.

[0044] 2. Take the femur: During the experiment, the mice were quickly killed by cervical dislocation, and the skin and muscles of the hind limbs were immediately cut off with scissors.

[0045] 3. Take bone marrow cells: draw 6ml of normal saline with a syringe, insert the needle of the syringe into one end of the bone marrow cavity, flush out the bone marrow cells with normal saline into a centrifuge tube, and rinse repeatedly until the bone cavity turns white. After balancing the collected bone marrow cell solution, put it into a centrifuge, centrifuge at 1000r / min for 10min, and suck off the supernatant.

[0046] 4. H...

Embodiment 3

[0058] This example is an example of clinical and pathological examination of mice in the cGVHD group.

[0059] 1. Carry out the clinical score of chronic GVHD to group D mice, and its scoring standard is shown in Table 1:

[0060] Table 1 Clinical scoring criteria for chronic GVHD

[0061]

[0062] Note: (a) 0.3 points are scored for each peeling or crusting of ears, tails, and soles. The lowest score for skin clinical manifestations is 0 points, and the highest score is 3.9 points. (b) GVHD was considered to have occurred if the skin score exceeded 0.6; GVHD was considered to have occurred even if the symptoms subsided, and the mice died naturally or caused by human factors; the asymptomatic death of mice was regarded as no GVHD.

[0063] Survival observed at least +60d. The dying mice were sacrificed, and the survival time was recorded until the day after the sacrifice.

[0064] figure 1 Schematic diagram of the clinical scores of mice in group D. (Scores at each ti...

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Abstract

The invention discloses a method for establishing a cGVHD model of a C5aR gene knockout mouse, which is realized through the following steps: (1) using an aged male mouse as a donor mouse; (2) using an aged female mouse as a recipient mouse; (3) After the recipient mice received whole body irradiation pretreatment, the bone marrow cell BMC suspension and splenocyte SpC suspension obtained from the donor mice were injected into the recipient mice. The C5aR gene knockout mouse cGVHD model established by the present invention involves many and typical diseased organs; the model tissue lacks the C5aR gene, which is a key tool for verifying the correlation between the C5a-C5aR complement pathway and cGVHD disease; model building With high efficiency and good repeatability, it provides a new experimental platform for the research on the prevention and treatment of human cGVHD.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for establishing a mouse cGVHD model. Background technique [0002] Allogeneic hematopoietic stem cell transplantation has become an important treatment strategy for various hematological malignancies, but the main complication after transplantation—chronic graft-versus-host disease has become the main reason restricting its efficacy, which not only seriously affects the quality of life of patients, but also causes death. The main reason is that it is urgent to explore the pathogenesis and prevention strategies of chronic graft-versus-host disease. C5a is the main potent mediator in the activation products of the complement system, which mediates important inflammatory responses of the body, and can also cause severe immune inflammatory damage while enhancing the body's defense function. Studies have found that C5a plays an important role in the occurrence of diseases such as...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071A01K67/02
CPCA01K67/02
Inventor 杜欣赖沛龙翁建宇王玉连陈晓梅黄欣
Owner GUANGDONG GENERAL HOSPITAL
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