Preparation method of retinal ganglion cells

A retinal ganglion and retinal cell technology, applied in the field of cells, can solve the problems of difficulty in in vitro culture, small number of cells, and low purity

Active Publication Date: 2021-01-22
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as one of the central neurons, primary retinal ganglion cells face many challenges in terms of extraction, purification and culture, such as low purity, small number of cells, and difficulties in in vitro culture.

Method used

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  • Preparation method of retinal ganglion cells
  • Preparation method of retinal ganglion cells
  • Preparation method of retinal ganglion cells

Examples

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preparation example Construction

[0041]The invention discloses a preparation method of retinal ganglion cells, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

[0042] RGCs are neurons located in the innermost ganglion cell layer of the retina and consist of multipolar ganglion cells. Dendrites are primarily associated with bipolar cells and can also ...

Embodiment 1

[0095] Example 1 Extracting the RGCs of 20-25 SD rat pups within 48-72h after birth

[0096] first day:

[0097] 1. Prepare Petri dishes for positive and negative screening (e.g. figure 1 )

[0098]1.1 Prepare two 50ml centrifuge tubes marked as "Negative Screening", and add 40ml of 50mM Tris-HCl (tris hydroxymethylaminomethane hydrochloride buffer) (pH 9.5) to each centrifuge tube, 120μL goat antibody Rabbit immunoglobulin (H+L) (Jackson Company 111-005-003), mix gently, set aside.

[0099] 1.2 Prepare four petri dishes for negative screening, marked as A1, A2, B1, and B2 respectively;

[0100] Add 20mL of antibody and Tris-HCl mixture to each 15cm petri dish, mix gently, seal with sterilized parafilm, and store in a 4-degree refrigerator overnight for later use.

[0101] 1.3 Take a 50ml centrifuge tube marked as "positive screening", add 20ml of 50mM Tris-HCl (pH 9.5), 60μl goat anti-mouse immunoglobulin IgM (H+L) (Jackson 115-005- 044), mix gently.

[0102] 1.4 Prepar...

Embodiment 2

[0145] Example 2 Immunofluorescence staining

[0146]After 24 hours of plating, the obtained cells were immunofluorescence stained with specific markers to determine the purity of the cells. After 24 hours of plating, the cell culture medium was discarded for the RGCs, rinsed three times with PBS (Solarbio P1020-500ml), and fixed with immunofixation solution (Beyotime P0098-100mL) at room temperature for 15 minutes. Immunostaining washing solution (Beyotime P0106), washing 3 times, 10 minutes each time. After rinsing, immunostaining blocking solution (Beyotime P0102) was used to block at room temperature for 1 h. This was followed by overnight incubation at 4°C with anti-β-tubulin III antibody (1:500, Abcam ab7751) or anti-BRN3A antibody (1:500, bs-3669R). Wash 3 times with immunostaining washing solution (Beyotime P0106), 5 minutes each time. Then incubate with the corresponding secondary antibody (donkey anti-mouse IgG H&L AlexaFluor488, diluted 1:1000, Abcam ab150105; do...

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Abstract

The invention relates to the field of cells, in particular to a preparation method of retinal ganglion cells (RGCs). According to the method, related methods for separating and purifying RGCs are improved, and multiple experiments prove that the number of RGCs obtained from an average single retinal tissue is obviously higher than that of RGCs obtained by an original two-step immune dissociation method, and the purity of the separated RGCs is also obviously higher than that of RGCs obtained by the original two-step immune dissociation method. Specific labeled antibodies beta-tubulin III and BRN3A are used for carrying out immunofluorescence staining on different batches of RGCs. Results show that the purity of the RGCs labeled by two antibodies has no statistical difference, and it is further verified that the improved two-step immune dissociation method can obtain the primary RGCs with relatively stable purity and yield. The research lays a cytological foundation for the large-scale deep exploration of the mechanism of visual impairment caused by RGCs necrosis and apoptosis.

Description

technical field [0001] The invention relates to the field of cells, in particular to a preparation method of retinal ganglion cells. Background technique [0002] It is estimated that the number of people with diabetes worldwide will increase to 592 million by 2035. Diabetic retinopathy (DR) is a complex complication of diabetes, characterized by progressive neurological dysfunction and retinal microvascular degeneration, often leading to vision loss and even blindness. However, a growing number of studies have shown that RGCs damage occurs early in retinopathy, possibly before visible retinal vasculopathy. Endoplasmic reticulum stress and mitochondrial oxidative stress further accelerated the apoptosis of RGCs in diabetic retinopathy. [0003] Glaucoma is an eye disease characterized by vision loss and visual field defects. Typical pathological features are changes in the optic nerve head and a decrease in RGCs. In addition to age, elevated intraocular pressure is consi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079
CPCC12N5/0621C12N2509/00
Inventor 马速佳党亚龙张珂朱豫王丽丽
Owner ZHENGZHOU UNIV
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