Effect of deuterium-depleted water on continuous growth of human mesenchymal stem cells

Abstract

The invention discloses an effect of deuterium-depleted water on continuous growth of human mesenchymal stem cells, and belongs to the field of biomedicine. The invention relates to a medicine for promoting the growth of human mesenchymal stem cells. The medicine comprises deuterium-depleted water with the concentration of 25-50 ppm. On the other hand, the invention further relates to application of the deuterium-depleted water in preparation of drugs for promoting growth of human mesenchymal stem cells. Compared with the prior art, the invention discloses the effect of the deuterium-depleted water on the human mesenchymal stem cells, specifically, the deuterium-depleted water is used for improving the proliferation capacity or proliferation activity of the human mesenchymal stem cells, and the deuterium-depleted water is used for preparing the deuterium-depleted water for prolonging the life of the human mesenchymal stem cells and promoting the single cell clone formation capacity of the human mesenchymal stem cells. And a new research and development direction is provided for repairing and culturing human mesenchymal stem cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the effect of low-deuterium water on the continuous growth of human mesenchymal stem cells. Background Art

[0002] Stem cells are cells in the human body with continuous proliferative activity and multi-directional differentiation potential, and are the key cells for human growth and development, maintaining youthfulness, and repairing injuries.

[0003] In the prior art, the research on the effect and mechanism of low-deuterium water on mesenchymal stem cells is still lacking. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention proposes the effect of low-deuterium water on the continuous growth of human mesenchymal stem cells.

[0005] The object of the present invention can be achieved by the following technical solutions:

[0006] In a first aspect of the present invention, there is provided a drug for promoting the growth of human mesenchymal stem cells, comprising low-deuterium water with a concentration of 25-50 ppm.

[0007] In a second aspect of the present invention, there is provided a culture medium for human mesenchymal stem cells, comprising low-deuterium water with a concentration of 25-50 ppm.

[0008] In a third aspect of the present invention, there is provided an application of low-deuterium water in the preparation of a drug for promoting the growth of human mesenchymal stem cells, wherein the concentration of deuterium in the low-deuterium water is 25-50 ppm.

[0009] In a fourth aspect of the present invention, there is provided an application of low-deuterium water in the preparation of a culture medium for human mesenchymal stem cells, wherein the concentration of deuterium in the low-deuterium water is 25-50 ppm.

[0010] In a fifth aspect of the present invention, there is provided an application of low-deuterium water in the preparation of a drug for improving the proliferation ability or proliferation activity of human mesenchymal stem cells, wherein the concentration of deuterium in the low-deuterium water is 25-50 ppm.

[0011] In a sixth aspect of the present invention, there is provided an application of low-deuterium water in the preparation of a drug for prolonging the lifespan of human mesenchymal stem cells, wherein the concentration of deuterium in the low-deuterium water is 25-50 ppm.

[0012] In a seventh aspect of the present invention, there is provided an application of low-deuterium water in the preparation of a drug for promoting the single-cell cloning ability of human mesenchymal stem cells, wherein the concentration of deuterium in the low-deuterium water is 25-50 ppm.

[0013] In an eighth aspect of the present invention, there is provided a method for constructing a cell model, comprising at least one of the following steps:

[0014] Step 1:

[0015] Human umbilical cord mesenchymal stem cells are inoculated into a culture flask, and a culture medium prepared with water of different deuterium concentrations is added for continuous passage culture. Record the growth and proliferation of human umbilical cord mesenchymal stem cells in culture media with different deuterium concentrations, and calculate the doubling times by cell counting.

[0016] Step 2:

[0017] Human umbilical cord mesenchymal stem cells continuously cultured in culture media with different deuterium concentrations are inoculated into a 96-well plate, with 20,000 cells per well, and 3 wells are set as replicates. After continuous culture, an MTS drug dye is added, and the OD values of the cells in each well are detected to determine the proliferation activity of the cells in each well.

[0018] Step 3:

[0019] Human umbilical cord mesenchymal stem cells continuously cultured in culture media with different deuterium concentrations are inoculated into a 96-well plate, with 5,000 cells per well, and 3 wells are set as replicates. The cells in 3 replicate wells are digested and counted separately every day, and digested and counted regularly once. The continuous growth of the cells is statistically analyzed, and a growth curve is plotted according to the cell quantity.

[0020] Step 4:

[0021] Human umbilical cord mesenchymal stem cells continuously cultured in culture media with different deuterium concentrations are digested and harvested when the cells grow to 60 - 70% confluence, and made into a single-cell suspension. The single cells in the suspension are fixed with ethanol and stained with propidium iodide, and then the cells are detected by a flow cytometer, and the DNA content of each cell is statistically analyzed for cell cycle analysis.

[0022] Step 5:

[0023] Human umbilical cord mesenchymal stem cells continuously cultured in culture media with different deuterium concentrations are inoculated into single wells of a 6-well plate at 50, 100, and 200 respectively. After continuous culture in culture media with different deuterium concentrations for 10 - 12 days, the single-cell clones in each well are stained with crystal violet dye, and the number of blue-purple monoclonal cells in each well is counted by photographing.

[0024] Step 6:

[0025] Human umbilical cord mesenchymal stem cells continuously cultured in culture media with different deuterium concentrations are inoculated into a 6-well plate. The cells are fixed with formalin, and then the cells are stained with X-Gal for β-galactosidase. After staining, photographs are taken under a microscope, and the number and proportion of blue-positive senescent cells of human umbilical cord mesenchymal stem cells in culture media with different deuterium concentrations are statistically analyzed.

[0026] Optionally, the culture medium contains α-MEM basal medium and serum-free additives.

[0027] Optionally, the different deuterium concentrations include 25 ppm, 50 ppm, and 150 ppm.

[0028] Advantages of the present invention:

[0029] The present invention reveals the effects of low-deuterium water on human mesenchymal stem cells from multiple aspects, specifically including enhancing the proliferation ability or proliferation activity of human mesenchymal stem cells, low-deuterium water in the preparation for extending the lifespan of human mesenchymal stem cells, and promoting the single-cell cloning formation ability of human mesenchymal stem cells, providing a new research and development direction for the repair and culture of human mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below with reference to the accompanying drawings.

[0031] Figure 1 Shows the continuous growth of MSCs of the present application in a low-deuterium water culture medium;

[0032] Figure 2 Shows the detection of the proliferation activity of MSCs of the present application in a low-deuterium water culture medium;

[0033] Figure 3 Shows the determination of the growth curve of MSCs of the present application in a low-deuterium water culture medium;

[0034] Figure 4 Shows the cell cycle detection results of MSCs of the present application after being cultured in different low-deuterium water culture media;

[0035] Figure 5 Shows the single-cell cloning formation determination of MSCs of the present application cultured in different low-deuterium waters;

[0036] Figure 6 Shows a schematic diagram of the determination results of the effect of low-deuterium water of the present application on the senescence of MSCs. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] In some embodiments of the present application, the effect of low-deuterium water on human mesenchymal stem cells (hMSCs)

[0039] on continuous growth

[0040] Research method: β-Galactosidase (β-GAL) staining;

[0041] To study the effects of low-deuterium water on the growth activity, continuous proliferation ability, senescence and lifespan of human umbilical cord mesenchymal stem cells during continuous culture

[0042] Research objectives

[0043] 1. Select two independently sourced human umbilical cord mesenchymal stem cells, UC-MSCs / 14 and MSCs / 71. Prepare cell culture media with purified water of three different deuterium concentrations (25 ppm, 50 ppm, and 150 ppm), and then conduct continuous in vitro culture for up to 80 days. Each strain of cells is passaged at least 20 times, with nearly 50 cell divisions. Detect the continuous growth, lifespan, and senescence of the three strains of cells during this period;

[0044] 2. After long-term acclimation culture in media with different deuterium concentrations, conduct the following experimental detections on each cell to determine the differences in cell health status and physiological functions. The specific experimental design is as follows:

[0045] A. Cell growth, proliferation activity, and lifespan

[0046] · MTS proliferation assay;

[0047] · Plot cell growth curves;

[0048] · Cell cycle;

[0049] · Monoclonal formation rate of stem cells;

[0050] B. Senescence state of stem cells

[0051] · β-Galactosidase (β-GAL) staining;

[0052] Example 1: Effects of low-deuterium water on the continuous growth and lifespan of MSCs

[0053] Experimental operation: Inoculate human umbilical cord mesenchymal stem cells (p3) MSCs / 14 and MSCs / 71 from different individuals into T-25 culture flasks, and add media (α-MEM basal medium and serum-free additives) prepared with water of different deuterium concentrations, namely 25 ppm, 50 ppm, and 150 ppm, for continuous passage culture. Passage once every 3 - 4 days and continuously culture for more than 70 days. Record the growth and proliferation of MSCs / 14 and MSCs / 71 in the three deuterium concentration media, and calculate the doubling times PD = log2 (number of harvested MSCs / number of inoculated MSCs) by cell counting.

[0054] Results: As Figure 1As shown, the continuous growth of human mesenchymal stem cells from different sources in media (α-MEM and serum-free additives) with different deuterium concentrations (25 ppm, 50 ppm, and 150 ppm) shows that the continuous growth ability of MSCs / 14 stem cells in 25 ppm and 50 ppm is slightly better than that in 150 ppm normal water; while MSCs / 71 stem cells show relatively similar growth characteristics. Conclusion: Media prepared with 25 ppm and 50 ppm low-deuterium water have a certain promoting effect on the continuous growth ability of stem cells, and can appropriately improve the activity and lifespan of stem cells.

[0055] Example 2: Effect of Low-Deuterium Water on the Proliferation Activity of MSCs

[0056] Experimental operation: MSCs / 14 and MSCs / 71 cells continuously cultured in media with three deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm were inoculated into 96-well plates, with 20,000 cells per well and 3 replicate wells. After culturing for 48, 72, and 96 hours respectively, MTS drug dye was added, and the OD values of the cells in each well were measured to determine the proliferation activity of the cells in each well.

[0057] Results: As Figure 2 shown, after continuous culture in media with different deuterium concentrations, the MTS proliferation activity of each group of stem cells was measured. Compared with 150 ppm normal water, the proliferation activity of stem cells cultured in 25 ppm and 50 ppm low-deuterium water media was significantly improved, and the results of the two independent stem cells were relatively consistent. *, significant difference; ***, extremely significant difference. Conclusion: 25 ppm and 50 ppm low-deuterium water can significantly improve the proliferation activity of stem cells.

[0058] Example 3: Effect of Low-Deuterium Water on the Short-Term Proliferation Ability of MSCs

[0059] Experimental operation: MSCs / 14 and MSCs / 71 cells continuously cultured in media with three deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm were inoculated into 96-well plates, with 5,000 cells per well and 3 replicate wells. The cells in 3 replicate wells were digested and counted every day for 192 hours (8 days), and counted every 24 hours. The continuous growth of the cells was statistically analyzed, and the growth curve was plotted according to the cell number.

[0060] Results: As Figure 3 shown, after continuous culture in media with different deuterium concentrations, the production curves of the two stem cells were measured. Compared with 150 ppm normal water, the growth rate of stem cells cultured in 25 ppm and 50 ppm low-deuterium water media was relatively fast, and the results of the two independent stem cells were relatively consistent. Conclusion: 25 ppm and 50 ppm low-deuterium water can increase the growth rate of stem cells.

[0061] Example 4: Effect of Low-Deuterium Water on the Cell Cycle of MSCs

[0062] Experimental operation: MSCs / 14 and MSCs / 71 cells continuously cultured in media with three deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm were digested and harvested when the cells grew to 60 - 70% confluence, made into single-cell suspensions, fixed with ethanol, and stained with propidium iodide (PI). Then, the cells were detected by flow cytometry, and the DNA content of each cell was counted for cell cycle analysis.

[0063] Results: After continuous culture in media with different deuterium concentrations, the cell cycles of the two groups of stem cells were detected. Compared with normal water at 150 ppm, the overall proportions of the G2 phase and S phase of stem cells cultured in low-deuterium water at 25 ppm and 50 ppm were higher, indicating that the proliferation activity of stem cells cultured in low-deuterium water was more vigorous. Conclusion: Low-deuterium water at 25 ppm and 50 ppm can improve the proliferation activity of stem cells and increase the proportion of cells in the G2 phase and S phase.

[0064] Example 5: Effect of Low-Deuterium Water on the Single-Cell Cloning Ability of MSCs

[0065] Experimental operation: MSCs / 14 and MSCs / 71 cells continuously cultured in media with three deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm were seeded at 50, 100, and 200 cells per well in 6-well plates respectively, and then continuously cultured in media with three deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm for 10 - 12 days. The single-cell clones in each well were stained with crystal violet solution, and the number of blue-violet monoclonal cells in each well was counted by taking pictures. The cloning efficiency of each cell was calculated by the formula: Cloning efficiency = number of cell clones in the well / number of seeded cells × 100%.

[0066] Results: After continuous culture with different deuterium concentrations, a single-cell cloning experiment was carried out on the two strains of stem cells. The single-cell cloning ability of the MSCs / 71 cell line cultured in low-deuterium water at 25 ppm was significantly better than that in normal water at 150 ppm, but the cloning efficiencies of the MSCs / 14 cell line in the three media were similar, with no significant difference (*, significant difference; ***, extremely significant difference). Conclusion: Low-deuterium water at 25 ppm has an obvious promoting effect on the single-cell cloning ability of MSCs / 71 cells.

[0067] Example 6: Effect of Low-Deuterium Water on the Senescence of MSCs Stem Cells

[0068] Experimental operation: MSCs / 14 and MSCs / 71 cells that had been continuously cultured in media with three deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm respectively were seeded into 6-well plates. After 48 hours, the cells were fixed with formalin, and then β-galactosidase staining was performed on the cells using X-Gal. After staining, photographs were taken under a microscope, and the number and proportion of blue-positive senescent cells of MSCs / 14 and MSCs / 71 under media with different deuterium concentrations were counted.

[0069] Results: After continuous culture in media with different deuterium concentrations, β-galactosidase senescence staining was performed on the two types of stem cells. The cells showing blue were positive senescent cells. The results showed that media prepared with low-deuterium water at 25 ppm and 50 ppm could significantly reduce the senescence proportion of human mesenchymal stem cells (the statistical results are shown in Figure 6 B) The difference was extremely significant. *, the difference was significant; ***, the difference was extremely significant. Conclusion: Low-deuterium water at 25 ppm and 50 ppm can significantly reduce the senescence proportion of stem cells. In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0070] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art of this industry should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A drug for promoting the growth of human mesenchymal stem cells, characterized in that: Including deuterium-depleted water with a concentration of 25 to 50 ppm.

2. A human mesenchymal stem cell culture medium, characterized in that: Including deuterium-depleted water with a concentration of 25 to 50 ppm.

3. Use of deuterium-depleted water in the preparation of a drug for promoting the growth of human mesenchymal stem cells, characterized in that: The concentration of deuterium in the deuterium-depleted water is 25 to 50 ppm.

4. Use of deuterium-depleted water in preparing a culture medium for human mesenchymal stem cells, characterized in that: The concentration of deuterium in the deuterium-depleted water is 25 to 50 ppm.

5. Use of deuterium-depleted water in the preparation of a drug for improving the proliferation ability or proliferation activity of mesenchymal stem cells, characterized in that: The concentration of deuterium in the deuterium-depleted water is 25 to 50 ppm.

6. Use of deuterium-depleted water in the preparation of a drug for prolonging the life span of human mesenchymal stem cells, characterized in that: The concentration of deuterium in the deuterium-depleted water is 25 to 50 ppm.

7. Use of deuterium-depleted water in the preparation of a drug for promoting the single-cell cloning ability of human mesenchymal stem cells, characterized in that: The concentration of deuterium in the deuterium-depleted water is 25 to 50 ppm.

8. A method for constructing a cell model, characterized in that: At least one of the following steps is included: Step 1: Human umbilical cord mesenchymal stem cells were inoculated into culture bottles, and medium prepared with water of different deuterium concentrations was added for continuous subculture. The growth and proliferation of human umbilical cord mesenchymal stem cells in medium of different deuterium concentrations were recorded, and the doubling times were calculated by cell counting; Step 2: Human umbilical cord mesenchymal stem cells cultured in different deuterium concentration medium were seeded into 96-well plates, with 20,000 cells per well, and 3 wells were set up for replication. After continuous culture, MTS drug dye was added, and the OD value of the cells in each well was detected to determine the proliferation activity of the cells in each well. Step 3: Human umbilical cord mesenchymal stem cells cultured in different deuterium concentration medium were inoculated into 96-well plates, with 5,000 cells per well. Three wells were set up for replication. The cells in the three replicate wells were digested and counted every day. The cells were digested and counted regularly to count the continuous growth of the cells, and a growth curve was drawn according to the number of cells. Step 4: Human umbilical cord mesenchymal stem cells were continuously cultured in medium with different deuterium concentrations, and digested and harvested when each cell grew to 60-70% confluence to prepare a single cell suspension. The single cells in the suspension were fixed with ethanol and stained with propidium iodide. The cells were then detected by flow cytometry, and the DNA content of each cell was counted for cell cycle analysis. Step 5: Human umbilical cord mesenchymal stem cells cultured in different deuterium concentration medium were seeded into single wells of a 6-well plate at 50, 100, and 200, respectively, and then cultured in different deuterium concentration medium for 10-12 days, and then the single cell clones in each well were stained with crystal violet stain, and the number of blue-purple monoclonal cells in each well was counted by taking pictures; Step 6: Human umbilical cord mesenchymal stem cells continuously cultured in culture medium with different deuterium concentrations were inoculated into 6-well plates, fixed with formalin, and then stained with β-galactosidase using X-Gal. After staining, the cells were photographed under a microscope, and the number and proportion of blue-positive senescent cells of human umbilical cord mesenchymal stem cells in culture medium with different deuterium concentrations were counted.

9. The method for constructing a cell model according to claim 8, characterized in that: The culture medium contains α-MEM basic fluid and serum-free supplements.

10. The method for constructing a cell model according to claim 8, characterized in that: The different deuterium concentrations include 25 ppm, 50 ppm and 150 ppm.

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