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siRNA targeting psmc5 gene and application of psmc5

A gene and targeting technology, applied in DNA/RNA fragments, genetic engineering, recombinant DNA technology, etc., to achieve significant interference effects, correct learning and memory, and regulate balance

Active Publication Date: 2022-04-05
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, there are no relevant reports on the effect of PSMC5 on the activation of microglia, improvement and blocking the progress of AD

Method used

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  • siRNA targeting psmc5 gene and application of psmc5
  • siRNA targeting psmc5 gene and application of psmc5
  • siRNA targeting psmc5 gene and application of psmc5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Screening for the best sequence of efficient silencing of PSMC5 siRNA

[0055] The expression of PSMC5mRNA was detected by fluorescence quantitative PCR, and the interference efficiency was analyzed; the Griess reagent method was used to detect the NO in the supernatant of BV2 microglial cells (purchased from the Cell Resource Center of Peking Union Medical College, cell number: 3111C0001CCC000063) 2- The content of IL-1β in BV2 cell supernatant was determined by ELISA kit competition method, and the expression of PSMC5 protein in BV2 cells was detected by Western blot method, and the best interference sequence was screened out.

[0056] 1. BV2 cell culture

[0057] BV2 cells were cultured in complete DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO 2 , cultured in a cell culture incubator under saturated humidity conditions.

[0058] 2. Selection, design and synthesis of PSMC5 siRNA targets

[0059] According to the known sequence of mouse PSMC...

Embodiment 2

[0092] Example 2 Establishment of AD Animal Model and Determination of PSMC5siRNA Treatment Dose and Time

[0093] C57 / 6J male mice aged 9-11 weeks were used, and lipopolysaccharide (Lipopolysaccharide, LPS) was injected intraperitoneally at 500 μg / kg or 750 μg / kg. The cognitive function of the mice was evaluated by the Morris water maze test and the dark avoidance test, and the motor coordination of the mice was detected by the rod climbing test.

[0094] 1. The effect of intraperitoneal injection of LPS on mouse positioning and navigation experiments and space exploration experiments

[0095] We set up the following groups and observed the latency to find the platform to evaluate the memory ability and spatial cognition ability of mice.

[0096] ①For the normal control group, normal saline (0.1 mL / 10 mg / d) was intraperitoneally administered for 7 consecutive days, and behavioral tests were performed 6 hours after administration.

[0097] ② LPS (500 μg / kg) group was adminis...

Embodiment 3

[0117] Example 3 PSMC5siRNA applied to activated BV2 microglial cells

[0118] 20 x 10 BV2 microglia 4 The cells / well were inoculated in 6-well plates, and the experiments were divided into the following five groups:

[0119] (1) Control group: no processing;

[0120] (2) LPS group: the concentration of LPS is 1000ng / mL;

[0121] (3) PSMC5siRNA+LPS group: transfected into PSMC5siRNA 1+LPS;

[0122] (4) NC+LPS group: negative control sequence PSMC5siRNA NC+LPS was transfected.

[0123] (5) NC group: transfect negative control sequence PSMC5siRNA NC.

[0124] The transfection method was the same as in Example 1.

[0125] The LPS-induced NO release from BV2 cells was detected by the Griess reagent method, and the specific operation steps and methods were the same as in Example 1.

[0126] The content of IL-1β, PGE2 and TNF-α in the cell supernatant was measured by enzyme-linked immunosorbent assay (ELISA): 20×10 BV2 microglial cells 4 Cells / well were seeded in 6-well plate...

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Abstract

The invention provides siRNA targeting PSMC5 gene and application of PSMC5. The present invention provides a technical solution for 26S proteasome regulatory subunit 8 (PSMC5) as a new target for the treatment of Alzheimer's disease. Effective treatment of Alzheimer's disease can be achieved by inhibiting the expression of PSMC5, which can be used for screening and developing anti-AD Medicines offer new ideas and new approaches. The present invention also provides a siRNA targeting PSMC5 gene, which can inhibit the excessive activation of microglial cells by inhibiting the signal pathway mediated by activation of TOLL-like receptors, thereby protecting hippocampal neurons and effectively improving and treating AD; It can protect and treat people with learning, memory and cognitive dysfunction diseases induced by potential microglial overactivation. The invention can be widely used in the fields of neurodegenerative diseases and geriatric medicine, and can generate huge social and economic benefits.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to the application of siRNA targeting PSMC5 gene and PSMC5. Background technique [0002] Alzheimer's disease (AD) is the most common type of senile dementia, and its main clinical manifestations are neurodegenerative diseases of cognitive function decline, life function decline and abnormal mental behavior. At present, the world is increasing at a rate of 4.6 million new cases per year (1 new case every 7 seconds), and by 2040, the number of AD patients worldwide will reach 81.1 million. AD has become the main cause of disability and inability to live independently in the elderly after heart disease, cancer, and stroke. It has brought serious harm and heavy burdens to families and the whole society, and the prevention and treatment situation is very grim. Therefore, it is of great significance to study the pathogenesis of AD in depth and explore feasible treatment methods to improve the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68C12N15/113C12N15/63A61K45/00A61K31/713A61P25/28A61P25/16
CPCG01N33/68C12N15/113C12N15/63A61K45/00A61K31/713A61P25/28A61P25/16C12N2310/14G01N2333/4703G01N2800/285G01N2800/2835G01N2800/2821
Inventor 朱丽红毕伟陆大祥肖书赵佳仪
Owner JINAN UNIVERSITY
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