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Construction method of retina bipolar cytopathy model and application thereof

A bipolar cell and construction method technology, applied in the field of medical engineering, can solve problems such as blindness, obstacles to clinical diagnosis and treatment of diseases, affecting the visual transmission function of bipolar cells, etc., and achieve the effect of broad application prospects.

Active Publication Date: 2020-10-16
SICHUAN PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These lesions of bipolar cells will also affect the visual transmission function of bipolar cells, leading to the aggravation of related retinal diseases and even blindness
[0004] However, the role of bipolar cell lesions in the occurrence and development of related retinal diseases is still unclear, and the specific molecular mechanism of the disease phenotype caused by bipolar cell lesions is still unclear, which brings new challenges to the clinical diagnosis and treatment of such diseases. Therefore, it is necessary to conduct in-depth research on the detailed pathogenic mechanism of bipolar cytopathy
[0005] Currently, there is a lack of corresponding bipolar cytopathic models

Method used

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  • Construction method of retina bipolar cytopathy model and application thereof
  • Construction method of retina bipolar cytopathy model and application thereof
  • Construction method of retina bipolar cytopathy model and application thereof

Examples

Experimental program
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Embodiment 1

[0043] This embodiment provides a method for constructing a retinal bipolar cell lesion model. In this example, mouse is used as the target animal, and the method for constructing the retinal bipolar cell lesion model provided by the present invention is described, and the route of Yap1 gene knockout is referred to figure 1 As shown, the specific operation is as follows:

[0044] 1) Obtain the first mouse with knockout of Yap1 gene, the knockout mouse has loxP sites arranged in the same direction at both ends of exon 1-2 of Yap1 gene;

[0045] 2) mating and breeding the first knockout mice obtained in step 1) with wild-type mice, and screening out the heterozygous mice for Yap1 gene knockout in the offspring;

[0046] 3) mating and breeding the Yap1 gene conditional knockout heterozygous mice obtained in step 2) to obtain Yap1 gene conditional knockout homozygous mice;

[0047] 4) The Yap1 gene conditional knockout homozygous animal obtained in step 3) is mated with the Chx1...

Embodiment 2

[0070] Immunohistochemical (IHC) assay was used to analyze the gene knockout efficiency in the retina of Chx10-Cre knockout mice.

[0071] Immunostaining of retinal frozen sections: Take 3-month-old retinal bipolar cell-specific knockout Yap1 mice constructed in Example 1, kill them by neck dislocation, quickly remove eyeballs, put them in 4% PFA, and fix them on ice for 15 minutes Finally, an incision was made on the cornea, and the fixation on ice continued. After 2 hours, the eyeballs were rinsed with PBS buffer for 3 times, and then dehydrated in 30% sucrose solution for 2 hours. Then, the cornea and crystals were cut off under a dissecting microscope, embedded in OCT and quickly placed in a -80°C refrigerator for freezing. After about 10 minutes, the OCT-embedded eyeballs were taken out, placed in a cryostat at -25°C for about 30 minutes, and sliced. The slice thickness was 12 μm.

[0072] After slicing, select a high-quality slice and place it in a 37°C oven for 30 min...

Embodiment 3

[0087] Carry out ERG visual acuity detection to the Yap1 gene knockout mouse of embodiment 1 of 12 months old:

[0088] 1) Dark-adapted animals should be dark-adapted all night, and the environment should be absolutely dark;

[0089] 2) Anesthesia the next day: weighing, intraperitoneal injection; deep anesthesia is appropriate;

[0090] 3) Animal immobilization and mydriasis: After the anesthesia is completed, fix the mouse with adhesive tape in front of the animal test platform under dark red light illumination: it is necessary to ensure that the mouse is "lying", that is, relative to the stimulation port of the flash stimulator, The eyes are at the same height, fully exposed, and mydriatics are added.

[0091] 4) Electrode installation: After preheating the electroretinograph (Espion Visual Electrophysiology System, Diagnosys LLC, Lit-tleton, MA, USA), apply conductive paste to the electrode, clamp the tail of the mouse, and insert it into the "ground" interface of the ampli...

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Abstract

The invention discloses a construction method and application of a retina bipolar cytopathy model, and relates to the technical field of medical engineering. Knocking out Yap1 gene, i.e., silencing its expression, in retinal bipolar cells of a target animal can result in the target animal exhibiting characteristics associated with a retinal bipolar cytopathic disease, such as bipolar cell death, primarily manifested by bipolar cell damage, degeneration, progressive thinning of retinal inner nuclear layer, and corresponding pathological changes in retinal outer nuclear layer and other associated cellular layers. Therefore, the target animal knocking out the Yap1 gene can be used as a retinal bipolar cytopathic model, the model can be used for researching pathogenetic process and pathogenesis of the retinal bipolar cytopathic diseases, and provide a basis for in-depth understanding of such diseases.

Description

technical field [0001] The invention relates to the technical field of medical engineering, in particular to a method for constructing a retinal bipolar cell lesion model and its application. Background technique [0002] Retinal bipolar cells (Bipolar cells) are glutamate interneurons in the vertebrate retina, which receive signal input from photoreceptor cells and transmit it to amacrine cells and ganglion cells after integration. The process is in a key position like a transfer station. [0003] It has been found that the occurrence and development of various retinal diseases are accompanied by bipolar cell lesions. Retinitis pigmentosa (RP) is a progressive, hereditary blinding retinal disease that causes vision loss, usually caused by abnormal retinal photoreceptors, and the clinical manifestations are chronic progressive visual field loss, night blindness, and pigmented retina Lesions and electroretinogram abnormalities can eventually lead to blindness. Studies on R...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N15/12A01K67/027A61K49/00
CPCA01K67/0276A01K2217/075A01K2227/105A01K2267/03A61K49/0008C07K14/4702C07K14/82C12N15/8509C12N15/907C12N2800/107C12N2800/30
Inventor 周波朱献军杨业明王璐瑶李潇田万里
Owner SICHUAN PROVINCIAL PEOPLES HOSPITAL
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