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A method for separating and purifying cerebral microvessels

A purification method and micro-technology, applied in the field of cell biology, can solve the problems of no approved treatment method and inability to obtain amyloid-β protein in brain capillary blood vessels

Active Publication Date: 2021-09-10
HUNAN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the etiology and pathogenesis of Alzheimer's disease have not yet been elucidated, resulting in no approved treatment methods. The main reason is that it is impossible to obtain a complete and sufficient amount of brain capillaries without destroying the content of amyloid-β protein in the blood vessels.

Method used

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  • A method for separating and purifying cerebral microvessels
  • A method for separating and purifying cerebral microvessels
  • A method for separating and purifying cerebral microvessels

Examples

Experimental program
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Effect test

Embodiment 1

[0021] see figure 1 , which is the effect of different homogenization times in the embodiment of the present invention on the separation and extraction of brain microvessels.

[0022] After the mouse brain tissue fragments were transferred to the Dounce homogenizer, push the grinding rod up and down 12-15 times to obtain tissue homogenization. The grinding process is required to be very gentle. The first few pushes of the homogenizer will be difficult, but as the tissue breaks down and the pieces are ground, the homogenizer pushes become easier. Make sure that no air bubbles are generated during the homogenization process. Use a sufficient amount of autoclaved sterile glass magnetic beads (~2g) to place in a 70μm nylon cell strainer, fill the nylon cell strainer, and install the nylon cell strainer in the mouth of a 50ml conical tube. Wash the sterile glass magnetic beads twice with 1ml of reagent [4]. Drop the cerebral blood vessel and microvascular tissue suspension obta...

Embodiment 2

[0026] see image 3 and Figure 4 , which is the content of cerebrovascular and microvascular target protein CD31 obtained by different separation and purification methods using Western blot in the embodiment of the present invention.

[0027] After the cerebrovascular and microvascular tissues obtained in Example 1 were centrifuged at 1000 g at 4° C. for 10 minutes, 200-300 ul of reagent [5] was used to resuspend the cerebrovascular and microvascular pellets. Aliquot and add 2x SDS buffer to make 100 μl 1x SDS w / 1x protease and 1x phosphatase inhibitor. The specific operation steps are as follows:

[0028]1. Use animal cell (tissue) total protein extraction reagent, add protease inhibitor mixture at a ratio of 1:99; add tissue protein extraction reagent at a ratio of 1:10 (g / ml), homogenate and sonicate. Take the purified material of mouse cerebrovascular and microvascular tissue stored at -80°C and place it in a 2ml centrifuge tube, add pre-cooled tissue protein extract ...

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Abstract

The invention discloses a method for separating and purifying brain microvessels, which comprises the following steps: (1) taking fresh whole brain tissue of mice, and grinding it up and down for 12-15 times with a Dounce homogenizer to prepare tissue homogenate; (2) homogenizing the tissue The slurry was double-filtered with sterile glass magnetic beads and a 70 μm nylon cell filter to obtain brain blood vessels and microvascular tissues. The method of the invention can not only effectively improve the integrity, quantity and purity of the isolated cerebral microvessels, but also not destroy the content of each target protein in the cerebral microvessels.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a method for separating and purifying brain microvessels. Background technique [0002] Alzheimer's disease (AD), namely senile dementia, is a progressive neurodegenerative disease that seriously affects the quality of life of the public. The characteristic pathological changes of Alzheimer's disease are brain tissue degeneration and aging, resulting in changes in the capillary permeability of arterioles, pia mater and cerebral cortex, which promote the deposition of amyloid-β protein in serum in brain tissue and on blood vessel walls, causing cerebral amyloid angiopathy (CAA). In recent years, vascular factors have been widely concerned in the occurrence and development of Alzheimer's disease. At present, the etiology and pathogenesis of Alzheimer's disease have not yet been elucidated, resulting in no approved treatment methods. The main reason is that it is impossible t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
CPCC12N5/069C12N2500/84C12N2509/00
Inventor 刘文锋大卫霍夫曼
Owner HUNAN NORMAL UNIVERSITY
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