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Method for regulating and controlling blood brain barrier permeability and application of method for regulating and controlling blood brain barrier permeability

A blood-brain barrier and permeability technology, applied in the field of medical biology, can solve problems such as unclear and increased ubiquitination of Mfsd2a

Active Publication Date: 2021-09-24
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A recent study showed that Zika virus increases Mfsd2a ubiquitination and leads to its proteasome-dependent degradation in human brain microvascular endothelial cells (HBMECs) (Zhou et al., 2019), implying that Mfsd2a may be Polyubiquitination is regulated, whereas the mechanism by which ubiquitin ligases cause Mfsd2a ubiquitination is unknown

Method used

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  • Method for regulating and controlling blood brain barrier permeability and application of method for regulating and controlling blood brain barrier permeability
  • Method for regulating and controlling blood brain barrier permeability and application of method for regulating and controlling blood brain barrier permeability
  • Method for regulating and controlling blood brain barrier permeability and application of method for regulating and controlling blood brain barrier permeability

Examples

Experimental program
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Effect test

Embodiment 1

[0071] Example 1. Knocking out the PTEN gene in cerebral blood vessels promotes transcytosis and increases the permeability of the blood-brain barrier

[0072] 1.1 Preparation of PTEN knockout mice specific for cerebrovascular endothelial cells

[0073] 1.1.1 PTEN conditional gene targeting mice (PTEN fl / fl mouse) construction

[0074]PTEN is a dual lipid / protein phosphatase that dephosphorylates the lipid phosphatidylinositol (3,4,5)-triphosphate and inhibits Akt activity (Song et al., 2012). Mouse homologous phosphatase-tensin (PTEN) is a protein whose amino acid sequence is shown in GenBank Accession No.NP_038796 (Update Date 2021-01-03), mouse PTEN gene (wild-type allele, PTEN + / + ) includes 9 exons, and its genome sequence is the sequence shown in the 32734977-32803560th nucleotide of GenBank Accession No.NC_000085.7 (Update Date 2020-09-22), and the 32734977th nucleotide is recorded as is the first nucleotide of the PTEN gene, wherein, 32734977-32735824 is the first e...

Embodiment 2

[0168] Example 2. Overexpression of NEDD4-2 in brain ECs leads to impaired blood-brain barrier integrity

[0169] 2.1 Construction of NEDD4-2 overexpression vector pAAV-Myc-NEDD4-2 in brain ECs

[0170] In order to ectopically overexpress the E3 ubiquitin ligase NEDD4-2 gene of brain Ecs, the present invention adopts the restructured brain endothelial-specific AAV-BR1 (pAAV-EGFP) system to obtain the recombinant expression vector pAAV-Myc-NEDD4- 2 overexpress NEDD4-2 gene in brain endothelial cells, and construct the overexpression vector of tdTomato gene and tdTomato-2A-Mfsd2a gene at the same time: Myc-NEDD4-2 (SEQ ID No.3 in the sequence listing), and tdTomato, The tdTomato-2A-Mfsd2a gene is cloned into pAAV-EGFP (comprising the CAG promoter, EGFP gene and SV40 Poly-A sequence) (SEQ ID No.4 in the sequence listing), respectively generating pAAV-Myc-NEDD4-2 plasmid, and pAAV-tdTomato, pAAV-tdTomato-2A-Mfsd2a plasmid (SEQ ID No. 5 in the sequence listing).

[0171] 2.1.1 Co...

Embodiment 3

[0211] Example 3. Research on the mechanism of action of the PTEN / Akt / NEDD4-2 / Mfsd2a axis in the PTEN / Akt signaling pathway

[0212] 3.1 Knockout of PTEN gene in cerebral blood vessels promotes transcytosis

[0213] In order to solve the problem of how to destroy the integrity of the blood-brain barrier after knocking out the PTEN gene in the cerebral vascular endothelium, the present invention studies its influence on the tight junction and transcytosis of endothelial cells. Electron microscope observation showed that PTEN fl / fl and PTEN fl / + Endothelial cells of littermates had typical features of capillary endothelial cells and were surrounded by astrocytes and pericytes. Consistent with the findings of the above examples, the expression level and localization of tight junction protein in brain endothelial cells of PTEN knockout mice were normal. Electron microscopy failed to detect 8 PTENs of P60 fl / fl Mice and PTEN fl / + Any obvious abnormalities in the ultrastructure...

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Abstract

The invention discloses a method for regulating and controlling blood brain barrier permeability and application of the method for the regulating and controlling blood brain barrier permeability. Through specific knockout of a fifth exon, a sixth exon and a seventh exon of a PTEN gene in a mouse cerebrovascular endothelial cell, increase of the blood brain barrier permeability and blood brain barrier leakage of a mouse can be observed; recombinant adeno-associated viruses expressing NEDD4-2 protein are constructed and injected into the mouse, it is found that the number of vesicles in cerebral vascular endothelial cells of the mouse is increased, the increase of the vesicles also leads to increase of the blood brain barrier permeability and the blood brain barrier leakage of the mouse. it is proved that PTEN deletion can cause blood brain barrier leakage caused by overexpression of NEDD4-2-Mfsd2a, therefore, the blood brain barrier permeability can be regulated and controlled by regulating and controlling the content or activity of the PTEN protein or the NEDD4-2 protein, and the PTEN protein or the NEDD4-2 protein can be applied to clinical preparation or development of related medicines for regulating and controlling the blood brain barrier permeability or cerebrovascular related medicines.

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to a method for regulating the permeability of the blood-brain barrier and its application Background technique [0002] Cerebral endothelial cells (ECs) located in the blood vessels of the central nervous system (CNS) form the blood-brain barrier (BBB), which strictly controls the entry and exit of substances into and out of the CNS, thereby maintaining the homeostasis of the CNS microenvironment (Zhao et al., 2015). The BBB's ability to restrict material access depends on unique features of brain ECs, including specialized tight junctions and adherens junctions, low levels of transcytosis, nonselective fenestrae, and pinocytosis (Andreone et al., 2015 ). The blood-brain barrier maintains the homeostasis of the central nervous system and is also one of the biggest hurdles to overcome in the development of drugs that act on the central nervous system. How to make therapeutic d...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12N15/864
CPCA01K67/0276C07K14/47C12N9/93C12N15/86C12Y603/0201A01K2227/105A01K2267/0375C12N2750/14143Y02A50/30
Inventor 王俊杨晓崔亚雄王燕晓
Owner ACADEMY OF MILITARY MEDICAL SCI
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