Application of cycloastragenol in delaying cell senescence and increasing passage times of cell senescence

Abstract

The invention relates to the technical field of biology, in particular to application of cycloastragenol in delaying cell senescence and increasing passage times of the cell senescence, and the cycloastragenol can delay the cell senescence and increase the passage times of the cell senescence by improving the relative telomerase activity of the cell. Cell experiments prove that the natural telomerase activator cycloastragenol used in the invention has no toxic or side effect on cells under the conditions of low-concentration (25-100 [mu] M) and short-time (12-24 h) incubation. Finally, a culture scheme of cycloastragenol is determined through drug toxicity and anti-aging tests: P3 and P6 generation cells are cultured for 24 hours by using 100 [mu] M cycloastragenol, P9 generation cells are cultured for 12 hours by using 100 [mu] M cycloastragenol, and under the culture condition, the relative telomerase activity of the cells can be remarkably improved, the aging level of the cells can be delayed, and the passage frequency of the cells can be remarkably prolonged.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of cycloastragenol in delaying cell senescence and increasing the number of cell passages. Background Art

[0002] Many local pigs are on the verge of extinction due to the introduction of new breeds, viral infections, habitat destruction and climate change. Establishing a somatic cell gene bank for endangered pig breeds has become one of the important ways to preserve their genetic information for future recovery or utilization. Primary cell lines can only divide a limited number of times when cultured in vitro due to the lack of telomerase activity. When the donor animal is old or the culture process is improperly operated, it will also cause premature aging of cells in early cultures, which seriously limits the number of cell generations and research applications in the later stages.

[0003] Currently, the degree of cell aging can be delayed through a variety of ways, including gene editing or adding telomerase activators to activate cell telomerase activity, adding antioxidants or growth factors to the cell culture medium, inhibiting the secretory phenotype associated with cell aging, or activating the apoptosis pathway of aging cells. The specific operation method is: immortalized cells can be obtained by lentivirus infection of human telomerase reverse transcriptase (hTERT), so that the cell division ability is not restricted. Adding Senolytics drugs such as BCL-2 family inhibitors or HSP90 inhibitors to the cell culture medium activates the apoptosis pathway of aging cells and selectively eliminates aging cells. Adding growth factors and hormones such as EGF, FGF, etc., and antioxidants such as vitamins C and E, etc. to the cell culture medium can reduce the damage of oxidative stress to cells and delay cell aging.

[0004] There are many types of anti-aging drugs on the market. Among them, antioxidants such as vitamin C and vitamin E have no significant anti-aging effects. AMPK activators such as emodin and metformin; Sirtuin activators such as rapamycin (Resveratrol). These drugs can slow down the aging of cells and keep cells healthy for a longer time. However, these drugs may have potential toxic side effects, and their safety and stability for long-term use have not yet been fully determined. Different species and even individuals have different responses to drugs. At the same time, some new drugs may have problems in terms of economy and accessibility, which limits their widespread application. Using gene transfection and other technologies to introduce exogenous immortal genes into cells can solve the problems of cell aging and proliferation, but when immortalized cells are used as donor cells for somatic cell nuclear transplantation, very few embryos can develop normally, and chromosomal abnormalities and other problems are prone to occur.

[0005] Cycloastragenol is a triterpenoid saponin component originally extracted from the traditional herb Astragalus. Based on previous studies, cycloastragenol can upregulate the telomerase activity of senescent cells both in vitro and in vivo, promote telomere extension, and delay cell aging. For example, in in vivo studies, injection of cycloastragenol restored short telomeres in female mice and alleviated age-related dysfunction in the ovarian tissue of mice. In in vitro studies, cycloastragenol activated telomerase and extended telomeres mainly in a telomerase-dependent manner, reducing the percentage of very short telomeres in cells. However, there is no research in the prior art on the effect of cycloastragenol on delaying the aging of ear fibroblasts and the number of passages. Summary of the invention

[0006] The purpose of the present invention is to provide an application of cycloastragenol in delaying cell senescence and increasing the number of cell generations. The cycloastragenol used in the present invention has no potential toxic side effects on cells, can delay cell senescence and the number of cell generations, has low application cost and high feasibility, and provides an optimal use scheme.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] The invention provides application of cycloastragenol in delaying cell senescence and increasing the number of cell passages.

[0009] Furthermore, the cycloastragenol delays cell senescence and increases the number of cell generations by increasing the relative telomerase activity of the cells.

[0010] Furthermore, the effective dose of cycloastragenol is 25-100 μM, and the treatment time is 12-24 hours.

[0011] Furthermore, the effective dose of cycloastragenol for P3 and P6 generation cells is 100 μM, and the treatment time is 24 h; the effective dose of cycloastragenol for P6 generation cells is 100 μM, and the treatment time is 12 h.

[0012] Furthermore, the cells are porcine somatic cells.

[0013] Furthermore, the porcine somatic cells are porcine ear marginal fibroblasts.

[0014] The beneficial effects of the present invention are:

[0015] Since the primary cell line lacks telomerase activity, the cells are prone to aging during the passage process, accumulating genetic mutations and functional disorders, which seriously limits the number of cell passages and later research applications, and causes great difficulties for the protection of animal somatic cell resources. The present invention accurately detects the toxicity of cycloastragenol, proposes different cycloastragenol culture schemes for cells of different generations (aging degrees), and reduces the toxic effects of drugs on cells. The final test results prove that cycloastragenol can delay cell aging. In addition, cycloastragenol can also activate the telomerase activity of fibroblasts and extend the number of cell passages. The natural telomerase activator cycloastragenol used in the present invention has been proved by cell experiments that it has no toxic side effects on cells under the conditions of low concentration (25-100 μM) and short time (12-24h) incubation. Finally, through drug toxicity and anti-aging tests, the culture scheme of cycloastragenol was determined as follows: P3 and P6 cells were cultured with 100 μM cycloastragenol for 24 hours, and P9 cells were cultured with 100 μM cycloastragenol for 12 hours. Under this culture condition, the relative telomerase activity of the cells can be significantly improved, the aging level of the cells can be delayed, and the number of cell generations can be significantly extended. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing cell viability when PMEF was incubated for 4 h using different concentrations of CAG (25-200) μM as detected by CCK8 method;

[0017] Figure 2 This is a graph showing the proliferation inhibition of PMEFs of different passage times by CAG of different concentrations and culture times;

[0018] Figure 3 The results of the cell senescence level test after PMEF of different generations were treated with different concentrations of CAG for 12h and 24h;

[0019] Figure 4 This is a graph showing the effect of CAG on the PMEF cell culture ability, where the vertical axis is the number of cells per square centimeter. DETAILED DESCRIPTION

[0020] Example 1

[0021] The present invention first uses CCK8 (cell viability detection kit, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) to preliminarily screen the concentration of CAG (cycloastragenol, purchased from MCE Company). After the pig ear edge fibroblasts (PEMF) were cultured with (25μM~200μM) CAG for 4 hours, compared with the blank control group, the PEMF cultured with low concentration (25μM~100μM) CAG did not show a significant decrease in viability (P>0.05). When the CAG concentration was increased to 125μM~200μM, the PMEF viability was significantly reduced (P<0.0001). Based on the above findings, the culture concentration range of CAG was narrowed to 25μM~100μM for the next experiment. Based on this, the present invention proves through cell experiments that it has no toxic side effects on cells under the conditions of low concentration (25~100μM) and short time (12~24h) incubation.

[0022] P3, P6 and P9 PMEF were cultured continuously for 168 h with 25 μM to 100 μM CAG. The specific subculture steps are as follows:

[0023] (1) Cut the sample into pieces smaller than 1 mm 3 Transfer the fragments to the cell culture dish (flask) with a Pasteur pipette so that they are evenly distributed on the bottom of the dish (flask);

[0024] (2) Invert the culture dish (bottle) and place it in a 37°C carbon dioxide incubator for 4 to 6 hours, observing every 2 hours.

[0025] (3) After the sample block adheres to the wall, add 4 mL of DMEM preheated to 37°C to the culture dish (bottle). After 12 hours, observe whether it is contaminated. If contaminated, discard it or wash it again;

[0026] (4) Observe cell migration and growth every 1 day;

[0027] (5) Observe the confluence of primary cells under a microscope. When the confluence reaches 70% to 90%, subculture is performed. The main process is:

[0028] S1. Remove the sample block from the culture dish (bottle) in the clean bench, wash the cells with DPBS 2-3 times, and discard the waste liquid. Add 1mL of trypsin to the culture dish (bottle), put it in the incubator for digestion for 3 minutes, take out the culture dish (bottle), observe the cell wall detachment under a microscope, and when the cells become round and most of the cells are detached, immediately add 2mL of DMEM to stop digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000rpm for 5 minutes, and keep the cell pellet.

[0029] S2. Add DMEM to resuspend the cells, take the cell suspension and evenly inoculate it into 2 cell culture dishes (bottles), mark them as P1 cells, place them in a carbon dioxide incubator and continue to culture, changing DMEM every 2 days.

[0030] S3. When the cell confluence is 70% to 90%, the next subculture is performed, and the next subculture steps are the same as above.

[0031] The results show that Figure 1 ), compared with the blank control group, when PMEF was cultured with CAG for 12 hours, all concentrations of CAG had no significant effect on the proliferation ability of PMEF of each generation (P>0.05). When the culture time was extended to 24 hours, all concentrations of CAG had no significant effect on the proliferation of PMEF of P3 and P6 generations (P>0.05), but 75μM and 100μM CAG significantly inhibited the proliferation ability of PMEF of P9 generation (P<0.0001). As the action time prolonged, the inhibitory effect of CAG on the proliferation of PMEF showed concentration and time dependence. The proliferation inhibition of PMEF of different passages with different concentrations and culture times is shown in the figure Figure 2 .

[0032] In order to intervene in aging without affecting the cell proliferation ability, the culture time of CAG was set to 12h and 24h in this experiment, and the aging level of cells in each generation was detected using SA-β-Gal probe. Figure 3 ) showed that CAG's effect on reducing the level of cellular senescence was dose- and time-dependent. More details:

[0033] After 12h of CAG culture, the cell senescence level of P3 generation PMEF in each concentration group had no significant difference with the blank control group (P>0.05). After 24h of culture, 50μM, 75μM and 100μM CAG significantly reduced the cell senescence level (P<0.01 vs P<0.001 vs P<0.0001).

[0034] After 12h of CAG culture, 75μM and 100μM CAG significantly reduced the level of cell senescence in P6 generation PMEF (P<0.01). After 24h of culture, 50μM, 75μM and 100μM CAG significantly reduced the level of cell senescence (P<0.05vsP<0.0001vsP<0.0001).

[0035] After 12h of CAG culture, 50μM, 75μM and 100μM CAG significantly reduced the level of cell senescence (P<0.01vsP<0.001vsP<0.0001). After 24h of culture, 25μM, 50μM, 75μM and 100μM CAG significantly reduced the level of cell senescence (P<0.01vsP<0.0001vsP<0.0001vsP<0.0001). Therefore, this experiment determined that the optimal concentration and time of CAG for PMEF senescence intervention were 100μM and 24h. However, due to the high degree of senescence of P9 generation PMEF, long-term culture would affect the viability and proliferation of cells, so the action time of CAG was shortened to 12h.

[0036] So far, this patent has clarified the application scheme of CAG in culturing cells of different generations, namely: P3 and P6 cells are cultured with 100μM cycloastragenol for 24 hours, and P9 cells are cultured with 100μM cycloastragenol for 12 hours. Under this culture condition, cycloastragenol can significantly increase the relative telomerase activity of cells, delay the aging level of cells, and significantly extend the number of cell generations. This provides a technical guarantee for accelerating the preservation of pig somatic cell genetic resources. In order to study the effect of CAG on extending the number of cell generations, the present invention selected P6 generation pig ear fibroblasts as representative cells, cultured them with 100μM cycloastragenol for 24 hours, and set up blank control group cells. The two groups of cells were continuously passaged at the same time in a ratio of 1:2, and the number of cells per square centimeter and the degree of cell confluence were counted 24 hours after each passage. The results showed that after the pig ear fibroblasts were cultured with cycloastragenol, the number of cells per unit area ( Figure 4 ) and the confluence of cells per unit time were significantly higher than those of the blank control group (P<0.01). The cells in the blank control group had lost the ability to pass on at P15, while the cells cultured with cycloastragenol still had the ability to continue to pass on.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. Application of cycloastragenol in delaying cell senescence and increasing its passage times.

2. The use according to claim 1, characterized in that The cycloastragenol delays cell senescence and increases the number of cell generations by increasing the relative telomerase activity of the cells.

3. The use according to claim 1, characterized in that The effective dosage of the cycloastragenol is 25-100 μM, and the treatment time is 12-24 hours.

4. The use according to claim 3, characterized in that The effective dose of cycloastragenol used for P3 and P6 generation cells is 100 μM, and the treatment time is 24 h; the effective dose of cycloastragenol used for P6 generation cells is 100 μM, and the treatment time is 12 h.

5. The use according to claim 1, characterized in that The cells are porcine somatic cells.

6. The use according to claim 5, characterized in that The porcine somatic cells are porcine ear marginal fibroblasts.

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