Effect of deuterium-depleted water for inhibiting growth of tumor cells

Abstract

The invention discloses an effect of deuterium-depleted water on inhibiting growth of tumor cells, and belongs to the technical field of tumor treatment. According to the application of the deuterium-depleted water in preparation of the anti-tumor medicine, the concentration of deuterium in the deuterium-depleted water is 25 ppm to 50 ppm. The tumor comprises human cervical cancer cells, human laryngeal cancer epithelial cells or human malignant embryonic rhabdomyoma cells. Compared with the prior art, the invention discloses the effect of the deuterium-depleted water on inhibiting the growth of the tumor cells, and verifies the effects of the deuterium-depleted water on inhibiting the continuous proliferation capacity of the tumor cells, inhibiting the growth of the tumor cells, promoting the apoptosis of the tumor cells, promoting the aging of the tumor cells and inhibiting the formation rate of monoclonal cells of the tumor cells.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and specifically relates to the effect of low-deuterium water on inhibiting the growth of tumor cells. Background Art

[0002] Stem cells are cells in the human body with continuous proliferation activity and multi-directional differentiation potential, and are the key cells for human growth and development, maintaining youthfulness, and repairing injuries. There have been news reports that low-deuterium water has an obvious promoting effect on human vitality and youthfulness, and domestic and foreign studies have also shown that the promoting effect of low-deuterium water on the growth and proliferation activity of human stem cells is the internal mechanism beneficial to human health and youthfulness. Therefore, human stem cells are selected as the primary object of this research.

[0003] The role and mechanism of low-deuterium water in anti-tumor drugs still need to be further studied. Summary of the Invention

[0004] In view of the above technical problems, the present invention proposes the following technical solutions:

[0005] In a first aspect of the present invention, it relates to the use of low-deuterium water in the preparation of anti-tumor drugs, and the concentration of deuterium in the low-deuterium water is: 25 ppm to 50 ppm.

[0006] Optionally, the tumor includes human cervical cancer cells, human laryngeal cancer epithelial cells, or human malignant embryonal rhabdomyosarcoma cells.

[0007] In a second aspect of the present invention, it relates to the use of low-deuterium water in the preparation of drugs for inhibiting the continuous proliferation ability of tumor cells, and the concentration of deuterium in the low-deuterium water is: 25 ppm to 50 ppm.

[0008] In a third aspect of the present invention, it relates to the use of low-deuterium water in the preparation of drugs for inhibiting tumor cell growth and promoting tumor cell apoptosis, and the concentration of deuterium in the low-deuterium water is: 25 ppm to 50 ppm.

[0009] In a fourth aspect of the present invention, it relates to the use of low-deuterium water in the preparation of drugs for promoting tumor cell senescence, and the concentration of deuterium in the low-deuterium water is: 25 ppm to 50 ppm.

[0010] In a fifth aspect of the present invention, it relates to the use of low-deuterium water in the preparation of drugs for inhibiting the monoclonal cell formation rate of tumor cells, and the concentration of deuterium in the low-deuterium water is: 25 ppm to 50 ppm.

[0011] In a sixth aspect of the present invention, it relates to an anti-tumor drug, including low-deuterium water, and the concentration of deuterium in the low-deuterium water is: 25 ppm to 50 ppm.

[0012] The seventh aspect of the present invention relates to a method for constructing a cell model, including any one or more of the following steps:

[0013] Step 1: Take tumor cells and continuously culture them in media with three deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm; when the cells reach a confluence of 85%-90%, digest and passage them, and record the number of cells passaged and inoculated each time; continuously culture and passage for more than 50-80 days, record the time and number of cells continuously proliferating, and calculate the number of cell divisions of each cell during the whole process;

[0014] Step 2: Take tumor cells, after continuously culturing for 50-80 days under different deuterium concentration culture conditions, harvest and count them, inoculate them into a well plate, and culture them in media with three deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm respectively; regularly harvest the cells in 3 replicate wells for counting until each cell enters the growth plateau phase; respectively count the average number of cells per well of each tumor cell under different deuterium concentration culture conditions, and draw a growth curve;

[0015] Step 3: Take tumor cells, after continuously culturing for 50-80 days under different deuterium concentration culture conditions, harvest and count them, then inoculate them into a well plate, and culture them with 25 ppm, 50 ppm, and 150 ppm respectively; use X-gal β-galactosidase staining to detect the senescence of each tumor cell, observe the blue positive senescent cells after photographing, and count the proportion of positive senescent cells in the total number of cells in different fields of view of the tumor cells;

[0016] Step 4: Take tumor cells, after continuously culturing for 50-80 days under different deuterium concentration culture conditions, harvest and count them, then inoculate them into a well plate, and culture them with 25 ppm, 50 ppm, and 150 ppm respectively; stain the cell colonies in each well with crystal violet, observe and photograph them respectively, and count the number of cell colonies in each well. Take the number of cell colonies / inoculated cell number × 100% as the colony formation rate of the tumor cells;

[0017] Step 5: Take tumor cells, after continuously culturing for 50-80 days under different deuterium concentration culture conditions, harvest and count them, then inoculate them into a well plate, and conduct a single cell colony formation culture test in the upper layer of soft agar; through crystal violet staining, photograph and count the number of single cell colonies in each well, and calculate the soft agar colony formation rate.

[0018] Optionally, the medium used for culturing is: MEM powder medium supplemented with 10% FBS.

[0019] Optionally, the tumor cells include one or more of human cervical cancer cells, human laryngeal cancer epithelial cells, and human malignant embryonal rhabdomyosarcoma cells. Description of the Drawings

[0020] Figure 1 Detection of continuous proliferation of tumor cells under three deuterium concentration culture conditions in Example 1 of this application;

[0021] Figure 2 Growth curve of tumor cells under three deuterium concentration culture conditions in Example 2 of this application;

[0022] Figure 3 Proportion of senescence staining of tumor cells under three deuterium concentration culture conditions in Example 3 of this application;

[0023] Figure 4 Detection of colony formation rate of tumor cells under three deuterium concentration culture conditions in Example 4 of this application;

[0024] Figure 5 Detection of soft agar monoclonal formation of tumor cells in Example 5 of this application. Detailed implementation mode

[0025] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with 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. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the scope of protection of the present invention.

[0026] The materials used in the following embodiments of this application include:

[0027] Cell materials:

[0028] 1. Hela: Human cervical cancer cells, from Xunrui Laboratory (tumor cells, initial passage number unknown);

[0029] 2. HEP-2: Human laryngeal cancer epithelial cells, from Xunrui Laboratory (tumor cells, initial passage number unknown);

[0030] 3. RD: Human malignant embryonal rhabdomyosarcoma cells, from Xunrui Laboratory (tumor cells, initial passage number unknown).

[0031] Culture media:

[0032] 1. For Hela, HEP-2 and RD, MEM powder culture media are used and cultured with 10% FBS (fetal bovine serum) added;

[0033] Specifically, the cell culture medium used in the embodiment can be selected to be prepared from powder and pure water with three different deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm (ordinary pure water), and 10% FBS of the same batch is added before use.

[0034] Reagents and consumables:

[0035] The digestive juice (trypsin), washing solution (PBS), cryopreservation solution, and culture consumables (culture flasks, culture plates, cryopreservation tubes, centrifuge tubes, pipettes, pipette tips, etc.) are the same and are all conventional reagent consumables.

[0036] For the remaining materials, unless otherwise specified, they are all commercially available materials known to those skilled in the art.

[0037] Research methods:

[0038] 1. Select 3 strains of human tumor cells (hela, Hep-2, RD), and use the culture media prepared with pure water of three different deuterium concentrations (25 ppm, 50 ppm, and 150 ppm) to continuously culture them in vitro for 50 - 80 days, and record the proliferation and division of the cells during this period;

[0039] 2. After culturing and subculturing the 3 strains of cells in the culture solution with different deuterium concentrations for 50 - 80 days, respectively detect the proliferation viability, growth rate, tumor formation ability, and senescence of the 3 strains of cells;

[0040] A. Detection of MTS cell proliferation viability;

[0041] B. Detection of cell growth curve;

[0042] C. Clonogenic ability of cells;

[0043] D. Clonogenic ability of tumor cells in soft agar;

[0044] E. Detection of cell senescence by β-Gal staining;

[0045] Example 1

[0046] Experimental operation:

[0047] 1. Inoculate the tumor cells Hela and Hep-2 into T25 culture flasks respectively, and continuously culture them with the culture media of three deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm;

[0048] 2. According to the actual growth of the cells, when the cells reach 85% - 90% confluence, digest and subculture them, and record the number of cells subcultured and inoculated each time;

[0049] 3. Continuously culture and subculture for more than 50 - 80 days, record the time and number of continuous cell proliferation, and calculate the number of cell divisions of each cell during the whole process.

[0050] Experimental results, as Figure 1 shown, at the initial stage of culture, the proliferation differences of tumor cells at different deuterium concentrations are not obvious. After one month, the proliferation rate of Hela cells under normal deuterium concentration culture conditions is significantly higher than that of cells cultured with low-deuterium water (Figure 1 In A) of [description], after 60 days of culturing, the proliferation activity of Hep-2 began to decline under the culture with 25 ppm low-deuterium water ( Figure 1 In B) of [description]. From the above steps and results, it can be seen that the low-deuterium water culture medium has a significant inhibitory effect on the continuous proliferation ability of tumor cells.

[0051] Example 2

[0052] Experimental operation:

[0053] 1. After continuously culturing 3 strains of tumor cells, Hela, Hep-2 and RD, for 50 - 80 days under the culture medium conditions with deuterium concentrations of 25 ppm, 50 ppm and 150 ppm respectively, harvest and count them, then inoculate 5000 cells per well into a 48-well plate, and then culture them using the corresponding culture media with deuterium concentrations of 25 ppm, 50 ppm and 150 ppm;

[0054] 2. Every 24 hours, harvest the cells in 3 replicate wells for counting, and continuously harvest for 8 days until each cell enters the growth plateau phase;

[0055] 3. According to different time points, respectively count the average number of cells per well of each tumor cell under different deuterium concentration culture conditions, and draw a growth curve graph;

[0056] Experimental results, as Figure 2 shown, during the growth latent period and the early growth stage after inoculation, the growth rates of each tumor cell under different deuterium concentration conditions have no obvious difference, but in the late growth stage, after the cell density is relatively high, each tumor cell under the low-deuterium water culture condition will be significantly inhibited, accelerating the apoptosis of tumor cells ( Figure 2 In A - C) of [description]. From the above steps and results, it can be seen that the low-deuterium water culture medium has an inhibitory and apoptosis-promoting effect on the late growth of tumor cells.

[0057] Example 3

[0058] Experimental operation:

[0059] 1. After continuously culturing 3 strains of tumor cells, Hela, Hep-2 and RD, for 50 - 80 days under the culture medium conditions with deuterium concentrations of 25 ppm, 50 ppm and 150 ppm respectively, harvest, count and then inoculate them into a 12-well plate, and then culture them using the corresponding culture media with deuterium concentrations of 25 ppm, 50 ppm and 150 ppm for 2 - 3 days;

[0060] 2. Use X-gal β-galactosidase staining to detect the senescence of each tumor cell, take pictures and observe the blue positive senescent cells, Figure 3In A - C, the pictures are of Hela cells after senescence staining following continuous culture at 25 ppm, 50 ppm, and 150 ppm respectively. The proportion of positive senescent cells to the total number of cells in different fields of view for the three cell lines, Hela, Hep - 2, and RD, was statistically analyzed, that is, the number of blue cells in the field of view / the total number of cells in the field of view × 100% = the proportion of senescent cells ( Figure 3 in D).

[0061] The experimental results, as Figure 3 shown, under the culture conditions of low - deuterium water, the senescence ratios of the three tumor cell lines, Hela ( Figure 3 in A), Hep - 2 ( Figure 3 in B), and RD ( Figure 3 in C), were all higher than those under the culture conditions of normal deuterium concentration, indicating that low - deuterium water has a certain promoting effect on the senescence of different tumor cells. From the above steps and results, it can be seen that the low - deuterium water medium can promote the senescence of tumor cells.

[0062] Example 4

[0063] Experimental operation:

[0064] 1. After continuously culturing the three tumor cell lines, Hela, Hep - 2, and RD, in media with deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm for 50 - 80 days, harvest and count them, and then inoculate them into 6 - well plates at a quantity of 30, 60, and 120 cells per well respectively, and then culture them for 11 - 12 days using the corresponding media with deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm;

[0065] 2. Stain the cell colonies in each well with crystal violet, observe and take pictures respectively ( Figure 4 in A), and count the number of cell colonies in each well. The colony - forming rate of tumor cells = the number of cell colonies / the number of inoculated cells × 100% ( Figure 4 in B).

[0066] The experimental results, as Figure 4 shown, the single - cell colony - forming rates of the three tumor cell lines, Hela, Hep - 2, and RD, were all inhibited to a certain extent under the condition of low - deuterium culture water ( Figure 4 in A), and the specific statistical numbers of colony formation can be seen in Figure 4 B. From the above steps and results, it can be seen that the low - deuterium water medium has an inhibitory effect on the single - cell colony - forming rate of tumor cells.

[0067] Example 5

[0068] Experimental operation:

[0069] 1. After culturing the three tumor cell lines Hela, Hep-2, and RD continuously for 50 - 80 days under the conditions of media with deuterium concentrations of 25 ppm, 50 ppm, and 150 ppm, harvest and count the cells, and then inoculate them into 6-well plates at a density of 50 and 100 cells per well respectively for single-cell clone formation culture experiments in upper-layer soft agar.

[0070] 2. After 14 days of culture, stain with crystal violet, take pictures, count the number of single-cell clones in each well, and calculate the soft agar clone formation rate.

[0071] The single-cell clone formation rates of the three tumor cell lines Hela, Hep-2, and RD were all inhibited to a certain extent under the condition of low-deuterium culture water. The photographed images of Hep-2 are shown in Figure 5 A in, and the statistical numbers of clone formations of the three tumor cell lines are shown in Figure 5 B in. From the above steps and results, it can be seen that the low-deuterium water medium has an inhibitory effect on the soft agar single-clone formation rate of tumor cells.

Claims

1. The use of deuterium-depleted water in the preparation of anti-tumor drugs, characterized in that: The concentration of deuterium in the deuterium-depleted water is 25 ppm to 50 ppm.

2. The use of deuterium-depleted water in the preparation of anti-tumor drugs according to claim 1, characterized in that: The tumor includes human cervical cancer cells, human laryngeal cancer epithelial cells or human malignant embryonic rhabdoid tumor cells.

3. The use of deuterium-depleted water in the preparation of a drug for inhibiting the sustained proliferation of tumor cells, characterized in that: The concentration of deuterium in the deuterium-depleted water is 25 ppm to 50 ppm.

4. Use of deuterium-depleted water in the preparation of a drug for inhibiting tumor cell growth and promoting tumor cell apoptosis, characterized in that: The concentration of deuterium in the deuterium-depleted water is 25 ppm to 50 ppm.

5. Use of deuterium-depleted water in the preparation of a drug for promoting tumor cell senescence, characterized in that: The concentration of deuterium in the deuterium-depleted water is 25 ppm to 50 ppm.

6. Use of deuterium-depleted water in the preparation of a drug for inhibiting the monoclonal cell formation rate of tumor cells, characterized in that: The concentration of deuterium in the deuterium-depleted water is 25 ppm to 50 ppm.

7. An anti-tumor drug, characterized in that: It includes deuterium-depleted water, wherein the concentration of deuterium in the deuterium-depleted water is 25ppm to 50ppm.

8. A method for constructing a cell model, characterized in that: Includes any one or more of the following steps: Step 1: Take tumor cells and continuously culture them in culture media with three deuterium concentrations of 25ppm, 50ppm and 150ppm; digest and passage the cells when they grow to 85%-90% confluence, and record the number of cells in each passage and inoculation; continue to culture and passage for more than 50-80 days, record the time and number of continuous cell proliferation, and calculate the number of divisions of each cell in the whole process; Step 2: Tumor cells were taken and cultured continuously for 50-80 days under different deuterium concentrations. The cells were harvested and counted, inoculated into well plates, and cultured with culture media with three deuterium concentrations of 25 ppm, 50 ppm and 150 ppm respectively; cells in three replicate wells were harvested and counted regularly until each cell entered a growth plateau; the average number of tumor cells per well under different deuterium concentrations was counted, and a growth curve was drawn; Step 3: Tumor cells were taken and cultured continuously for 50-80 days under different deuterium concentrations. After harvesting and counting, they were inoculated into well plates and then cultured at 25ppm, 50ppm and 150ppm respectively. The senescence of each tumor cell was detected by X-galβ-galactosidase staining. Blue positive senescent cells were observed after taking photos, and the proportion of positive senescent cells to the total number of cells in different fields of view of tumor cells was counted. Step 4: Take tumor cells, culture them continuously under different deuterium concentrations for 50-80 days, harvest and count them, and then inoculate them into well plates, and then culture them at 25ppm, 50ppm and 150ppm respectively; stain the cell colonies in each well with crystal violet, observe and take pictures respectively, and count the number of cell colonies in each well, and take the number of cell colonies / the number of inoculated cells×100% as the cloning rate of tumor cells; Step 5: Take tumor cells, culture them continuously under different deuterium concentrations for 50-80 days, harvest and count them, and then inoculate them into well plates, and perform a single cell clone formation culture test in the upper soft agar; use crystal violet staining, take pictures and count the number of single cell clones in each well, and calculate the soft agar clone formation rate.

9. The method for constructing a cell model according to claim 8, characterized in that: The culture medium used in the culture is: MEM powder culture medium supplemented with 10% FBS.

10. The method for constructing a cell model according to claim 8, characterized in that: The tumor cells include one or more of human cervical cancer cells, human laryngeal cancer epithelial cells, and human malignant embryonic rhabdomyoma cells.

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