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Specific thymus lymphocyte injury and regeneration model and construction method thereof

A technology of lymphocytes and construction methods, applied in the field of specific thymus lymphocyte injury and regeneration models and their construction, can solve the problems of animal models lacking T lymphocyte regeneration and immune system recovery thymus injury models

Pending Publication Date: 2022-07-08
润康生物医药(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention intends to provide a method for constructing a specific thymus lymphocyte injury and regeneration model to solve the technical problem in the prior art that there is no thymus injury model animal model for T lymphocyte regeneration and immune system recovery research

Method used

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  • Specific thymus lymphocyte injury and regeneration model and construction method thereof
  • Specific thymus lymphocyte injury and regeneration model and construction method thereof
  • Specific thymus lymphocyte injury and regeneration model and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Construction of transgenic zebrafish lines

[0037] 1. Zebrafish Tg(coro1a:Dendra2-NTR) strain:

[0038] The coro1a gene is a marker of leukocytes, and T lymphocytes are a type of leukocytes. Mature T lymphocytes develop in the thymus and are the main lymphocytes present in the thymus. This transgenic line uses the promoter of the coro1a gene to control the expression of the Dendra2-NTR fusion protein gene, where Dendra2 is a fluorescent protein gene, and NTR is a nitroreductase gene. NTR can make non-toxic metronidazole reduced by NTR As a cytotoxic metabolite, it can specifically induce apoptosis in NTR-expressing cells controlled by the coro1a gene promoter. The construction method of this strain is as follows:

[0039] (1) Amplify the promoter of the coro1a gene (the nucleotide sequence is shown in SEQ ID NO. 1) and the Dendra2-NTR fusion protein gene (the nucleotide sequence is shown in SEQ ID NO. 2) by conventional PCR.

[0040] Among them, for the p...

Embodiment 2

[0064] Example 2: Construction of thymic lymphocyte injury and regeneration animal model based on Tg(coro1a:Dendra2-NTR) transgenic line

[0065] Through the created Tg(coro1a:Dendra2-NTR) zebrafish transgenic line, based on the principle of nitroreductase / metronidazole system-mediated lymphocyte elimination, an animal model of thymic lymphocyte injury was constructed through the following steps:

[0066] This protocol uses a Tg(coro1a:Dendra2-NTR) zebrafish transgenic line. The promoter of the coro1a gene drives the expression of Dendra2-fused NTR protein in leukocytes (including lymphocytes). NTR, as a nitroreductase, can make non-toxic The metronidazole is reduced by NTR to a cytotoxic metabolite capable of specifically inducing coro1a + Cells undergo apoptosis. Tg(coro1a:Dendra2-NTR) zebras were immersed with a series of different concentrations of metronidazole (MTZ, M3761, Sigma-Aldrich) during zebrafish Tg(coro1a:Dendra2-NTR) development to day 5 post-fertilization (5d...

Embodiment 3

[0070] Example 3: Construction of thymic lymphocyte injury and regeneration animal model based on Tg (coro1a:Dendra2-NTR; rag2:DsRed) transgenic line

[0071] The establishment of an animal model for regeneration of thymic lymphocytes after injury is achieved through the following steps:

[0072] This protocol uses a Tg(coro1a:Dendra2-NTR; rag2:DsRed) zebrafish transgenic line, and 2 mM metronidide is used when the zebrafish Tg(coro1a:Dendra2-NTR; rag2:DsRed) develops to day 5 after fertilization (5dpf). Tg (coro1a:Dendra2-NTR; rag2:DsRed) zebrafish larvae were soaked in azole (the control group was DMSO-treated group), and the drug was withdrawn after 12 hours of treatment. The fish were placed in embryo culture medium (which added 0.003% PTU, P7629, Sigma-Aldrich) for 12 hours to recover, and continued to culture. Confocal live imaging was used to continuously observe the fluorescent signal of thymic lymphocytes in juvenile fish to observe whether the development of zebrafis...

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Abstract

The invention relates to the technical field of disease animal models, in particular to a specific thymus lymphocyte injury and regeneration model and a construction method thereof. The construction method of the specific thymus lymphocyte injury and regeneration model sequentially comprises the following steps: culturing fertilized eggs of transgenic zebrafish to obtain juvenile fish to be treated; the transgenic zebrafish is used for expressing nitroreductase, and the expression of the gene of the nitroreductase is regulated and controlled by a promoter of the coro1a gene; 2 mM metronidazole is used for treating juvenile fish to be treated, and juvenile fish treated by metronidazole is obtained; and cleaning the metronidazole treated juvenile fish to obtain the specific thymus lymphocyte injury and regeneration model. According to the scheme, the technical problem that a thymus injury model animal model for research on lymphocyte regeneration and immune system recovery is lacked in the prior art is solved, and conditions are created for research on immune system recovery conditions and related drug screening under acquired immune system injury.

Description

technical field [0001] The invention relates to the technical field of disease animal models, in particular to a specific thymic lymphocyte injury and regeneration model and a construction method thereof. Background technique [0002] T lymphocytes play an important role in the immune system, and the receptors they express can recognize a variety of antigens, maintain immune memory and self-tolerance, and play a key role in anti-infection, anti-tumor and immune regulation. As the lymphoid organ of the body, the thymus is an important source of T lymphocytes in the acquired immune system, and plays a central role in the human immune system. In recent years, cases of thymic organ damage, lymphopenia, and cellular immunodeficiency have increased dramatically in the presence of radiation exposure, anticancer drugs, bacterial or viral infection, and DNA damage. In addition, the thymus is easily affected by the secretion of hormones by adrenal glands, thyroid glands, gonads, pine...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N9/04C12N15/113A01K67/027A01K61/10
CPCC12N15/8509C12N9/0006A01K67/0278A01K61/10C12Y101/01284A01K2207/15A01K2207/20A01K2217/072A01K2227/40A01K2267/03Y02A40/81
Inventor 李礼赵方莹逯亚芳贺蒋勇崔念飞李智繁
Owner 润康生物医药(苏州)有限公司
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