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Application of a lentivirus carrying neuritin gene in the preparation of drugs for repairing retinal pigment epithelial degeneration

A technology of retinal pigment and lentivirus, which is applied in the field of genetic engineering and retinal damage, can solve the problems that have not yet been seen in the expression changes and functions of Neuritin, and achieve the effect of improving survival and enhancing internal regeneration ability

Inactive Publication Date: 2017-02-22
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

[0017] At present, there are no research reports on the expression changes and effects of Neuritin after RP

Method used

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  • Application of a lentivirus carrying neuritin gene in the preparation of drugs for repairing retinal pigment epithelial degeneration
  • Application of a lentivirus carrying neuritin gene in the preparation of drugs for repairing retinal pigment epithelial degeneration
  • Application of a lentivirus carrying neuritin gene in the preparation of drugs for repairing retinal pigment epithelial degeneration

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Construction of Neuritin gene lentiviral expression vector

[0044] (1) Digestion of lentiviral expression vector and target gene

[0045] The pLenO-RIP (purchased from Addgene) vector was digested with Not I and Mlu I, and the digested vector was prepared for vector construction ( figure 1 ). 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 Not I and Mlu I, and the vector and target gene DNA were recovered after identification by agarose gel electrophoresis.

[0046] (2) Ligation reaction

[0047] The digested lentiviral vector and the PCR product of the NRN1 gene were ligated according to the reaction system in Table 1.

[0048] Table 1: Ligation reaction system

[0049]

[0050] After the above reagents were mixed, they were ligated at 4°C for 12 hours to prepare the cloning ligation solution for transformation.

[0051] (3) Transformation, identification o...

Embodiment 2

[0065] 1. In vitro transfection of retinal pigment epithelial cells with lentivirus and overexpression of Neuritin

[0066] Under sterile conditions, the eyes of 19-day pregnant rats were incised and the cornea, lens, vitreous body and retinal nerve layer were discarded. After washing with PBS phosphate buffer, add 0.25% trypsin and 0.02 ethylenediaminetetraacetic acid (EDTA), digest and collect cells at 37°C, discard the supernatant, add DMEM medium containing fetal bovine serum in C0 2 Cultured in an incubator, the cells were subcultured and identified by mouse anti-human cytokeratin monoclonal antibody and fluorescein labeling. Indomethacin (Indomethacin, IN) induced apoptosis model of RPE cells was established, adding recombinant Neuritin with a final concentration of 10, 30, 50, 100, 200, 300ng / ml, and LV-RFP, LV-Neuritin, the latter two The lentivirus group was transfected into RPE cells 2 days before injury and its expression was verified. The effects of Neuritin on c...

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Abstract

The invention belongs to the technical field of gene engineering and retinal damage, and in particular relates to application of lentivirus carrying neuritin gene in preparation of drugs for restoration of retinal pigment epithelial degeneration. The constructed lentivirus carrying the neuritin gene is used in a retinal pigment epithelial cell degeneration animal model for detection of related indexes, and confirms that the neuritin lentiviral vector can successfully transfect and highly express rat RPE (retinal pigment epithelium) so as to protect the retina retinal pigment epithelium, visual cells and the like and improve functional recovery.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and retinal damage, and specifically relates to the application of a lentivirus carrying Neuritin gene in preparing a medicine for repairing retinal pigment epithelial degeneration. Background technique [0002] The retinal pigment epithelium (RPE) is located between the retina and the choroid, and has multiple functions such as phagocytosis, barrier and ion transport. RPE degeneration is a group of diseases that end in the progressive apoptosis and death of retinal photoreceptor cells and RPE, causing vision loss and visual field defects as common manifestations. It has a genetic predisposition, and more than 100 related genes have been identified. Find. The incidence rate of the disease is relatively high, about 1 / 4000, and it is the key link of blinding diseases. (Reference [1] Sung CH, Chuang JZ. The cell biology of vision. J Cell Biol 2010; 190:953-63). Moreover, there is no ef...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61P27/02
Inventor 许家军刘芳曹文珞蔺海燕
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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