CN109762784B

CN109762784B - Application of clozapine in delaying aging of cultured mesenchymal stem cells

Info

Publication number: CN109762784B
Application number: CN201910242111.6A
Authority: CN
Inventors: 嵐山芮; 魏伟; 李祎
Original assignee: Guangzhou Ruiplatinum Health Technology Co Ltd
Current assignee: Guangzhou Ruiplatinum Health Technology Co Ltd
Priority date: 2019-03-27
Filing date: 2019-03-27
Publication date: 2021-05-11
Anticipated expiration: 2039-03-27
Also published as: CN109762784A

Abstract

The invention discloses an application of clozapine in delaying senescence of cultured mesenchymal stem cells, belonging to the technical field of cell culture.

Description

Application of clozapine in delaying aging of cultured mesenchymal stem cells

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to application of clozapine in delaying the aging of cultured mesenchymal stem cells.

Background

Mesenchymal Stem Cells (MSCs) have strong self-renewal and multi-directional differentiation potential and have been widely used in the field of tissue engineering and regenerative medicine. At present, after extensive research, MSC can be differentiated into osteoblasts, chondrocytes, adipocytes, nerve cells and the like, and has a wide application prospect in regenerative medicine.

In order to obtain sufficient numbers of MSCs for clinical treatment, MSCs need to be expanded in large amounts in vitro, however replicative senescence phenomena inevitably occur during subculture proliferation of stem cells. This was widely explored by research teams worldwide in order to slow down MSC aging that occurs during the culture process and better maintain stem cell characteristics. At present, the aging in the cell culture process is delayed mainly by methods of regulating the concentration and oxygen content of growth factors, antioxidants and nutrients, and the like, so that the proliferation of MSCs is promoted. Ito et al demonstrated that fibroblast growth factor 2 can slow down replicative senescence that occurs during long-term culture of human MSCs by reducing the expression levels of the P21, P53, P16 genes. Melatonin (N-acetyl-5-methoxy-tryptamine) can protect cell nucleus, mitochondria and DNA from oxidative stress damage by scavenging free radicals, so the melatonin also has the potential of resisting aging. In addition, histone deacetylase inhibitors, sirolimus, single-cell seaweed extract, etc. have also been demonstrated to slow down the aging of human umbilical cord MSCs.

If the aging of MSCs can be slowed down in the culture process, the application prospect of MSCs in the fields of regenerative medicine and tissue engineering can be wider, which can benefit thousands of patients. In addition, the research on aging slowing in the MSC culture process and the safe and effective method for seeking early intervention of the MSC culture process also have important reference significance for delaying aging of human bodies.

There are several deficiencies with current agents for delaying the senescence of MSCs: 1) high cost, such as growth factors, nutrients; 2) the components are unstable and often contain different types of impurities, which carries a series of unpredictable risks for clinical grade MSCs, such as single cell seaweed extracts.

Clozapine is widely used clinically for controlling insomnia, depression, epilepsy and other diseases, is only a representative atypical antipsychotic drug, and has better curative effect compared with other antipsychotic drugs. Currently, clozapine research is focused mainly on the therapeutic effects and the associated mechanisms of action in neurological disorders.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, provides the application of clozapine in delaying the aging of cultured mesenchymal stem cells, can safely and repeatedly realize the delaying of the aging of the mesenchymal stem cells, and has the advantages of low cost and simple and reliable process.

In order to achieve the purpose, the invention adopts the technical scheme that: the use of clozapine for delaying senescence in cultured mesenchymal stem cells.

As an improvement of the technical scheme, clozapine is firstly dissolved in ethanol and then diluted by a cell culture medium to prepare a clozapine diluent, and the clozapine diluent is added into the cell culture medium of the mesenchymal stem cells.

As a further improvement of the technical scheme, the concentration of clozapine in the clozapine dilution is 12.5-50 mug/mL.

As a further improvement of the technical scheme, the concentration of clozapine in the cell culture medium of the mesenchymal stem cells is 12.5-50 mu g/mL.

As an improvement of the technical scheme, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

In addition, the invention also provides application of clozapine in preparation of a medicine for delaying mesenchymal stem cell senescence.

The invention has the beneficial effects that: the invention provides the application of clozapine in delaying the aging of cultured mesenchymal stem cells, and the clozapine diluent used in the invention is simpler, safer and more efficient in the process of delaying the aging of mesenchymal stem cells, and has low cost; in addition, the clozapine storage solution prepared by the invention is prepared by dissolving clozapine in absolute ethyl alcohol; when the clozapine storage solution is used, the culture medium is used as a solvent, and the clozapine storage solution is diluted to 12.5-50 mu g/mL to a great extent, so that the clozapine storage solution has almost no toxic effect on cells, and the effect of the clozapine in the cell culture process is ensured.

Drawings

FIG. 1 shows the results of the expression of surface antigens of P8 umbilical cord mesenchymal stem cells by clozapine; wherein a is control, b is 12.5 mu g/mL clozapine diluent; c is a clozapine dilution of 25. mu.g/mL, d is a clozapine dilution of 50. mu.g/mL;

FIG. 2 is a line graph showing the expression of surface antigens of P8 umbilical cord mesenchymal stem cells under the action of clozapine;

FIG. 3 is a staining pattern for beta-galactosidase from umbilical cord mesenchymal stem cells; wherein a is control, b is 12.5 mu g/mL clozapine diluent; c 25. mu.g/mL clozapine dilution and d 50. mu.g/mL clozapine dilution.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed description and accompanying drawings.

Preparation of umbilical cord mesenchymal stem cells

1) Cleaning umbilical cord with sterile normal saline, cutting into small segments, placing into an empty culture dish, tearing the umbilical cord with tissue forceps, stripping two arterial blood vessels, and tearing off Walton's gel distributed around the blood vessels; putting the separated Wharton's jelly into a 50mL centrifuge tube, adding 2mL culture medium, shearing the Wharton's jelly into fragments with the size of 1mm by using scissors, adding 6mL primary culture medium, uniformly mixing, and uniformly putting into 6T 25 culture bottles;

2) placing the T25 culture flask into an incubator at 37 deg.C and 5% CO₂Culturing;

3) when 2-3 colonies grow out from each T25 culture bottle and each colony has at least hundreds of cells, pouring out the culture medium and the tissue blocks in the culture bottles, and adding 2mL of culture medium for passage into each culture bottle; placing into an incubator at 37 deg.C and 5% CO₂And (5) culturing.

4) After culturing for 2 days, adding physiological saline into each culture bottle, washing for 2 times, pouring out the physiological saline as much as possible after washing, then adding pancreatin, digesting for one minute, enabling cells to be visible to naked eyes to be separated from the bottom of the bottle in a flaky manner, tapping the culture bottle, observing under a microscope, and adding 2-3 mL of culture medium to stop digestion when all the cells are separated from the wall;

5) pouring the culture medium into a centrifuge tube, centrifuging at the rotation speed of 600g for 4 minutes at the temperature of 20 ℃;

6) discarding supernatant of centrifuged cells, resuspending the culture medium, inoculating the suspension into a T75 culture flask, adding 15mL of the culture medium, shaking, placing the mixture into an incubator at 37 ℃ and 5% CO₂Culturing, wherein the cells are P1 generation;

7) when the flask was full of cells, subculture was continued.

Clozapine-treated mesenchymal stem cells

1) Dissolving clozapine in absolute ethyl alcohol to prepare a clozapine storage solution;

2) diluting the clozapine stock solution with cell culture medium to form clozapine dilutions of 12.5. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL;

3) the P3 generation cells were inoculated into 12T 25 flasks, one group in each of three flasks, and specifically grouped as follows: (I) the method comprises the following steps Control group (containing no clozapine); (II): adding 3mL of clozapine dilution with the concentration of 12.5 mu g/mL; (III): adding 3mL of clozapine dilution with the concentration of 25 mug/mL; (IV): adding 3mL of clozapine with a concentration of 50. mu.g/mL; shaking, adding 5% CO at 37 deg.C₂Culturing in an incubator;

4) when the cells in the bottle grow full, the cells are passaged, the number of the passaged cells in each bottle is consistent, and the clozapine diluent added in each group is as described in the step 3); the above steps are repeated until generation P8.

Flow assay

1) Taking out 2 bottles of T25 cells growing completely from each group, pouring out the culture solution, washing with normal saline, and digesting with pancreatin;

2) putting each group of cells into a centrifuge tube, centrifuging at the rotating speed of 600g for 4 minutes at the temperature of 20 ℃;

3) centrifuging, removing supernatant, adding physiological saline, blowing, mixing uniformly, centrifuging again at the rotation speed of 600g for 4 minutes at the temperature of 20 ℃;

4) after centrifugation, adding 8mL of physiological saline into each group, blowing, beating and uniformly mixing, counting cells of each group, and adjusting to make the number of the cells of each group consistent; and (5) sending the flow to detect.

As shown in FIG. 1 and FIG. 2, the ratios of CD90 positively expressed in MSCs cells were 98.64%, 99.47%, 99.13% and 99.42% respectively after the samples were treated with Control, 12.5. mu.g/mL, 25. mu.g/mL and 50. mu.g/mL clozapine dilutions; the proportion of positively expressed CD105 in the MSCs cells is 86.26%, 91.59%, 91.72% and 92.31% respectively; the proportion of positively expressed CD73 in MSCs cells was 90.80%, 93.60%, 95.51% and 95.01%, respectively. The MSCs with high expression of CD90, CD105 and CD73 have the function of delaying cell senescence, and the proportion of MSCs positively expressing CD90, CD105 and CD73 is obviously increased due to the fact that the MSCs are cultured by the clozapine diluent, so that the clozapine can be used for delaying the senescence of cultured mesenchymal stem cells.

Staining with beta-galactosidase (beta-gal)

1) After digesting and centrifuging the remaining one bottle of just full T25 cells, each group of cells was mixed with a culture medium containing clozapine, and the cells of each group were counted and adjusted so that the number of cells of each group was uniform (5X 10)⁵one/mL), then inoculated in 6-well plates, two wells for each group, 2mL for each well, blown evenly, placed at 37 ℃ in 5% CO₂Culturing in an incubator;

2) the beta-galactosidase staining kit is put in a refrigerator at 4 ℃ for unfreezing 4 hours in advance;

3) after the iron wall of the cells in the 6-well plate is removed, the culture solution of each group of cells is sucked out, washed once by using normal saline, added with 1mL of beta-galactosidase staining fixing solution and fixed for 15min at room temperature;

4) removing the fixing solution by suction, and washing with normal saline for 3 times;

5) absorbing normal saline, and adding 1mL of dyeing working solution into an enzyme hole;

6) incubating at 37 ℃ overnight, and wrapping a 6-hole plate with a preservative film during incubation so as to prevent the staining solution from evaporating;

7) after incubation was complete, photographs were taken under the microscope.

Senescent cells have a beta-galactosidase with high enzymatic activity at pH 6.0. Beta-gal is the earliest and most widely used one of the lysosomal-derived markers of aging, capable of increasing lysosomal biosynthesis in aging cells and progressively accumulating in vivo as the cells age. After culturing from P3 to P8, as shown in FIG. 3, the cells in the Control group are observed to be large in volume under an optical microscope, and show a large amount of blue (expressing a large amount of beta-galactosidase) and show a more obvious aging characteristic; after each sample was treated with 12.5, 25, 50 μ g/mL dilutions of clozapine, the cell volume was relatively small and the blue color was light, indicating that clozapine could be used to delay senescence of cultured mesenchymal stem cells.

Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of clozapine in preparing a medicine for delaying the aging of mesenchymal stem cells; the preparation method is characterized in that clozapine is dissolved in ethanol firstly and then diluted by a cell culture medium to prepare clozapine diluent, and the clozapine diluent is added into the cell culture medium of mesenchymal stem cells; the concentration of clozapine in the clozapine diluent is 12.5-50 mug/mL; the mesenchymal stem cells are umbilical cord mesenchymal stem cells.