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A type Ⅱ adeno-associated virus carrying neuritin gene and its application in repairing optic nerve damage

A gene and virus technology, applied in the repair of optic nerve damage, in the field of adeno-associated virus type II, to improve survival and regeneration, enhance intrinsic regeneration ability, and promote optic nerve regeneration.

Inactive Publication Date: 2015-12-09
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no literature report that the Neuritin gene is related to the repair of optic nerve damage, and there is no literature report on the construction of a type Ⅱ adeno-associated virus vector carrying the Neuritin gene to treat optic nerve damage

Method used

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  • A type Ⅱ adeno-associated virus carrying neuritin gene and its application in repairing optic nerve damage
  • A type Ⅱ adeno-associated virus carrying neuritin gene and its application in repairing optic nerve damage
  • A type Ⅱ adeno-associated virus carrying neuritin gene and its application in repairing optic nerve damage

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1. Construction of Neuritin gene type Ⅱ adeno-associated virus expression vector

[0045] (1) Type II adeno-associated virus expression vector and enzyme digestion of target gene

[0046] The pAOV-CAG-eGFP vector (purchased from Addgene) was used to carry out enzyme digestion with NotI and MluI, and the digested vector was prepared for use in vector construction. Using the cDNA clone of the CDS region of the NRN1 gene as a template, the NRN1 gene fragment was amplified by PCR, and the product was digested with NotI and MluI, and the vector and target gene DNA were recovered after identification by agarose gel electrophoresis.

[0047] (2) Ligation reaction

[0048] The digested type II adeno-associated virus vector and the PCR product of the NRN1 gene were ligated according to the reaction system in Table 1.

[0049] Table 1: Ligation reaction system

[0050]

[0051] After the above reagents were mixed, they were ligated at 4°C for 12 hours to prepare the cloning l...

Embodiment 2

[0066] 1. Culture and induction of RGC5 differentiation

[0067] RGC5 is the retinal ganglion cell line of the one-day-old rat (see [20] KrishnamoorthyRR, AgarwalP, PrasannaG, etal.Characterizationofatransformedradretinalganglioncellline.BrainResMolBrainRes.2001;86(1-2):1-12.), immature , first induced by staurosporine to differentiate into mature neuron-like cells ( figure 1 ). RGC5 before induction was cultured in high-glucose DMEM medium and 5% serum in a place containing 5% CO 2 , in an incubator at 37°C, and the cells were passaged once a day. When used, 326nM staurosporine was used to induce for 24 hours.

[0068] 2. Overexpression of Neuritin gene and establishment of RGC5 injury model

[0069] When RGC5 was cultured to 80% confluence, add the appropriate amount of type II adeno-associated virus and a certain amount of polybrene at a pre-measured concentration, and replace it with normal medium after 24 hours, observe the expression of RFP with a fluorescence micros...

Embodiment 3

[0081] 1. In vivo transfection of retinal ganglion cells with type Ⅱ adeno-associated virus and overexpression of Neuritin

[0082]Adeno-associated virus type II was injected into the vitreous body, and mice were perfused whole body with 4% paraformaldehyde one week later. Retinal slices were taken, and immunohistochemical β-Ⅲ-tubulin staining or cholera toxin B subunit (CTB) injection was used to co-localize the retinal ganglion cells to observe the transfection effect, the transfection efficiency is about 90% or more (such as Figure 5 shown).

[0083] Western blot and immunohistochemical methods were used to observe the overexpression and localization of Neuritin protein after Neuritin type Ⅱ adeno-associated virus transfection.

[0084] 2. Establishment of Optic Nerve Injury Model

[0085] The present invention adopts the optic nerve crush model, using Yasargil cerebral aneurysm clip on the optic nerve of adult male rats at about 1mm behind the eyeball to crush for 9 sec...

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Abstract

The invention belongs to the technical field of gene engineering and nerve injuries. Optic nerve injuries are common eye diseases and external injuries, and cause the optic nerve atrophia and blindness of patients when the optic nerve injuries are severe, and at present, the clinic actual curative effect is unsatisfactory. The invention provides a type-II adeno-associated virus carrying neuritin genes and a building method thereof, the invention also further provides the application of the type-II adeno-associated virus carrying neuritin genes in preparing medicaments for restoring optic nerve injuries.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and nerve injury, and specifically relates to a type II adeno-associated virus carrying Neuritin gene and its application in repairing optic nerve injury. Background technique [0002] Optic nerve injury is a common link of many eye diseases and traumas, such as glaucoma, optic nerve ischemia, blunt trauma, optic canal fracture, etc., leading to visual function damage, and in severe cases, it will cause optic nerve atrophy and blindness in patients. Although a lot of research work has been carried out on the treatment of optic nerve injury, its clinical efficacy is unsatisfactory, and it is difficult to restore vision to patients according to the current medical technology level. The low intrinsic regenerative ability of retinal ganglion cells (RGCs) is the key factor for optic nerve atrophy and blindness caused by regeneration failure after optic nerve injury. How to carry out effecti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864C12N15/66A61K48/00A61P25/02A61P27/02
Inventor 许家军曹文珞刘芳蔺海燕
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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