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A zebrafish brain trauma model and its preparation method and application

A zebrafish and brain trauma technology, applied in the field of zebrafish brain trauma model and its preparation, can solve problems such as non-traumatic brain trauma model, achieve good prospects and value, and promote the effect of repair

Active Publication Date: 2021-08-27
AFFILIATED HOSPITAL OF GUANGDONG MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] But currently there is no suitable model of traumatic brain injury for medical research
Although a series of craniocerebral trauma models have been developed using mice and other animals as research objects at home and abroad, there are relatively few studies on the establishment of brain trauma models using zebrafish juveniles as research objects. The brain injury model of fish larvae is of great significance for future research on drug screening and human brain injury repair mechanism

Method used

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  • A zebrafish brain trauma model and its preparation method and application
  • A zebrafish brain trauma model and its preparation method and application
  • A zebrafish brain trauma model and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction of Zebrafish Brain Injury Model

[0030] 1. Experimental method

[0031] 1. Zebrafish rearing and embryo reproduction

[0032] Commercially available enhanced green fluorescent protein-labeled macrophages (microglia distributed in the brain, hereinafter referred to as microglia) transgenic zebrafish (coro1α: EGFP zebrafish) were used as research objects; The Eisen zebrafish breeding system was used to feed brine shrimp twice a day, with 14 hours of light and 10 hours of darkness. The temperature is 28.5°C, the pH is 7.0-8.0, the dissolved oxygen value is 4.5-5.5 mg / l, and the conductivity is 550-650 μS / cm.

[0033] Coro1α: EGFP zebrafish were mated, male fish: female fish = 1: (1~2).

[0034] Embryos were collected and cultured in an embryo incubator at 28.5°C. Dead eggs were picked every day, embryo water was changed, and embryo water containing 0.003% (g / ml) PTU was changed at 1 dpf (days post fertilization) to inhibit pigment production. At...

Embodiment 2

[0051] Example 2 In situ hybridization method to detect the changes of inflammatory factors at different time points

[0052]1. Experimental method

[0053] Taking the animal model constructed in Example 1 as the object (trauma group), with untreated zebrafish of the same age as the control (non-trauma group), the embryos of 1hpl and 24hpl after the trauma were collected for the detection of IL1-β and IL-6 inflammatory factors. in situ hybridization. 30 embryos of 1 hpl (hourspost lesion, 1 hour after wounding) were collected from the non-trauma group (untreated zebrafish group of the same age) and the wound group (animal model constructed in Example 1) respectively, and fixed overnight at 4° C. with 4% PFA. The temporal and spatial changes of the transcriptional levels of inflammatory factors IL1-β and IL-6 were detected by in situ hybridization. Probe concentration: IL1-β: 2.4ng / μl, IL-6: 3.0ng / μl.

[0054] 2. Experimental results

[0055] see results figure 2 , IL1-β ...

Embodiment 3

[0056] Example 3 Real-time tracking of changes in microglia at the trauma site after intervention

[0057] 1. Experimental method

[0058] Taking the animal model constructed in Example 1 as the object (injury group) and untreated zebrafish of the same age as the control (non-injury group), confocal tracking of the aggregation of microglia at the wound site at 1hpl and 24hpl.

[0059] Video: Using FV3000 Olympus Confocal, the setting parameters are: Format: 1024×1024, Nr.ofsteps: 15, Z-step size: 2.92μm, Z-Volume: 40.804μm, Time Interval: 1min, Duration: 1h , stacks: 60.

[0060] Photographing: Use confocal to collect 1hpl and 24hpl images of non-trauma group and trauma group, setting parameters: Format: 1024×1024, Nr.of steps: 15, Z-step size: 2.92μm, Z-Volume: 40.804μm.

[0061] 2. Experimental results

[0062] Such as image 3 As shown, there was no microglial cell aggregation within 160 μm around the trauma site in the non-trauma group, but there was obvious microglial...

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Abstract

The invention discloses a zebrafish brain trauma model, its preparation method and application. A method for preparing a zebrafish brain injury model. For embryos at the 3dpf stage, an empty needle is poked in the hindbrain of the zebrafish, and the microglial cells are labeled with fluorescent proteins. In the invention of this method, after the intervention of clinical first-line anti-inflammatory drugs, more microglial cells can be activated to migrate and gather to the wound site, thereby promoting the repair of the post-traumatic brain. The microglial cell transgenic zebrafish brain trauma model can It is used for rapid drug screening, which provides an economical, convenient and reliable research model for enterprise drug development and clinical efficacy evaluation of anti-inflammatory drugs in diseases such as brain trauma and encephalitis, and has good prospects and value.

Description

technical field [0001] The invention relates to the technical field of animal model construction, and more specifically, to a zebrafish brain trauma model and its preparation method and application. Background technique [0002] Zebrafish (Daniorerio) is an internationally recognized experimental fish. It has many characteristics such as small size, easy breeding, in vitro fertilization, fast development, strong reproductive ability, transparent embryo, short sexual maturation cycle, and clear genetic background. It has become a life in the era of functional genomes. One of the important model animals in scientific research, and many characteristics such as high-throughput screening for therapeutic drugs, make it one of the important model vertebrates in life science research in the post-genome era. [0003] Traumatic brain injury (TBI) is the leading cause of death and disability worldwide in today's life, and it is also a major unresolved public health issue worldwide. Me...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01K61/10
CPCA01K2267/0356A01K61/10Y02A40/81
Inventor 张晶晶甘达青伍水龙李炎玉
Owner AFFILIATED HOSPITAL OF GUANGDONG MEDICAL UNIV
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