Zebrafish model of thrombocytopenia

A thrombocytopenia, zebrafish technology, applied in the biological field, can solve the problem of lack of animal models of thrombocytopenia

Active Publication Date: 2017-02-15
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, the prior art lacks a model of thrombocytopenia during embryonic development and an animal model suitable for large-scale stable screening of drug candidates for thrombocytopenia

Method used

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  • Zebrafish model of thrombocytopenia
  • Zebrafish model of thrombocytopenia
  • Zebrafish model of thrombocytopenia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] The acquisition of the mutant zebrafish of embodiment 1.mpl gene sequence deletion

[0091] In this experiment, the zebrafish mpl gene transcript mpl-001ENSDART00000124917 was designed, and a sequence TTTGGGATGCACCT on exon 1 was designed as the TALEN spacer region, and the target recognition TALEN module plasmids on the left and right sides were synthesized and knocked out in a targeted manner to obtain A mutant mpl with 8bp deletion and 48bp insertion of Mpl protein terminated prematurely smu40 (see figure 1 A. figure 1 B).

[0092] mpl gene expression

[0093] In order to test the effectiveness of the mutation, mpl was used as a marker gene to mark platelets and platelet precursor cells. Detection of wild-type sibling fish and mpl at 3dpf using in situ hybridization smu40 The mRNA expression of mpl gene in mutants.

[0094] The result is as figure 1 As shown in D, signal points represent the mRNA expression of mpl gene. The results showed that the mutant mp...

Embodiment 2

[0095] Embodiment 2. The mutant zebrafish of embodiment 1 of the present invention can be used as an animal model of thrombocytopenia

[0096] platelet count

[0097] Take zebrafish embryos at 5dpf and transfer the mutant mpl smu40 Mating with transgenic zebrafish Tg(CD41:eGFP) 4 , to obtain mutants with Tg(CD41:eGFP) transgenic background. The same treatment was carried out for wild-type sibling fish. For mpl with Tg(CD41:eGFP) transgenic background smu40 Immunochemical staining was performed on the mutant and wild-type compatriot fish, and the brightly fluorescent cells in the platelets flowing in the blood were observed under a fluorescent microscope, and the signal point was CD41:eGFP high The number of signal points will represent the number of platelet cells.

[0098] The result is as figure 1 C, mpl smu40 The number of platelets in mutants was significantly reduced compared with wild-type sibling fish.

[0099] mRNA expression of multiple platelet-related ge...

Embodiment 3

[0103] Example 3. Using the mutant zebrafish of Example 1 of the present invention to construct a model of coagulation dysfunction caused by thrombocytopenia.

[0104] due to mutant mpl smu40 It shows thrombocytopenia, so this mutant can be used to study the effect of platelets on zebrafish hemostasis, and then construct an experimental model of zebrafish coagulation dysfunction 19 . First, use 3dpf zebrafish embryos, at least 10 in each group, respectively for the wild-type sibling fish group and the mutant mpl smu40 The zebrafish in the group were subjected to needle-puncture injury to the tail blood vessels, and the size of the wound blood clot after the blood vessel injury was observed under an optical microscope at 20 times, and the time from the beginning of the injury to the bleeding stop time of each zebrafish embryo was recorded.

[0105] figure 2 A shows the size of the blood clot in the wound after vascular injury, the left picture is the wild-type sibling fish,...

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Abstract

The invention provides a zebrafish model of thrombocytopenia. Specially, the invention provides an application of an mplsmu40 mutant zebrafish in preparing an animal model of the thrombocytopenia. With the application of the zebrafish Mpl mutant provided by the invention, studies on human thrombocytopenia as well as large-scale screening of alternative medicines for treating the thrombocytopenia can be implemented.

Description

technical field [0001] The present application relates to the field of biotechnology, in particular, to a zebrafish model for platelet development research, thrombocytopenia research and its large-scale screening of drug candidates. Background technique [0002] Platelets are an indispensable part of the blood system. Its main function is to coagulate and stop bleeding, and to repair damaged blood vessels. When there is a problem with platelets, the body can develop a disease state, such as platelet disease, which is caused by poor formation of blood clots and bleeding due to decreased number (thrombocytopenia) or decreased function (thrombocytopenia) of platelets. Thrombocytopenia is a disease in which the decrease of platelets in peripheral blood leads to bleeding in the skin, mucous membranes and internal organs. It is a common clinical group of diseases characterized by coagulation dysfunction and bleeding. [0003] In order to better study the development and function ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/89A01K67/027A61K49/00
CPCA01K61/00A01K67/0276A01K2207/00A01K2217/075A01K2227/40A01K2267/03A61K49/0008C07K14/715C12N15/8509C12N15/89C12N2800/106C12N2800/80C12N2800/90Y02A40/81
Inventor 张译月张文清林青张阳萍
Owner SOUTHERN MEDICAL UNIVERSITY
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