WO2023241118A1

WO2023241118A1 - Anti-senescent drug screening method

Info

Publication number: WO2023241118A1
Application number: PCT/CN2023/080293
Authority: WO
Inventors: 张传茂; 王向阳
Original assignee: 北京大学
Priority date: 2022-06-13
Filing date: 2023-03-08
Publication date: 2023-12-21
Also published as: CN115044639A

Abstract

The present invention relates to the technical field of genetic engineering, and in particular to an anti-senescent drug screening method. The screening method comprises: treating a GFP-progerin protein overexpressing cell by using a drug to be screened; comparing the treated GFP-progerin protein overexpressing cell with a GFP-progerin protein overexpressing cell that is not treated by the drug to be screened; and determining, according to a change in cell morphology, an anti-senescent function of the drug to be screened. The present invention finds that a progerin protein positioned on a nuclear membrane can cause a cell to show obvious characteristics of senescence, including nuclear membrane shrinkage and nuclear membrane budding. However, a progerin protein positioned in a cell nucleus would not cause a cell to show obvious characteristics of senescence. If a drug has an anti-senescent effect, a progerin protein positioned on a nuclear membrane would be transferred into a cell nucleus. On this basis, the present invention provides an anti-senescent drug screening method, capable of accurately and efficiently screening out an anti-senescent drug.

Description

A screening method for anti-aging drugs

cross reference

This application claims the priority of Chinese patent application No. 2022106648794, which was submitted on June 13, 2022 and is titled "A screening method for anti-aging drugs". The entire disclosure content is incorporated herein by reference in its entirety.

Technical field

The invention relates to the technical field of genetic engineering, and in particular to a screening method for anti-aging drugs.

Background technique

Senescent cells exhibit a variety of typical characteristics, including increased DNA damage, increased p16, increased β-galactosidase activity, and increased senescence-related secretory phenotypes. The idea of developing traditional anti-aging drugs is to kill senescent cells, and the specific strategy is to develop drugs based on the typical characteristics of senescent cells. For example, specifically eliminate cells with high p16 expression, design lead drugs and use the activity of β-galactosidase to kill cells, inhibit the expression of aging-related secreted factors, etc. The anti-aging drugs selected by these methods can inhibit cellular aging and adult aging to a certain extent.

Typical characteristics of senescent cells also appear in normal cells. For example, senescence-related secreted factors can promote embryonic structure recognition, tissue remodeling and repair, etc. The inhibition of their expression will disrupt the normal functions of the body. β-Galactosidase is also expressed in non-senescent tissue cells, such as bone marrow. Therefore, anti-aging drug screening based on the characteristics of senescent cells has major side effects and affects the functions of normal tissue cells.

Contents of the invention

In order to solve the problems existing in the existing technology, the present invention provides a screening method for anti-aging drugs based on the principle that the positioning of progerin protein on the nuclear membrane in cells overexpressing GFP-progerin protein will change based on the anti-aging effect of the drug. , drugs with anti-aging effects can be screened accurately and quickly.

In a first aspect, the present invention provides a method for screening anti-aging drugs, including:

The cells overexpressing the GFP-progerin protein are treated with the drug to be screened and compared with the cells overexpressing the GFP-progerin protein that are not treated with the drug to be screened. The resistance of the drug to be screened is judged based on the changes in cell morphology. Aging effect.

Further, the cells overexpressing GFP-progerin protein are primary cells overexpressing GFP-progerin protein.

Further, the progerin protein includes the amino acid sequence shown in SEQ ID NO.1.

Further, the cells overexpressing the GFP-progerin protein are prepared by constructing the gene encoding the progerin protein into a lentiviral vector and then transfecting HK293T cells, collecting the viral fluid and then infecting primary cells.

Further, the gene encoding the progerin protein includes the nucleotide sequence shown in SEQ ID NO. 2.

Further, the primary cells are human primary skin fibroblasts.

Further, the lentiviral vector includes one or more of pCDH, pQCXIP, pLVX or pLenti6/TR.

Further, judging the anti-aging effect of the drug to be screened based on changes in cell morphology is:

When one or more of the phenomena of nuclear membrane budding, nuclear membrane shrinkage, heterochromatin abnormalities, or the appearance of micronuclei are inhibited in cells overexpressing GFP-progerin protein that have been treated with the drug to be screened, it is judged that the The drugs to be screened have anti-aging effects.

When the localization of progerin protein in cells overexpressing GFP-progerin protein is changed after treatment with the drug to be screened, it is judged that the drug to be screened has an anti-aging effect.

Further, the positioning changes from positioning on the cell nuclear membrane to positioning within the cell nucleus.

The invention has the following beneficial effects:

The present invention provides an anti-aging drug screening method based on the GFP-progerin senescent cell model, which can achieve high-throughput and high-precision anti-aging drug screening. The present invention provides an efficient, stable and accurate drug screening platform, which can realize the screening of up to 1536 small molecule compounds at a time, achieving high-throughput and high-precision drug screening. With the help of this screening method, the present invention has successfully screened a variety of candidate drugs with anti-aging functions, and it has been verified that they also have the effect of inhibiting aging on patient cells, which affects progeria, aging, vascular sclerosis, tissue and organ fibrosis, The treatment of aging inflammation and other diseases provides new possibilities.

Description of the drawings

Figure 1 is a diagram showing the results of cell immunofluorescence detection of abnormal cell nuclei caused by GFP-progerin provided in Example 1 of the present invention.

Figure 2 is a graph showing the proportional statistical results of nuclear membrane budding caused by GFP-progerin provided in Example 1 of the present invention.

Figure 3 is a schematic diagram of the impact of progerin protein localization on cells in the GFP-progerin senescent cell model provided in Example 1 of the present invention.

Figure 4 is an overall flow chart of anti-aging drug screening provided in Embodiment 1 of the present invention.

Figure 5 is a schematic flow chart of high-throughput screening of anti-aging drugs provided in Embodiment 1 of the present invention.

Figure 6 is a schematic diagram of the effect of drug candidate 1 provided in Example 2 of the present invention on progerin localization.

Figure 7 is a schematic diagram of the effects of candidate drugs 2 and 3 provided in Example 2 of the present invention on progerin localization.

Figure 8 is a schematic diagram of the effects of candidate drugs 1, 2 and 3 on nuclear membrane budding provided in Example 2 of the present invention.

Figure 9 is a schematic diagram of the effects of candidate drugs 1, 2 and 3 provided in Example 2 of the present invention on the localization of progerin in cells of patients with progeria.

Figure 10 shows the effect of candidate drugs 1, 2 and 3 on progeria patient cells provided in Example 2 of the present invention. Schematic representation of the effects of nuclear membrane budding.

Detailed ways

Exemplary embodiments of the invention are provided in the following examples. The following examples are given by way of example only and are intended to assist those of ordinary skill in using the present invention. The described examples do not limit the scope of the invention in any way.

Example 1

This embodiment provides a method for screening anti-aging drugs. The process is as follows:

1. Experimental materials

Senescent cell model: GFP-progerin senescent cell model;

Medium: high sugar Dulbecco’s modified Eagle medium (DMEM) + 5-20% fetal calf serum + 100 U/mL penicillin + 100 μg/mL streptomycin;

The GFP-progerin senescent cell model was prepared by the following method:

(1) Isolation of human skin fibroblasts

First, skin tissue with a diameter of 1 mm was removed from the volunteers' skin and washed three times with phosphate buffer solution (PBS); then, excess fat tissue was removed with ophthalmic scissors on a clean workbench, and the remaining fat tissue was cut into pieces. Tissues were washed three times with PBS; then add 0.1-0.25% trypsin to immerse all tissue blocks, place them in a cell culture incubator containing 5% CO2, and digest at 37°C for 5-10 minutes; after digestion, add 5- Digestion was terminated in DMEM medium with 20% fetal calf serum. Then use a pipette to blow gently and repeatedly to disperse the cell clumps. Finally, transfer the cell suspension to a 35 mm culture dish, add 4 mL of DMED medium containing 5-20% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin, and place it in a cell culture incubator at 5% CO2 , culture at 37°C for 4-6 days. As the culture progresses, the culture medium can be added appropriately according to the amount of culture medium in the petri dish.

Finally, the isolated cells are primary human dermal fibroblasts (HDF). During the entire cell separation process, aseptic operation must be ensured, and double antibodies must be added to the culture medium to avoid contamination. After the cells are separated, mycoplasma testing must be performed to confirm that there is no mycoplasma contamination before the cells can be used for subsequent experiments. In order to facilitate subsequent experiments, as many low-generation HDFs as possible should be frozen. cell.

The donor skin sample is cut into small pieces with ophthalmic scissors, digested with trypsin and cultured in an incubator. The freed cells are skin fibroblasts. Isolated primary human skin fibroblasts (passage 3) were used in this example.

(2) Construction vector

Cloning of human progerin coding sequence:

Total RNA was extracted from blood or skin samples of progerin patients, and then a cDNA library was obtained by RT-PCR. Finally, the coding sequence was amplified by PCR using progerin primers.

pCDH-GFP-progerin vector construction:

The coding sequence of GFP was cloned from the pEGFP-C1 vector by PCR, and then the pCDH-CMV-MCS-EF1 vector was cut with XbaⅠ and NotⅠ double enzyme digestion, and the coding sequences of GFP and progerin were inserted respectively. Among them, GFP is located at the N-terminus of progerin. The vector was used for subsequent experiments after successful sequencing.

The PCR primer sequence of progerin is as follows:

progerin F：5’-CAGTCTAGAATGGTGAGCAAGGGCGAGGA-3’

progerin R: 5’-CAGGCGGCCGCTTACATGATGCTGCAGTTCT-3’.

(3)Virus packaging

Passage HEK293T into a 10cm culture dish, add 10mL of culture medium, and then place it in a cell culture incubator containing 5% CO2 for culture. After the cell density reaches 40-70%, the constructed pCDH-GFP-progerin vector, psPAX2, and pMD2.G are transfected and expressed using PEI at a ratio of 2:1:1. After 48 hours, collect the cell culture supernatant, add the sedimentation solution at the ratio of supernatant: PEG-itTM virus sedimentation solution = 1:4, mix well, and place it in a 4°C refrigerator for overnight sedimentation. Then centrifuge at 1500g for 30 minutes at 4°C to enrich the virus particles, and resuspend the virus in 1 mL of PBS.

(4) Sorting senescent cells

Passage HDF cells into a 10cm culture dish and add 10mL of culture medium for culture. When the cell density reaches 40-70%, add an appropriate amount of virus to infect the cells; after 6 hours, replace Fresh culture medium. After continuing to culture for 24-48 hours, absorb the culture medium in the culture dish and wash it twice with PBS, 2 mL each time; then absorb the PBS, add 1 mL of trypsin with a concentration of 0.25%, and shake the culture dish repeatedly with both hands to make the pancreas The enzyme evenly infiltrates the bottom of the culture dish; finally, place it in a cell culture incubator containing 5% CO2 and digest it at 37°C for 1-2 minutes; take out the digested cells and add 0.5 mL of ordinary culture medium to the culture dish to stop digestion; then pipette Repeatedly pipette the cells to mix them evenly, transfer them to a 1.5mL centrifuge tube, and centrifuge at 1000rpm/min for 4 minutes. Aspirate off the supernatant, add 1mL DMEM, pipet gently and repeatedly, mix the cells, and analyze by flow cytometry. The GFP-positive cells are sorted out by a sorting instrument and inoculated for amplification. The cells obtained are GFP-progerin senescent cells.

2. Drug screening process

The screening process is shown in Figure 4, and the details are as follows:

(1)Seed cells

Passage GFP-progerin senescent cells into a 10cm culture dish and add 10ml of culture medium for culture. When the cell density reaches 90-100%, senescent cells are digested with 0.25% trypsin, the cells are blown evenly and then seeded into a 96-well plate for culture.

(2)Drug handling

When the cell density reaches 60-80%, small molecule compounds are added to single wells of the 96-well plate and treated for 24 hours.

(3) High-throughput screening

The treated GFP-progerin senescent cells were observed on a high-content fluorescence microscope and a large-field fluorescence image was captured. As shown in Figure 5, GFP-progerin senescent cells were seeded into a 96-well plate, treated with small molecule compounds for 24 hours, and then screened by high-content fluorescence microscopy. The screening criterion was that GFP-progerin was localized in the nucleus. Finally, the single wells in the 96-well plate that change the positioning of GFP-progerin are screened, and the corresponding small molecule compounds are candidate drugs.

(4)Patient cell verification

Treating skin fibroblasts from progeria patients with drug candidates to further verify the effectiveness of the drug candidates. A small molecule that is effective against GFP-progerin senescent cells and progeria patient cells The compound is the final candidate drug screened.

As shown in Figure 1, the positioning of GFP-progerin-WT on the nuclear membrane will cause nuclear membrane budding and nuclear membrane shrinkage (WT), while the mutant GFP-progerin-C661S is localized in the nucleus and does not cause nuclear abnormalities (C661S ).

As shown in Figure 2, compared with GFP-progerin, the mutant GFP-C661S caused a lower proportion of nuclear envelope budding. These results indicate that localization of progerin in the nucleus can improve nuclear membrane abnormalities.

As shown in Figure 3, the localization of GFP-progerin on the nuclear membrane will cause nuclear membrane budding and nuclear membrane shrinkage (left picture), while inhibiting the localization of GFP-progerin on the nuclear membrane will return the cells to normal (right picture).

As shown in Figure 4, the anti-aging drug screening process includes: inoculation of GFP-progerin senescent cells, drug treatment, high-content fluorescence microscopy screening, and progeria patient cell verification.

Example 2

In this example, the anti-aging drugs screened in Example 1 were verified through cellular immunofluorescence experiments.

1. The experimental method of cell immunofluorescence is as follows:

(1) Passage the cells into a 35mm culture dish covered with a coverslip, add 3ml of culture medium, place it in a cell culture incubator containing 5% CO2, and culture at 37°C;

(2) When the cell density reaches 70-100%, wash the coverslip three times with PBS and then fix it with 4% paraformaldehyde (PFA) for 20 minutes;

(3) After washing three times with PBS, punch holes with 2.5% Triton-100 for 5 minutes;

(4) After washing three times with PBS, the fixed cells were labeled with antibodies. Antibodies for progerin, emerin, lamin A/C, and lamin B1 were added to 3% BSA in PBS buffer at a ratio of 1:200. The fixed cells were inverted in primary antibody dilution and incubated overnight at 4°C.

(5) After incubation, gently dip and wash the cells in PBS buffer three times;

(6) According to the species properties corresponding to the primary antibody, dilute the fluorescent secondary antibody in 3% BSA buffer at a ratio of 1:200, and then incubate the fixed cells upside down in the secondary antibody diluent at room temperature for 1 hr.

(7) Heat the Mowiol mounting medium in a 55°C oven in advance, which contains 1 μg/mL DNA dye DAPI and 2.5% anti-fade agent DABCO. The cells incubated with the secondary antibody were washed three times with PBS, then placed upside down on 15 μL of melted Mowiol mounting medium, and dried at room temperature in the dark for 1 hour. After the mounting medium solidified, microscopy imaging was performed.

(8) Use a DeltaVision microscope (equipped with an Olympus IX-71 inverted microscope, 100×/1.4N.A. oil objective lens and CCD camera) for microscopy imaging; and use Volocity 6.1.1 software to process the images.

2. Experimental results

As shown in Figure 6, green fluorescence represents GFP-progerin, red fluorescence represents nuclear membrane protein emerin, purple fluorescence represents lamin B1, and blue fluorescence represents chromatin. The results showed that the screened candidate drug 1 (potential drug 1) can change the localization of GFP-progerin, causing it to form aggregates in the nucleus.

As shown in Figure 7, green fluorescence represents GFP-progerin, red fluorescence represents lamin A/C, purple fluorescence represents lamin B1, and blue fluorescence represents chromatin. The results showed that the screened candidate drugs 2 and 3 (potential drug 2 and 3) can change the localization of GFP-progerin, causing it to form aggregates in the nucleus.

As shown in Figure 8, compared with the control group, the proportion of nuclear membrane budding in the candidate drug 1, 2, and 3 treatment groups (potential drug 1, 2, and 3) decreased significantly. These results indicate that drug candidates 1, 2, and 3 can significantly inhibit nuclear envelope budding.

As shown in Figure 9, green fluorescence represents progerin, red fluorescence represents lamin B1, and blue fluorescence represents chromatin. The results showed that the screened candidate drugs 1, 2 and 3 (potential drug 1, 2 and 3) can change the localization of progerin in the cells of patients with progeria, causing it to form aggregates in the nucleus.

As shown in Figure 10, compared with the control group, the proportion of nuclear membrane budding in the candidate drug 1, 2, and 3 treatment groups (potential drug 1, 2, and 3) decreased significantly. These results indicate that drug candidates 1, 2, and 3 can significantly inhibit nuclear membrane budding of progeria patient cells.

Although the present invention has been described in detail above using general descriptions and specific embodiments, description, but on the basis of the present invention, some modifications or improvements can be made, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Industrial applicability

The invention provides a screening method for anti-aging drugs. The cells overexpressing the GFP-progerin protein are treated with the drug to be screened and compared with the cells overexpressing the GFP-progerin protein that are not treated with the drug to be screened. The resistance of the drug to be screened is judged based on the changes in cell morphology. Aging effect. The present invention provides an anti-aging drug screening method based on the GFP-progerin senescent cell model, which can achieve high-throughput and high-precision anti-aging drug screening. It provides new possibilities for the treatment of diseases such as progeria, aging, vascular sclerosis, tissue and organ fibrosis, aging inflammation, etc., and has good economic value and application prospects.

Claims

A method for screening anti-aging drugs, which is characterized by including:

The cells overexpressing the GFP-progerin protein are treated with the drug to be screened and compared with the cells overexpressing the GFP-progerin protein that are not treated with the drug to be screened. The resistance of the drug to be screened is judged based on the changes in cell morphology. Aging effect.
The screening method according to claim 1, wherein the cells overexpressing GFP-progerin protein are primary cells overexpressing progerin protein.
The screening method according to claim 1 or 2, wherein the GFP-progerin protein includes the amino acid sequence shown in SEQ ID NO.1.
The screening method according to claim 1, wherein the cells overexpressing GFP-progerin protein are prepared in the following manner:

The gene encoding the progerin protein is constructed into a lentiviral vector and then transfected into HK293T cells. The viral fluid is collected and then infected into primary cells.
The screening method according to claim 4, wherein the gene encoding the progerin protein includes the nucleotide sequence shown in SEQ ID NO. 2.
The screening method according to claim 4, wherein the primary cells are human primary skin fibroblasts.
The screening method according to claim 4, wherein the lentiviral vector includes one or more of pCDH, pQCXIP, pLVX or pLenti6/TR.
The screening method according to claim 1, wherein the judgment of the anti-aging effect of the drug to be screened based on changes in cell morphology is:

When one or more of the phenomena of nuclear membrane budding, nuclear membrane shrinkage, heterochromatin abnormalities, or the appearance of micronuclei are inhibited in cells overexpressing GFP-progerin protein that have been treated with the drug to be screened, it is judged that the The drugs to be screened have anti-aging effects.
The screening method according to claim 1, wherein the judgment of the anti-aging effect of the drug to be screened based on changes in cell morphology is:

When the cells overexpressing GFP-progerin protein were treated with the drug to be screened, When the localization of GFP-progerin protein changes in cells, it is judged that the drug to be screened has an anti-aging effect.
The screening method according to claim 9, characterized in that the positioning changes from positioning on the cell nuclear membrane to positioning in the cell nucleus.