CN113462650A

CN113462650A - Alzheimer's disease cell model and construction method thereof

Info

Publication number: CN113462650A
Application number: CN202110667453.XA
Authority: CN
Inventors: 赵海誉; 王琳娜; 边宝林; 王宏洁; 魏晓露; 司南; 周严严
Original assignee: Institute of Materia Medica of CACMS
Current assignee: Institute of Materia Medica of CACMS
Priority date: 2021-06-16
Filing date: 2021-06-16
Publication date: 2021-10-01

Abstract

The invention discloses an Alzheimer's disease cell model and a construction method thereof, belonging to the technical field of cell models. The cell model of the present invention is a human astroglioma cell carrying and expressing the APP695 gene and the PS1-DE9 gene, and the construction method of the cell model is to connect the APP695 gene and the PS1-DE9 gene with a lentivirus expression vector to construct a recombinant After the plasmid is introduced by lentivirus, a cell line capable of stably expressing the APP695 gene and PS1-DE9 gene is obtained through screening and culture. Research the pathogenesis and pathological process of Alzheimer's disease and screen new drugs to prevent and treat Alzheimer's disease.

Description

Alzheimer's disease cell model and construction method thereof

Technical Field

The invention relates to the technical field of cell models, in particular to an Alzheimer's disease cell model and a construction method thereof.

Background

Alzheimer's Disease (AD) is a central nervous system disorder characterized by progressive cognitive dysfunction and behavioral impairment, and there is no specific treatment, Senile Plaque (SP) and neurofibrillary tangle (NFT) are pathological changes characteristic of AD, while beta amyloid protein (a β) is a major component of senile plaque of AD patients, and a β is produced by hydrolysis of amyloid precursor protein (β -amyloid precursor protein, APP) by β -secretase and γ -secretase.

APP is a single transmembrane protein that is widely found in various tissues in the body, but is expressed at the highest level in brain tissue, with APP695 being most abundant in the brain, and produced primarily by neurons. Overexpression of APP increases a β levels and also inhibits the transmission of synaptic excitation. Mutations in the Amyloid Precursor Protein (APP), presenilin 1(presenilin 1, PS1), presenilin 2(presenilin 2, PS2) genes are implicated in patients with early-onset familial AD.

Currently, the cell models for studying alzheimer disease are mostly as follows: directly inducing nerve cells to generate protein aggregation by the Abeta to prepare an AD cell model; a lentivirus-stable single PS1 mutant AD cell model; a single APP mutant AD cell model with lentivirus stable transfer. The model simulation of the A beta direct induction nerve cells is an A beta injury nerve cell model, is suitable for researching medicaments with neuroprotection, but is not suitable for pharmacodynamic research and mechanism research on reducing the generation of the A beta; the AD symptoms expressed by the single transgenic model induced by lentivirus have limitation, and the over-expression amount of the Abeta is less than that of the double transgenic cells.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide an Alzheimer disease cell model for stably expressing APP695 gene and PS1-DE9 gene at the same time.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides an alzheimer's disease cell model U251MG/(human) APP695/PS1- (DE9), which is a mammalian cell carrying and capable of expressing the APP695 gene and the PS1-DE9 gene.

Further, the mammalian cell is a U251MG cell.

Furthermore, the nucleotide sequence of the APP695 gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the PS1-DE9 gene is shown as SEQ ID NO. 2.

In a second aspect, the present invention also provides a method for constructing a cell model of alzheimer's disease, comprising the following steps:

s1, PCR amplification of a target gene;

s2, homologous recombination and transformation of target genes;

s3, slow virus packaging and titer detection;

s4, lentivirus infection and sorting of stable strains.

The method for constructing the alzheimer' S disease cell model further includes a step S5 of evaluating a cell line: whether the cell is used as a cell model is evaluated according to the results of western detection, qPCR detection and Elisa detection.

Further, the target gene in step S1 is composed of three parts: a: APP695 fragment (SEQ ID NO: 1), B: ZYY1 fragment, C: PS1-DE9 fragment (shown in SEQ ID NO:2 in sequence), the part B is used as a bridge to connect the part A and the part C to obtain a target gene, and the primer sequence of the part B is as follows:

ZYY1-F1：GGATCCGGCGCAACAAACTTCTC；

ZYY1-R1：GGTTGTGGCCATATTATCATCGT。

further, the lentiviral expression vector in step S2 is a PLENTI-C-MGFP plasmid.

Further, the lentivirus packaging system in step S3 is a three-plasmid system.

Further, the virus MOI value of the lentivirus infection in step S4 is 20.

In a third aspect, the invention also provides an application of the Alzheimer disease cell model in screening of drugs for treating Alzheimer disease.

Compared with the prior art, the invention has the beneficial effects that:

the Alzheimer disease cell model can stably over-express APP695, PS1-DE9 and A beta 1-42 proteins at the same time, compared with a single transgenic model, the over-expression amount of the A beta is large, the A beta plaque can be enhanced and advanced, and the form and symptoms of the Alzheimer disease are more consistent.

In addition to over-expressing the above proteins, the alzheimer's disease cell model of the present invention has features of neuroinflammation. The invention can simulate the symptom of the Alzheimer disease and provides an effective tool for researching the pathogenesis and pathological process of the Alzheimer disease and screening new drugs for preventing and treating the Alzheimer disease.

Drawings

The invention is further illustrated by means of the attached drawings, but the embodiments in the drawings do not constitute any limitation to the invention, and for a person skilled in the art, other drawings can be obtained on the basis of the following drawings without inventive effort.

FIG. 1 is a morphological feature diagram of the Alzheimer's disease cell model under an optical microscope;

FIG. 2 is a morphological feature diagram of the Alzheimer's disease cell model under an optical microscope;

FIG. 3 is a Western blot analysis chart of the Alzheimer's disease cell model of the present invention;

FIG. 4 is a graph of the qPCR assay results for a model of Alzheimer's disease cells of the present invention;

FIG. 5 is a graph of the qPCR assay results for the Alzheimer's disease cell model of the present invention.

Detailed Description

The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1

Step 1: PCR amplification of target genes

According to the coding region (CDS) of the genes APP695 and PS1-DE9 provided by NCBI, the genes APP695 and PS1-DE9 are synthesized, and the target gene consists of three parts: a: APP695 fragment, the nucleotide sequence of which is shown in SEQ ID NO:1, B: ZYY1 fragment, C: the nucleotide sequence of the PS1-DE9 fragment is shown as SEQ ID NO:2, 3 pairs of specific primers are designed to respectively carry out PCR amplification on a target fragment A, B, C, wherein a part B serves as a bridge to connect a part A and a part C to obtain a target gene, and the primer sequence of the part B is as follows:

ZYY1-F1：GGATCCGGCGCAACAAACTTCTC；

ZYY1-R1：GGTTGTGGCCATATTATCATCGT。

the PCR reaction system was 50. mu.l containing 5 XBuffer Buffer 10. mu.l, 1 XDNTP (2.5mmol/L) 4. mu.l, forward and reverse primers (10. mu. mol/L) 1. mu.l each, PrimeSTAR 0.5. mu.l, and sterilized water to 50. mu.l.

The PCR reaction conditions are as follows: performing pre-denaturation at 98 ℃ for 3min, and then performing 30 cycles, wherein the main cycle comprises denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 60s, and after the completion, extension at 72 ℃ for 10min, and storing the product at 4 ℃.

The PCR products were identified by electrophoresis on 1.2% agarose gel, 50. mu.l of the mixture of the PCR products and the loading buffer was added to each well, 5. mu.l of Maker was added thereto, horizontal electrophoresis was performed for 30min, and the presence of bands was observed.

Step 2: homologous recombination and transformation of target gene

Recovering target fragments by gel purification, adding the target DNA fragments and a lentivirus expression vector PLENTI-C-MGFP which is digested by BamHI-HF and EcoRI-HF into a test tube in a molar ratio of 2: 1 for recombination reaction, uniformly mixing, incubating at 42 ℃ for 30min, transferring to ice, placing for 2-3min, taking 10 mul of reaction liquid, converting into competent cells, uniformly mixing the contents, placing in ice for 30min, placing the tube into a thermostatic waterbath kettle which is pre-warmed to 42 ℃ for 90s, quickly transferring the tube into an ice bath, cooling the cells for 2-3min, adding 900 mul of LB culture solution into each tube, transferring to a shaking bed at 37 ℃, incubating for 1h to recover the bacteria, taking just equivalent of the conversion liquid, coating the conversion liquid on an LB agar plate (containing antibiotics corresponding to the expression vectors), inverting the plate, and culturing for 16h at 37 ℃ in a thermostatic incubator. Transformants grown on the plate were picked and resuspended in 10. mu.l of LB medium, and 1. mu.l of the medium was used as a template for colony PCR identification. And inoculating positive clone after the identification is correct, preserving the seeds, subpackaging 100 mu l for sequencing, and inoculating the extracted plasmid after the sequencing is successful.

And step 3: lentiviral packaging and titer detection

293T lentivirus packaging cells (ATCC NO. CRL-3216) were stored in 10cm DMEM + 10% FBS, 1% Glutamax, 1% penicillin-streptomycin medium at 37 ℃ with 5% CO₂And culturing in a saturated humidity incubator, and observing that the transfection is carried out when the fusion degree under the microscope is about 80% -90%. 3 plasmid system for viral packaging: the vector plasmid 65. mu.g to which the target gene was ligated, the packaging plasmid dR8.965. mu.g and the VSVG plasmid 35. mu.g were put into a 15ml centrifuge tube and supplemented to 5ml with Opti-MEM culture medium. Another 15ml centrifuge tube was added with 500. mu.l of Trans-EZ solution and 4.5ml of Opti-MEM culture solution, and gently mixed by an electric pipette. The Trans-EZ diluent was added dropwise to the plasmid tube, and gently shaken well while adding. After incubation at room temperature for 20min, the cells were added to the cell culture dish. After 6h, the culture medium was changed with DMEM complete culture medium, and the culture supernatant was collected and observed after further 24h of culture. The culture was continued for 48h and the supernatant was collected again, dispensed into 50ml centrifuge tubes, centrifuged at 4 ℃ and 500 Xg for 10min to remove debris, filtered using a 0.22 μm PVDF filter, then dispensed into Ultra-clear SW 28 centrifuge tubes, centrifuged at 4 ℃ and 82700 Xg for 2h using an ultracentrifuge, the supernatant was discarded, the viral particles were resuspended using Opti-MEM, dissolved overnight at 4 ℃ and dispensed into EP tubes and stored in a-80 ℃ freezer.

The 24-well plates were seeded with 293T cells to a length of about 1X 10 cells per well⁵The virus was added dropwise for infection, and after 20h, the culture supernatant was removed and replaced with 500. mu.l of complete medium (high-sugar DMEM + 10% FBS), 5% CO₂The cultivation was continued for 48h in an incubator saturated with humidity. 0.2ml of a 0.25% pancreatin-EDTA solution was used to digest the cells, and the cells were collected by centrifugation. Extracting genome DNA according to the cell tissue genome extraction kit, and performing quantitative PCR analysis by using an ABI7500 quantitative system under the following reaction conditions: 5min at 95 ℃; 5s at 95 ℃; 34s at 65 ℃ for 40 cycles, and analyzing the data to determine the number of integrated viral copies per genome, the titer (integration units per ml, IU ml)^-1) The calculation formula of (2) is as follows: IU ml^-1＝(C×N×D×1000)/V。

Wherein: c-the average number of integrated viral copies per genome; n-the number of cells at the time of infection (approximately 1 × 10)⁵) (ii) a D ═ dilution factor of viral vector; v ═ the number of volumes of added diluted virus.

And 4, step 4: lentiviral infection and sorting of stable strains

U251MG cells were collected at 2X 10 per well⁵Cells, 3ml culture system, were seeded in 6-well plates. Adding concentrated lentivirus particles according to the MOI value of 20, setting a virus-free group as a negative control group, adding puro into the control group and an experimental group respectively according to the concentration of 2 mu g/ml after the virus is infected for 48 hours, observing every day after adding the medicine, adding puro into the control group and the experimental group according to the concentration of 1 mu g/ml every other day until all cells of the negative control group die, and displaying the phenotype of the stable strain obtained after screening the cell model under an optical microscope as shown in figures 1 and 2.

And 5: evaluation of cell lines

Selecting stable strains infected by lentivirus and screened: u251MG/(human) APP695/PS1- (DE9) -12(OE) cells and U251MG/(human) APP695/PS1- (DE9) -45(OE) cells were tested simultaneously with the control U251MG cells without added lentivirus.

The results of detection of APP695 protein, PS1-DE9 protein and A β 1-42 protein in lysates of 12(OE), 45(OE) and U251 cells using Western blotting are shown in FIG. 3, which concludes: the cell model contained APP, PS1 and A β 1-42 at higher levels than U251MG cells, with 12(OE) APP and A β 1-42 at higher levels than 45(OE) but PS1 at lower levels than 45 (OE). The cell model can stably over-express APP695, PS1-DE9 and A beta 1-42 protein.

The APP gene and PS1 gene expression levels in 12(OE) cells, 45(OE) cells and U251 cells were measured by qPCR method, wherein the APP gene expression results are shown in fig. 4 and the PS1 gene expression results are shown in fig. 5, and after quantification, the results are shown in table 1:

TABLE 1 fold expression of APP695 Gene and PS1-DE9 Gene in cells

Sample name	APP695 expression fold	Fold expression of PS1-DE9
			U251MG control
	1	1
			U251MG-12(OE)	98.7	5442.3
U251MG-45(OE)	44.48	1789.08

And (4) conclusion: through qPCR analysis, the gene expressions of APP695 and PS1-DE9 in U251MG-12(OE) and U251MG-45(OE) cells are all obviously improved. The cell model can stably over-express APP695 and PS1-DE9 genes.

Supernatants of U251MG-12(OE) cells and U251 cells in 24-well plates of 24H, 48H, 72H, respectively, were collected and assayed for inflammatory cytokine IL-6 levels using the Elisa assay. The results are shown in Table 2:

TABLE 2 content of inflammatory cytokine IL-6 in cells

And (4) conclusion: analysis of the results by Elisa experiments indicated that U251MG-12(OE) cells have inflammatory manifestations. The cell model is proved to have the characteristic of neuroinflammation.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Sequence listing

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<120> Alzheimer's disease cell model and construction method thereof

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Claims

1. An Alzheimer's disease cell model, characterized in that, the Alzheimer's disease cell model is a mammalian cell that carries and expresses the APP695 gene and the PS1-DE9 gene.

2 . The Alzheimer's disease cell model according to claim 1 , wherein the mammalian cells are U251MG cells. 3 .

3. The Alzheimer's disease cell model according to claim 1, wherein the nucleotide sequence of the APP695 gene is shown in SEQ ID NO: 1, and the nucleotide sequence of the PS1-DE9 gene As shown in SEQ ID NO:2.

4. a construction method according to the arbitrary described Alzheimer's disease cell model of claim 1-3, is characterized in that, comprises the following steps:

S1. PCR amplification of the target gene;

S2. Homologous recombination and transformation of the target gene;

S3, lentivirus packaging and titer detection;

S4. Lentivirus infection and sorting of stable strains.

5. the construction method of Alzheimer's disease cell model according to claim 4, is characterized in that, also comprises the step S5 that cell line is evaluated: according to western detection, qPCR detection, Elisa detection result to evaluate whether cell line is used as cell model.

6. The construction method of Alzheimer's disease cell model according to claim 4, wherein the target gene in the step S1 is composed of three parts: A: APP695 fragment, the nucleotide sequence is such as SEQ ID NO : 1, B: ZYY1 fragment, C: PS1-DE9 fragment, and the nucleotide sequence is shown in SEQ ID NO: 2; the B part acts as a bridge to connect the A part and the C part to obtain the purpose gene, the primer sequence of the B part is:

ZYY1-F1:GGATCCGGCGCAACAAACTTCTC;

ZYY1-R1: GGTTGTGGCCATATTATCATCGT.

7. The method for constructing Alzheimer's disease cell model according to claim 4, wherein the lentiviral expression vector of the homologous recombination of the target gene in the step S2 is a PLENTI-C-MGFP plasmid.

8 . The method for constructing Alzheimer's disease cell model according to claim 4 , wherein in the step S3, the lentiviral packaging system is a three-plasmid system. 9 .

9 . The method for constructing an Alzheimer's disease cell model according to claim 4 , wherein the MOI value of the lentivirus-infected virus in the step S4 is 20. 10 .

10. An application of the Alzheimer's disease cell model according to any one of claims 1-3 in screening for a drug for treating Alzheimer's disease.