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Application of REG1A protein in preparation of drugs for treating and/or preventing retinal cell apoptosis

A retinal cell and protein technology, applied in the field of biomedicine, can solve problems such as chronic rejection, gene therapy mechanism cannot be fully clarified, patients' physical and psychological trauma, etc., and achieve the effect of small dosage and prevention of retinal degeneration

Active Publication Date: 2017-09-15
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are many treatment methods for retinal cell apoptosis diseases, such as retinal transplantation, but this method may cause chronic rejection, and at the same time cause great physical and psychological trauma to the patient
In addition, gene therapy is the use of vectors, chemical or physical methods to introduce normal genes into corresponding cells of eye tissue for expression, but the mechanism of gene therapy is not yet fully clear, and there are problems such as effective transfection of target genes and long-term sustained expression of functional genes. It also needs to be solved, therefore, gene therapy methods still need to be studied in depth before they can be applied clinically

Method used

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  • Application of REG1A protein in preparation of drugs for treating and/or preventing retinal cell apoptosis
  • Application of REG1A protein in preparation of drugs for treating and/or preventing retinal cell apoptosis
  • Application of REG1A protein in preparation of drugs for treating and/or preventing retinal cell apoptosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Expression of neurosecretory factor reg1a gene in mouse retina

[0039] Various parts of mice were taken as experimental materials, including pancreas, kidney, heart, skin, small intestine tissue, brain, testis, choroid, liver, spleen, lung, subfrontal gland, cornea, retina and lens. The mRNA of each material was extracted from the above materials using the kit method.

[0040] After the quality of the extracted mRNA is tested, the mRNA with high integrity is subjected to semi-quantitative PCR detection. Specifically, a reverse transcription kit was used to reverse transcribe mRNA into cDNA, perform semi-quantitative PCR reaction on REG1A gene, and use GAPDH as an internal reference gene. The primer sequences of EG1A gene and mGAPDH are shown in Table 1, the PCR reaction system is shown in Table 2, and the PCR program is shown in Table 1. table 3.

[0041] Table 1 Primer sequences of REG1A gene and GAPDH

[0042]

[0043]

[0044] Table 2 PCR amplification syst...

Embodiment 2

[0051] Immunostaining steps:

[0052] Mouse retinas were dissected, fixed in 4% paraformaldehyde for 1 h, dehydrated with 15% and 30% glucose, embedded in OCT embedding medium, and frozen sections were prepared;

[0053] For washing, the retinal slices were put into DPBS, placed on a shaker and slowly rinsed 3 times, 5 minutes each time.

[0054] After rinsing, 500 μl of 0.5% TritonX-100 penetration solution prepared in DPBS was added to each well, and the cells were permeated for 10 minutes at room temperature, and the penetration solution was aspirated.

[0055] Repeat step 3, adding 500 μl of 3% HO in DPBS to each well 2 o 2 , 10min at room temperature to remove the influence of peroxidase.

[0056]Absorb H 2 o 2 , repeat step 3. Block with 4% BSA (prepared with DPBS) for 30 min at room temperature.

[0057] After aspirating the BSA, repeat step 3. Dilute the REG1A antibody at 1:100, add it to the culture dish, and incubate overnight at 4°C.

[0058] Recover the pr...

Embodiment 3

[0063] Expression and Purification of Recombinant REG1A Protein

[0064] The reg1a gene was subjected to agarose gel electrophoresis according to the PCR amplification method in Example 1 to obtain the reg1a gene fragment, and a large amount of reg1a gene fragments were obtained after gel recovery. Using the ligation kit, the recovered reg1a gene fragment was digested and ligated with the pET28a vector to obtain a recombinant vector. The recombinant vector was transformed into Escherichia coli strain BL-1, cultured on a shaker, and the cultured colonies were verified as positive bacteria by colony PCR. Add isopropylthiogalactopyranoside (IPTG) to the positive strains to induce expression, collect the bacteria by centrifugation, add buffer to make a suspension of bacteria, ultrasonically break, and collect the protein by centrifugation. Comparing the protein expression changes in bacteria before and after adding IPTG, the extracted protein was subjected to PAGE gel electrophor...

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Abstract

The invention provides an application of an REG1A protein in preparation of drugs for treating and / or preventing retinal cell apoptosis and belongs to the field of biological medicines. A novel acting protein for treating and / or preventing retinal cell apoptosis is provided by the application. The REG1A protein is capable of inhibiting retinal cell apoptosis through combination with a receptor EXTL3 on the surface of a retinal cell. The application provided by the invention is free of toxic or side effect, and has the advantages of being small in dosage, safe, stable in a long period and suitable for clinical treatment, and the organism is free of exclusion reaction.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to the application of REG1A protein in the preparation of drugs for treating and / or preventing retinal cell apoptosis. Background technique [0002] Light acts on the visual organs to excite the sensory cells, and the information is processed by the visual nervous system to produce vision. Through vision, humans and animals perceive the size, light and shade, color, and movement of external objects, and obtain various information that is important to the survival of the organism. At least 80% of external information is obtained through vision, and vision is the most important for humans and animals. Feel. Hundreds of millions of nerve cells in the retina of the visual organ are arranged in three layers and form a complex network for processing information through synapses. The first layer is photoreceptors, the second layer is interneurocytes, including bipolar cells, horizo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61P27/02A61P9/10
CPCA61K38/1709
Inventor 金子兵高美玲
Owner WENZHOU MEDICAL UNIV
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