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Method for improving blood-brain barrier permeability by regulating Swiprosin-1 expression

A blood-brain barrier and permeability technology, applied in the field of molecular biology, can solve problems such as the unclear role of Swiprosin-1, and achieve the effect of improving expression

Inactive Publication Date: 2015-11-11
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

At the same time, the role of Swiprosin-1 in other aspects is still unclear
[0005] There is no report on the relationship between Swiprosin-1 and blood-brain barrier permeability

Method used

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  • Method for improving blood-brain barrier permeability by regulating Swiprosin-1 expression
  • Method for improving blood-brain barrier permeability by regulating Swiprosin-1 expression
  • Method for improving blood-brain barrier permeability by regulating Swiprosin-1 expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Increased blood-brain barrier permeability in mice overexpressing Swiprosin-1

[0054] 1. Preparation of Swiprosin-1 overexpression mice

[0055] 1 material

[0056] Plasmid pcDNA-CAG (sequence shown in SEQIDNO.7) was provided by the Southern Model Organism Research Center, DH5α was purchased from Tiangen Biological Company, EFDH2cDNA was purchased from SourceBioscience; various restriction enzymes, T4DNA ligase, T4DNApolymerase, Taq enzyme Reagents related to PCR and PCR were purchased from Takara and NEB companies; Gel Recovery Kit was purchased from Tiangen Biological Company, Plasmid Extraction Kit was purchased from Qiagen Company; anti-goat Swiprosin-1 polyclonal antibody was purchased from Imgenex Company.

[0057] The sequences of primers for transgenic plasmid construction and primers for identification of transgenic positive mice are as follows:

[0058] efdhu1

catGGATCcggggagtgtcaggaagaggaag

SEQ ID NO.5

efdhl1

catAAGCTT...

Embodiment 2

[0101] Example 2 Decreased blood-brain barrier permeability in Swiprosin-1 knockout mice after MCAO

[0102] 1. Swiprosin-1 knockout mice (Swp - / - )preparation

[0103] 1 method

[0104] 1.1 Construction of transgenic plasmid

[0105] The long arm fragment and the short arm fragment were cloned from the Swiprosin-1 gene, and after connecting into the T vector to confirm the correctness of the above fragments, the short arm fragment was connected into the Neo vector to obtain the Neo-short arm vector, and then the long arm fragment was connected Insert the Neo-short arm vector to obtain the long arm-Neo-short arm vector, and finally connect the long arm-Neo-short arm vector to the TK vector to obtain the TK-long arm-Neo-short arm vector. The final vector was verified by enzyme digestion and sequencing.

[0106] 1.2 Preparation of transgenic mice

[0107] The TK-long arm-Neo-short arm vector was digested with Not1, and the DNA fragment containing the long arm sequence and s...

Embodiment 3

[0152] Example 3 Cell Experiment of Swiprosin-1 Gene Knockout

[0153] The effects of Swiprosin-1 gene knockout in rat brain microvascular endothelial cells rBMEC on the expression of tight junction protein ZO-1 were studied by oxygen-glucose deprivation (OGD).

[0154] 1 material

[0155] The lentiviral vector pLVTHM was purchased from Shenzhen Baozhu Biotechnology Co., Ltd., see the vector map Figure 10 ; Chloral hydrate and TTC were purchased from Sinopharm Chemical Reagent Co., Ltd.; PBS was purchased from Shanghai Boguang Biological Co., Ltd.; fetal bovine serum (FBS) and DMEM medium were purchased from Gibco.

[0156] 2 methods

[0157] 2.1 Swiprosin-1 interferes with lentiviral vector construction

[0158] The following siRNA molecules for Swiprosin-1 were designed:

[0159] Sense strand: 5'-AAGGGUGCCAAGAACUUCU-3' (SEQ ID NO.1);

[0160] Antisense strand: 5'-AGAAGUUCUUGGCACCCUU-3' (SEQ ID NO.2).

[0161] According to the above siRNA molecule design and synthesis ...

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Abstract

The invention relates to a method for improving blood-brain barrier permeability by regulating Swiprosin-1 expression, and specifically provides an application of Swiprosin-1 and a regulator thereof in preparing a drug. The drug is used for regulating the blood-brain barrier permeability, or preparing an animal model with increased or reduced blood-brain barrier permeability. The invention further provides a specific Swiprosin-1 overexpression vector, siRNA and shRNA. The method proves that a mouse with Swiprosin-1 overexpression has increased blood-brain barrier permeability, while a mouse with Swiprosin-1 genes knocked out after MCAO operation has decreased blood-brain barrier permeability, therefore, Swiprosin-1 and the regulator thereof can be used for treating diseases related to blood-brain barrier permeability, and establishing an animal model with increased or decreased blood-brain barrier permeability.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to regulating the expression of Swiprosin-1 and improving the permeability of the blood-brain barrier. Background technique [0002] The blood-brain barrier (bloodbrainbarrier) refers to the structure of brain capillaries that prevent certain substances (mostly harmful) from entering the brain tissue from the blood. A variety of solutes in the blood enter the brain tissue from the brain capillaries, which are difficult or easy. Some pass through quickly, some slowly, and some cannot pass through at all. This selective permeability phenomenon makes people imagine that there may be restrictions on solute permeability. This structure can make the brain tissue less or even not damaged by harmful substances in the circulating blood, so as to maintain the basic stability of the internal environment of the brain tissue and play an important biological role in maintaining the norm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K38/17A61K38/16A61P9/00C12N15/113C12N15/867
Inventor 苏定冯刘玮晔李玲王志斌王跃仝令畅张立超王荣美
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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