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Application of Spag6 in preparation of medicine for relieving cerebral arterial thrombosis reperfusion mediated synaptic injury

A technology for ischemic stroke and reperfusion, applied in application, drug combination, microorganism, etc., to achieve the effect of improving synaptic damage

Pending Publication Date: 2022-05-13
WUHAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of cerebral edema secondary to CIS / R, there is no relevant report on the role of Spag6 expression and its regulation of motor cilia function on brain injury.

Method used

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  • Application of Spag6 in preparation of medicine for relieving cerebral arterial thrombosis reperfusion mediated synaptic injury
  • Application of Spag6 in preparation of medicine for relieving cerebral arterial thrombosis reperfusion mediated synaptic injury
  • Application of Spag6 in preparation of medicine for relieving cerebral arterial thrombosis reperfusion mediated synaptic injury

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of Spag6 overexpression plasmid

[0037] a. Using the total RNA of Hela cells as a template to reverse transcription and amplify the cDNA chain (Hela cells are derived from ATCC)

[0038] 1) Collect Hela cultured cells, add 1ml TRIzo to prepare cell suspension.

[0039] 2) Transfer the cell suspension to an EP tube, add 0.2ml chloroform / phenol, mix well for 10 seconds, let stand for 2-3 minutes, and centrifuge at 12000g for 15 minutes (2°C-8°C).

[0040] 3) Aspirate the water phase, add 0.5ml of isopropanol, and centrifuge at 12000g for 10 minutes (2°C-8°C).

[0041] 4) After centrifugation, discard the supernatant and add 1ml of 75% ethanol to wash, vortex and mix well, and then centrifuge at 7500g for 5 minutes (2°C-8°C).

[0042] 5) Add 20ul RNA-free water to dissolve to obtain total RNA.

[0043]b. Prepare cDNA by reverse transcription, and configure the following reaction system with takara reverse transcription reagent:

[0044] ...

Embodiment 2

[0061] Example 2 Spag6 lentiviral vector construction and packaging

[0062] (1) 24 hours before transfection, digest the 293T cells in the logarithmic growth phase with trypsin, and adjust the cell density to about 5x10 with the medium containing 10% serum. 6 Cells / 15ml, reseeded in 10cm cell culture dish, cultured in 37°C, 5% CO2 incubator. After 24 hours, when the cell density reaches 70%-80%, it can be used for transfection;

[0063] (2) Replace with serum-free medium 2 hours before transfection;

[0064] (3) Add the prepared DNA solutions (20 μg of Spag6 overexpression plasmid, 15 μg of pHelper1.0 vector plasmid, and 10 μg of pHelper2.0 vector plasmid) into a sterilized centrifuge tube, and mix the corresponding volume of GeneKy transfection reagent evenly. Adjust the total volume to 1ml and incubate at room temperature for 15min;

[0065] (4) Slowly add the mixture to the culture medium of 293T cells, mix well, and culture in a 37°C, 5% CO2 cell incubator; note: the a...

Embodiment 3

[0073] Example 3 Construction of arterial embolism (MCAO) reperfusion injury model

[0074] Repeatedly immerse one end of the 0.23mm-diameter nylon thread in molten paraffin about 5 times. After the paraffin on the surface of the nylon thread solidifies, soak the thread in heparin for 30 minutes and dry it for later use. After the rat was anesthetized (0.35mL / 100g) by intraperitoneal injection of 10% chloral hydrate, it was fixed supine on the operating table with a rubber band, a median incision was made in the neck, the fascia was cut, and the right sternocleidomastoid was bluntly separated The right common carotid artery (Common Carotid artery, CCA) and vagus nerve were exposed and separated. Along the bifurcation of the right external carotid artery (ECA) and internal carotid artery (Internal Carotid Artery, ICA), the ECA and ICA were separated, and the ECA was ligated. Clamp the CCA and ICA with an arterial clip, cut a small hole with ophthalmic scissors at the place 5mm...

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Abstract

The invention provides an application of Spag6 in a medicine for treating cerebral arterial thrombosis reperfusion mediated synaptic injury. The constructed Spag6 lentiviral vector verifies that the Spag6 can promote the expression of synaptic protein and relieve encephaledema from the animal level. The compound is expected to become a medicine for clinically treating cerebral arterial thrombosis reperfusion mediated synaptic injury.

Description

technical field [0001] The invention belongs to the field of new application of drugs, and in particular relates to the application of Spag6 in drugs for alleviating synaptic damage mediated by ischemic stroke. Background technique [0002] Cerebral stroke, also known as "stroke", is the most common disease with high mortality and high disability rate. Stroke includes ischemic stroke (Cerebral ischemic stroke, CIS) and hemorrhagic stroke (Cerebralhemorrhagic stroke, CHS). According to available data, about 15 million people around the world suffer from stroke every year, which poses a serious burden to family and society. At present, the treatment for CIS is mainly to implement thrombolytic therapy within the effective time window to restore the blood supply as soon as possible to prevent the spread of ischemic areas. Once the effective time window for re-thrombolytic therapy is exceeded, cerebral ischemic stroke-reperfusion (CIS / R) injury will occur, and its impact on the...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K48/00A61P9/10C12N15/867C12N15/12C12N7/01
CPCA61K38/1709A61K48/005A61K48/0008A61P9/10C12N15/86C07K14/47C12N7/00C12N2740/15043C12N2740/15021C12N2740/15032
Inventor 曹晓璐胡一鸣张玲柳赟昊石玉琴付国庆周婷全超
Owner WUHAN UNIV OF SCI & TECH
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