Method for constructing Alzheimer's disease (AD) cell model, and application of Alzheimer's disease (AD) cell model

An Alzheimer's disease, cell model technology, applied in the field of biotechnology and genetic engineering, can solve the problem that the degree of amyloid plaques and neurofibrillary tangles cannot be standardized, and AD cell models have uneven secretion of Aβ and Tau phosphorylation ability , not for a long time, etc., to achieve stable and continuous tau protein phosphorylation, stable and continuous Aβ deposition, and simple and fast technology.

Pending Publication Date: 2021-06-18
SHANGHAI UNIV +1
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Problems solved by technology

[0004] At this stage, AD cell models have been widely used, but there are still some shortcomings and disadvantages. The main problems are: (1) The ability of AD cell models to secrete Aβ and Tau phosphorylation is uneven, resulting in the degree of amylo...

Method used

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  • Method for constructing Alzheimer's disease (AD) cell model, and application of Alzheimer's disease (AD) cell model
  • Method for constructing Alzheimer's disease (AD) cell model, and application of Alzheimer's disease (AD) cell model
  • Method for constructing Alzheimer's disease (AD) cell model, and application of Alzheimer's disease (AD) cell model

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Embodiment 1

[0063] This example provides a method for constructing an Alzheimer's disease cell model (ADcell model) that can be used for a long time and has stable properties. The main technical process can be divided into four steps: 1. Vector construction; 2. Cell construction; 3. Matrigel culture; 4. Congo red amyloid staining and Western blotting to detect the pathological features of AD.

[0064] Each step is described in detail below:

[0065] 1. Vector construction (using molecular biology techniques to construct PLVX-APP recombinant vector)

[0066] (1) Selection of suitable carrier skeleton. The present invention selects the lentiviral vector pLVX-IRES-Puro (hereinafter referred to as PLVX) as the basic skeleton of the recombinant vector.

[0067] (2) National Center of Biotechnology Information (NCBI) website ( https: / / www.ncbi.nlm.nih.gov / ) search for the coding sequence (codingsequence, CDS) of the APP gene.

[0068] (3) Use the Primer5 primer design software to design p...

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Abstract

The invention discloses a method for constructing an Alzheimer's disease (AD) cell model, and application of the Alzheimer's disease (AD) cell model. The method comprises the following steps of: constructing a recombinant vector containing a coding gene of APP protein, and integrating the coding gene of the APP protein into a genome of a host cell by using the recombinant vector, so as to construct and form the cell model. According to the invention, CHO (Chinese Hamster Ovary) and HFF (Human Foreskin Fibroblast) cell lines overexpressing APP genes are constructed through a gene technology to serve as the AD cell model; a lentivirus transfection mode is adopted, so that it can be guaranteed that the AD cell model has the durability and stability of AD pathological characteristics; moreover, the AD cell model disclosed by the invention can stably express and secrete A beta, can be used for a long time, has stable and continuous A beta deposition and tau protein phosphorylation characteristics, and can well simulate pathological characteristics of the Alzheimer's disease; and an in-vitro cell research platform possibility is provided for research, development and screening of Alzheimer's disease related drugs and research of mechanisms.

Description

technical field [0001] The present invention relates to a method for constructing an Alzheimer's disease cell model, in particular to a method for constructing an Alzheimer's disease cell model (AD cellmodel) capable of secreting Aβ and Tau phosphorylation and its optimization , and its application in the detection of secreting Aβ and Tau phosphorylation ability in pathology in vitro, belonging to the technical field of biotechnology and genetic engineering. Background technique [0002] Alzheimer's disease (Alzheimer's Disease, hereinafter referred to as AD) is one of the most common and most destructive systemic degenerative diseases in the elderly. At present, it has two main pathological features: 1. Intracellular amyloid precursor protein is wrongly sheared to produce amyloid bata, and Aβ is secreted to the outside of the cell to form amyloid plaques; 2. Hyperphosphorylation of tau protein leads to neuronal hyperphosphorylation Adhesion between each other hinders nerve...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/12C12N5/10C12Q1/02
CPCC12N15/86C07K14/4711C12N5/0682C12N5/0656G01N33/5008C12N2740/15043C12N2800/107C12N2510/00C12N2503/02G01N2500/10
Inventor 索广力于延珍梁瑜乔勇张书忙肖同乾
Owner SHANGHAI UNIV
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