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A lentivirus carrying neuritin gene and its application in repairing optic nerve damage

An optic nerve injury and lentivirus technology, applied in applications, gene therapy, genetic engineering, etc., can solve the problem of no literature report Neuritin gene, etc., to improve survival and regeneration, enhance intrinsic regeneration ability, and promote the effect of 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 lentiviral vector carrying the Neuritin gene to treat optic nerve damage

Method used

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  • A lentivirus carrying neuritin gene and its application in repairing optic nerve damage
  • A lentivirus carrying neuritin gene and its application in repairing optic nerve damage
  • A lentivirus carrying neuritin gene and its application in repairing optic nerve damage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1. Construction of Neuritin gene lentiviral expression vector

[0045] (1) Digestion of lentiviral expression vector and target gene

[0046] The pLenO-RIP (purchased from Addgene) vector was digested with NotI and MluI, and the digested vector was prepared for 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 lentiviral 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 ligation solution for transformation.

[0052] (3) Transformation, identification of picked clones

...

Embodiment 2

[0066] 1. Culture and induction of RGC5 differentiation

[0067] RGC5 is the retinal ganglion cell line of one-day-old rats (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 is cultured to 80% confluence, add a suitable amount of lentivirus and a certain amount of polybrene at a pre-measured concentration, replace it with normal medium after 24 hours, and observe the expression of RFP with a fluorescence microscope after 96 hours. The tran...

Embodiment 3

[0081] 1. In vivo transfection of retinal ganglion cells with lentivirus and overexpression of Neuritin

[0082] The neuritin lentivirus was injected into the vitreous body, and the mice were perfused with 4% paraformaldehyde one week later. The retinal slices were taken, and immunohistochemical βⅢ-tubulin staining or cholera toxin B subunit (CTB) injection of co-localized retinal ganglion cells were used to observe the transformation. Transfection effect, the transfection efficiency is about 20-30% (such as Figure 7 shown).

[0083] Western blot and immunohistochemical methods were used to observe the overexpression and expression localization of Neuritin protein after Neuritin lentivirus 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 seconds, the specific operation ...

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Abstract

The invention belongs to the technical field of genetic engineering and nerve injuries. Optic nerve injuries are a lot of eye diseases and eye traumas, the optic nerve atrophy and blindness of patients can be caused when optic nerve injuries are severe, and at present, the clinical practical curative effect is not satisfactory. The research of the invention finds that the Neurithin gene is related to optic nerve injury repair, so the invention provides a Neurithin gene-carrying lentivirus vector and a construction method thereof and also further provides the application of the Neurithin gene-carrying lentivirus vector in the preparation of medicines for treating and repairing optic nerve injuries.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and nerve injury, and in particular relates to a lentivirus 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 effective reversal therapy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/12C12N15/66A61K48/00A61P25/00
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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