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Construction method and application of retinal neovascular disease model

A new blood vessel and disease model technology, applied in the biological field, can solve the problems of lack of animal models and achieve the effect of broad application prospects

Active Publication Date: 2019-07-12
SICHUAN PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is a lack of relevant animal models that can be used for the study of retinal neovascular diseases

Method used

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  • Construction method and application of retinal neovascular disease model
  • Construction method and application of retinal neovascular disease model
  • Construction method and application of retinal neovascular disease model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] In this example, mice are used as target animals, and the method for constructing the retinal neovascular disease model provided by the present invention is described. The route of knocking out the TMEM30A gene is as follows: figure 2 As shown in A, the specific operation is as follows:

[0058] 1. Refer to the method of the Chinese patent application No. 2017103803265, named the construction method and application of the mouse model of pancreatic beta cell conditional knockout of Tmem30a gene, to obtain the conditional knockout of Tmem30a gene (exon No. 3) homozygous small mouse;

[0059] 2. The Tmem30a gene conditional knockout homozygous mice were mated with PDGFβ-Cre ER transgenic mice to obtain Tmem30a gene conditional knockout mice for vascular endothelial cells.

[0060] When PDGFβ-Cre ER transgenic mice were crossed with Tmem30a knockout homozygous mice, half of the offspring carried both PDGFβ-Cre ER and Tmem30a conditional knockout. This animal was mated wi...

Embodiment 2

[0062] 1 RT-PCR method was used to detect the expression of TMEM30A gene in three kinds of vascular endothelial cells.

[0063] Methods: The total RNA of three kinds of vascular endothelial cells human retinal vascular endothelial cells (HREC), human umbilical vein vascular endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC) were extracted respectively, and then cDNA synthesis kit (Invitrogen, Waltham, MA, USA) to synthesize cDNA. Primers were designed according to the cDNA sequence of TMEM30A:

[0064] TMEM30A-H-F: 5'-TCGGTCTCATCTTCATTCCC-3';

[0065] TMEM30A-H-R: 5'-GGAAGGCTCTGTTCCGGTAT-3'.

[0066] Using the extracted cDNA as a template, RT-PCR was performed. After amplification, electrophoresis was carried out, and the results were shown in figure 1 .

[0067] see results figure 1 A, it can be seen that the expression of TMEM30A gene in human retinal vascular endothelial cells (HREC), human umbilical vein vascular endothelial cells (HUVEC) and...

Embodiment 3

[0081] Identification of the genotype of the mouse that specifically knocked out the Tmem30a gene in the vascular endothelial cells in Example 1.

[0082] method:

[0083] (1) Cut a little tissue sample from the mouse tail and place it in a clean 1.5ml centrifuge tube;

[0084] (2) Add 100μl of lysate (40mM NaOH, 0.2mM EDTA solution) to the centrifuge tube, and heat in a metal bath at 100°C for 1h; (3) Take out the centrifuge tube, cool to room temperature, and add 100μl of neutralizing solution (40mM Tris-HCl, pH 5.5), centrifuged at 10000 g for 2 min, and the supernatant was taken for mouse genotype identification.

[0085] (4) PCR amplification: configure the PCR reaction system according to the following system

[0086]

[0087] The primer sequences are as follows:

[0088] Tmem30a-Forward sequence: 5'-ATTCCCCTTCAAGATAGCTAC-3';

[0089] Tmem30a-Reverse sequence: 5'-AATGATCAACTGTAATTCCCC-3';

[0090] PDGFβ-Forward sequence: 5'-GCCGCCGGGATCACTCTCG-3';

[0091] PDGFβ...

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Abstract

The invention discloses a construction method and application of a retinal neovascularization disease model, and relates to the field of biotechnology. The construction method knocks out the Tmem30a gene sequence in the genome of the target animal vascular endothelial cells, so that the target animal exhibits the typical retinal neovascularization disease characteristics. The target animal can beused as a model of retinal neovascularization disease, which can help to clarify the pathogenesis and mechanism of retinal neovascularization disease, and provide a new target for the treatment or prevention of retinal neovascularization disease.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a retinal neovascular disease model and its application. Background technique [0002] Retinal neovascularization (RNV)-related eye diseases seriously endanger human vision health, such diseases mainly include retinopathy of prematurity (ROP), proliferative diabetic retinopathy (proliferative diabetic retinopathy, PDR), Retinal vein occlusion (central retinal vein occlusion, RVO), etc. Pathological retinal neovascularization is the main pathological feature of these diseases. Globally, the incidence of retinal neovascular diseases is high, the treatment effect of the late stage of the disease is poor, and the blindness rate is high. [0003] At present, laser photocoagulation and intraocular injection of anti-vascular endothelial growth factor (VEGF) drugs are the main means of clinical treatment of RNV. However, a large number of studies have shown that ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52A01K67/027
CPCA01K67/0276A01K2217/075A01K2267/03C12N9/00
Inventor 朱献军张琳张珊珊刘文静杨业明孙宽祥
Owner SICHUAN PROVINCIAL PEOPLES HOSPITAL
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