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Alzheimer's disease model as well as establishment method and application thereof

A technology for Alzheimer's disease and method establishment, which is applied in the field of Alzheimer's disease model and its establishment, can solve problems such as lack of AD models, and achieve high practicality, reliable method establishment, and good repeatability

Pending Publication Date: 2022-06-28
中山大学深圳 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, reliable AD models for mutations at this site are still lacking

Method used

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  • Alzheimer's disease model as well as establishment method and application thereof
  • Alzheimer's disease model as well as establishment method and application thereof
  • Alzheimer's disease model as well as establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Construction and validation of a CLU mutant AD cell model.

[0080] 1. Establishment of CLU mutant AD cell model

[0081] 1.1 Cell reprogramming

[0082] The flow chart of reprogramming cells into human-derived inducible neural precursor cells (AD hiNPC) is shown in figure 1 shown, including the following steps:

[0083] (1) After signing the consent form for the collection of samples from the medical biobank, collect blood samples from AD patients, screen samples from AD patients with CLU site mutations by conventional PCR resequencing, and isolate their peripheral blood mononuclear cells (PBMNC).

[0084] (2) The PBMNCs obtained above were cultured in mononuclear cell proliferation medium (MNC), and OCT3 / 4, SOX2, KLF4, c-MYC and Sendai virus were added for transfection when the PBMNCs were expanded and cultured for 14 days.

[0085] In the transfection system, the concentration of transcription factor OCT3 / 4 (POU class 5homeobox 1, source: Invitrogen) was 2×10 5 T...

Embodiment 2

[0103] Establishment and validation of AD mouse model.

[0104] 1. Establishment of CLU mutant AD mouse model

[0105] (1) 8-week-old C57BL / 6J male mice were taken and anesthetized by intraperitoneal injection of 3% sodium pentobarbital saline solution (1.5 ml / kg).

[0106] (2) Fix the anesthetized mice on the brain stereotaxic apparatus, prepare the skin, make a longitudinal incision of about 3 cm along the top of the midline, separate the skin and the skull, and determine the right hippocampal dentate gyrus (DG area). location, drill holes.

[0107] (3) The micro-syringe is inserted through the hole, the depth is 2mm below the skull, and each animal is injected with 5 × 10 6 The AD MGE cells (ie Alzheimer's disease model cells) containing CLU site mutation constructed in Example 1.

[0108] (4) Suture the wound, disinfect the wound surface, and subcutaneously inject cyclosporine A (7 mg / kg) every day from the day before the operation. After about 3 months, the Alzheimer's...

Embodiment 3

[0128] Experiment of the effect of Gabrα2 antagonist on AD mouse model established by the present invention.

[0129] 1. Method.

[0130] The AD mouse model was established according to Example 2, and pentylenetetrazole (1 mg / kg) was administered by gavage at 6.5 months after modeling, once a day for 2 consecutive weeks. At the end of administration, the behavioral changes of mice were observed by Morris water maze and Y maze.

[0131] 2. Results.

[0132] The result is as Figure 41-47 As shown in the figure, WT in each figure is the wild-type Control group (C57 / 6J mice), AD hiNPC-derived MGE-WT is C57 / 6J mice injected with AD MGE in the hippocampus for 6 months, AD hiNPC derived MGE-WT-Tetrapentazole is C57 / 6J mice 6J mice were injected with AD MGE in the hippocampus for 6 months and then treated with tetrapentazole for 1 month.

[0133] in, Figure 41 In order to locate the time when each group of mice reached the platform in the cruise experiment, Figure 42 In order t...

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Abstract

The invention relates to an Alzheimer's disease model and an establishment method and application thereof, and belongs to the technical field of disease models. The method comprises the following steps: cell reprogramming: acquiring a peripheral blood sample of an AD patient with CLU site mutation, and separating to obtain mononuclear cells; carrying out multiplication culture on the mononuclear cells, adding OCT3 / 4, SOX2, KLF4, c-MYC and Sendai virus for transfection, and culturing to obtain human induced neural precursor cells; and cell differentiation: selecting the human induced neural precursor cells for cloning, adding PDL and Laminin for multiplication culture, performing culture for a preset time, enabling the human induced neural precursor cells to differentiate into gamma-aminobutyric acid energy neural precursor cells, and performing differentiation culture to obtain the MGE cells of the Alzheimer's disease, namely the Alzheimer's disease cell model. The model obtained by the method can better restore the pathogenesis process of the Alzheimer's disease, and provides an important basis for exploring the pathogenesis of the AD and screening drugs.

Description

technical field [0001] The present invention relates to the technical field of disease models, in particular to an Alzheimer's disease model and its establishment method and use. Background technique [0002] Alzheimer's disease (AD) is the most common cause of dementia, accounting for an estimated 60-80% of dementia cases. Its most common pathological features are the accumulation of extraneuronal β-amyloid (Aβ) and intraneuronal tau protein. Recently, increasing evidence supports the involvement of γ-aminobutyric acid (GABA)-ergic signaling in the development of AD. Abnormal GABAergic signaling is involved in the imbalance of excitatory and inhibitory signaling (E / I) and is considered to be an important driver of AD pathogenesis. The vicious cycle between Aβ aggregation, tau hyperphosphorylation, and GABAergic dysfunction exacerbates the pathogenesis of AD. [0003] With the application of genome-wide association studies to large-scale populations, CLU mutations were sc...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/0793C12N5/0797A01K67/027A61K49/00
CPCC12N5/0619C12N5/0623A01K67/0271A61K49/0008C12N2501/603C12N2501/602C12N2501/604C12N2501/606C12N2510/00C12N2501/00C12N2533/32C12N2533/52C12N2500/32A01K2207/12A01K2207/20A01K2227/105A01K2267/0312
Inventor 邓文斌陈春霞郭莹
Owner 中山大学深圳