Telomerase and application thereof in anti-aging

Abstract

The invention provides telomerase and application thereof in anti-aging, the telomerase is a site-directed modified high-activity anti-aging telomerase variant, and after the telomerase is prepared into a composition, the composition has a good anti-aging effect and is wide in application field.

Description

Technical Field

[0001] The invention belongs to the biological field, and more specifically relates to providing a telomerase and its use in anti-aging. Background Art

[0002] Cell aging and carcinogenesis are important cellular life phenomena in organisms. The activity of telomerase plays a key role in these two processes. Telomerase is an enzyme responsible for protecting the ends of chromosomes. It consumes telomeres during cell division to prevent the gradual shortening and rupture of chromosome telomeres. Each time a cell divides, the telomeres shorten by a small section. When the telomeres shorten to a certain extent, the cells cannot maintain the normal chromosome structure, thus triggering the cell aging mechanism. Telomerase can extend the life of chromosomes by adding new telomere sequences to the ends of chromosomes, thereby maintaining the viability of cells.

[0003] Past studies have shown that telomerase activity is affected by a variety of regulatory factors. The most commonly studied factors are telomere length and telomerase expression. Studies have found that cells with shorter telomeres tend to have lower telomerase activity, which means that telomere length may play an important role in regulating cell aging and carcinogenesis. In addition, changes in chromosome structure and function, DNA damage and repair, and intracellular signal transduction pathways can also affect telomerase activity. Among the existing treatments, inhibitors against telomerase have become a hot topic of research. These inhibitors can inhibit the growth and division of cancer cells by blocking the activity of telomerase. Some drugs have entered the clinical trial stage and have shown potential anti-tumor activity. In addition, researchers are also exploring gene therapy methods for telomerase to restore or inhibit the activity of telomerase and further explore its role in cell aging and carcinogenesis.

[0004] Because telomerase has this magical anti-aging effect, the existing technology has developed many telomerase series products, all of which have the effect of delaying aging. The product mainly enhances the activity of telomerase and promotes cell division after taking enough effective telomerase, thereby delaying aging. Scientific experiments have also shown that when 3% concentration of telomerase is applied to skin care products, you can gradually see that the basal layer of the skin begins to delay aging, achieving what we call the anti-aging effect. It is also an experiment done by American scientists. After using skin care products containing telomerase for 4 weeks, the user's eyes, mouth corners and other places prone to aging have been significantly improved, and the wrinkles in these two places have been tightened by 20% and more than 50% respectively.

[0005] Although there are now many documents proving that telomerase does have the above effects to some extent, the amount of telomerase used in skin care products is still relatively large, the cost is high, and it is not suitable for large-scale popularization. Summary of the invention

[0006] Telomerase is composed of telomere reverse transcriptase (TERT) and telomerase RNA component (TERC). As the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT) plays a decisive role in the activation of telomerase. The expression of hTERT represents the activation of telomerase, and increasing the activity of hTERT can promote the activity of telomerase.

[0007] Based on the prior art, the present invention provides an improved telomerase, which is obtained by mutating the 252nd amino acid sequence from G to H and the 594th amino acid sequence from K to S based on the amino acid sequence shown in SEQ ID NO: 1.

[0008] The present invention also provides an anti-aging composition, which comprises improved telomerase and stem cell lysate.

[0009] Furthermore, the composition also contains a pharmaceutically acceptable carrier.

[0010] The compositions used according to the invention may be in various forms acceptable for topical application, in particular solid or liquid form, depending on the physiologically acceptable medium used.

[0011] The topical compositions may be in the form of water-in-oil (W / O) or oil-in-water (O / W) emulsions, suspensions, oils, gels, pastes, creams, lotions, solutions, slurries, e.g. aqueous, alcoholic, hydro-alcoholic or fatty solutions, powders.

[0012] The polyol included as an embodiment may refer to a polyol, for example, a fatty alcohol with two or more hydroxyl groups (-OH). The alcohol with two hydroxyl groups is referred to as a diol or a glycol, the alcohol with three hydroxyl groups is referred to as a triol (for example, glycerol), and the alcohol with four hydroxyl groups is referred to as a tetraol (for example, pentaerythritol). According to an embodiment, the polyol may include one or more selected from polyethylene glycol, polypropylene glycol, dipropylene glycol, propylene glycol, butylene glycol, glycerol, polyglycerol-3, propylene glycol, sorbitol, erythritol, xylitol, maltitol, ethyl hexanediol, 1,2-hexanediol, PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol and pentanediol. The amount of the polyol included in the cosmetic composition of the present invention is not limited and can be appropriately adjusted by the user. According to one embodiment, the polyol can be present in an amount of 1 wt.% or more, 2 wt.% or more, 3 wt.% or more, 4 wt.% or more, 5 wt.% or more, 6 wt.% or more, 7 wt.% or more, 8 wt.% or more, 9 wt.% or more, 10 wt.% or more, 11 wt.% or more, 12 wt.% or more, 13 wt.% or more, 14 wt.% or more, 15 wt.% or more, 16 wt.% or more, 17 wt.% or more, 18 wt.% or more, 19 wt.% or more, 20 wt.% or more, 21 wt.% or more, 22 wt.% or more, 23 wt.% or more, 24 wt.% or more, 25 wt.% or more, 26 wt.% or more, 27 wt.% or more, 28 wt.% or more, 29 wt.% or more, or 30 wt.% or more, relative to the total weight of the composition. In addition, according to one embodiment, the polyol can be present in an amount of 30 wt % or less, 29 wt % or less, 28 wt % or less, 27 wt % or less, 26 wt % or less, 25 wt % or less, 24 wt % or less, 23 wt % or less, 22 wt % or less, 21 wt % or less, 20 wt % or less, 19 wt % or less, 18 wt % or less, 17 wt % or less, 16 wt % or less, 15 wt % or less, 14 wt % or less, 13 wt % or less, 12 wt % or less, 11 wt % or less, 10 wt % or less, 9 wt % or less, 8 wt % or less, 7 wt % or less, 6 wt % or less, 5 wt % or less, 4 wt % or less, 3 wt % or less, 2 wt % or less, or 1 wt % or less, relative to the total weight of the composition. Alternatively, according to one embodiment, the composition may comprise a polyol in an amount of 1 wt.% to 30 wt.%, such as 15 wt.% to 25 wt.%, relative to the total weight of the composition. According to one embodiment, the composition may comprise a polyol in an amount of 5 wt.% to 35 wt.%, such as 20 wt.% to 30 wt.%, relative to the total weight of the external phase.

[0013] Specifically, the stem cells are mesenchymal stem cells. Mesenchymal stem cells are a group of heterogeneous cells derived from the matrix, which contain a large number of active ingredients, such as stem cell growth factor, fibroblast growth factor, endothelial cell growth factor, epidermal cell growth factor, collagen, hyaluronic acid, etc., which can promote the growth of epidermal keratinocytes, repair damaged cells, and improve cell metabolism; the cell lysate is obtained by using adipose tissue-derived mesenchymal stem cells after cell recovery, subculture, and cell lysis. After the cell lysate is lipid-embedded, sterilized, and freeze-dried, the active shelf life of the growth factor in the cell lysate can be increased, and the water in the cell lysate is sublimated in a vacuum sterile environment, which causes less damage to the structure and characteristics of biological tissues and cells, allowing them to quickly enter a dormant state, thereby effectively prolonging their biological activity and improving the anti-aging effect.

[0014] The present invention also provides a cosmetic, which consists of 5-10 parts by weight of water, 0.5-1 parts by weight of glycerol, 0.2-0.6 parts by weight of propylene glycol, 0.1-0.3 parts by weight of sodium hyaluronate, 0.05-0.2 parts by weight of p-hydroxyacetophenone, 0.05-0.2 parts by weight of olive oil, 0.05-0.01 parts by weight of vitamin E, 0.05-0.1 parts by weight of dicaprylyl carbonate and 0.01-0.02 parts by weight of hydrogenated lecithin, 0.05-0.2 parts by weight of adipose mesenchymal stem cell lysate and 0.05-0.2 parts by weight of hTERT252 / 594 protein.

[0015] The present invention also provides a cosmetic, which consists of 0.7 parts by weight of water, 0.07 parts by weight of glycerol, 0.035 parts by weight of propylene glycol, 0.015 parts by weight of sodium hyaluronate, 0.01 parts by weight of p-hydroxyacetophenone, 0.01 parts by weight of olive oil, 0.008 parts by weight of vitamin E, 0.005 parts by weight of dicaprylyl carbonate, 0.001 parts by weight of hydrogenated lecithin, 0.01 parts by weight of adipose mesenchymal stem cell lysate and 0.01 parts by weight of hTERT252 / 594 protein.

[0016] Beneficial effects:

[0017] (1) Stem cell lysate is rich in cytokines, small nucleic acids, DNA and other growth factors and signaling molecules produced by stem cells, which can activate cells or tissues to achieve anti-aging purposes.

[0018] (2) The latest medical research has found that telomerase can repair damaged chromosome telomeres and improve the regeneration capacity of collagen, elastic fibers, and hyaluronic acid, which can achieve the effects of wrinkle removal, firming, and enhancing skin elasticity. On this basis, the present invention provides an improved telomerase variant, the activity of which is further enhanced compared to the original telomerase, and the improved telomerase variant can more significantly improve the anti-aging effect.

[0019] (3) The present invention provides a composition, which is essentially an anti-aging, wrinkle-removing and beauty cosmetic composition, which comprises highly active telomerase and stem cell lysate and a specific auxiliary composition. The composition can overcome the problem that conventional telomerase cosmetics need to be stored at low temperatures and are costly. The composition of the present invention can maintain its activity for a long time at room temperature, has a low storage cost, and is suitable for marketization. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Western-Blot identification results of expressed hTERT protein DETAILED DESCRIPTION

[0021] The following description is merely exemplary in nature and is not intended to limit the present disclosure, application, and use. It should be understood that in all of these figures, the same or similar figure numbers indicate the same or similar parts and features. The various figures only schematically represent the concepts and principles of the embodiments of the present disclosure, and do not necessarily show the specific dimensions and proportions of the various embodiments of the present disclosure. Specific parts in specific drawings may use exaggerated methods to illustrate the relevant details or structures of the embodiments of the present disclosure. The various publications, patents, and published patent specifications cited herein are incorporated herein by reference in their entirety. The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention.

[0022] Example 1 Design, expression and purification of telomerase mutants

[0023] According to the sequence of the hTERT gene (NCBI accession number: NM_198253.2), the inventors conducted preliminary analysis of the amino acid sequence structure of hTERT through computer simulation and three-dimensional modeling, and found that the telomerase mutant obtained by mutating the amino acid sequence of SEQ ID NO: 1 from G to H and from K to S at position 252 had improved activity.

[0024] Based on the NM_198253.2 sequence, the amino acid sequence at position 252 was H and the amino acid sequence at position 594 was S modified to the corresponding codons, and Shanghai Yubo Biotechnology Co., Ltd. was commissioned to perform artificial synthesis and construct the corresponding pFastBacHTA-hTERT252 / 594. The control unmutated pFastBacHTA-hTERT was also synthesized. The plasmid was transformed into DH10BacTM E.coli competent cells for homologous recombination, and the Bacmid recombinant shuttle plasmid containing hTERT252 / 594 or hTERT was extracted and transfected into Sf9 cells. The cells in the 6-well plate transfected for 192 hours were taken and IFA was used to identify the positive wells. The positive wells were expressed in large quantities, purified by affinity chromatography using Akata, concentrated with an ultrafiltration tube, and the protein concentration was adjusted to 50 mg / mL for standby use.

[0025] Western-Blot identification: Take 5μl of the two purified proteins and the control empty vector sf9 lysate, run the gel by SDS-PAGE electrophoresis, and perform Western-Blot experimental identification. The specific method is as follows: Activate the PVDF membrane with formaldehyde for 1min and soak it in Bio-Red reagent for 1min; Load the sample: Mix the sample in a 1:1 ratio and cook it in a microwave for 3min. The loading volume is 20ul; Transfer the protein from the gel to the PVDF membrane using the Bio-Red electrotransfer instrument. The transfer time is 7min; Block: After the transfer is completed, block the PVDF membrane with 5% skim milk. F membrane, time is 1 hour; PBST (0.05% Tween-20) wash PVDF membrane, 5 minutes each time, wash 3 times, incubate with 5% skim milk at a dilution of 1:3000 times 6*His monoclonal antibody, shake at 4°C overnight; the next day, PBST (0.05% Tween-20) wash PVDF membrane, 5 minutes each time, wash 3 times, incubate with 5% skim milk at a dilution of 1:8000 times HRP goat anti-mouse antibody at room temperature for 1 hour, PBST (0.05% Tween-20) wash PVDF membrane 3 times, 5 minutes each time; use TMB-Blotting Solution to develop color and observe the results. Figure 1 It can be seen that the control empty vector sf9 lysate did not produce any bands, while the purified hTERT and hTERT252 / 594 proteins both showed relatively pure bands, indicating that relatively pure recombinant proteins were obtained by purification.

[0026] Example 2 Identification of hTERT252 / 594 Activity

[0027] HaCaT cells in the logarithmic growth phase were digested with 0.25% trypsin, washed several times with PBS (pH 7.2), counted, and then the cell concentration was adjusted to 2×10 in RPMI1640 culture medium containing 10% calf serum. 8 / L. 1 Add 4 ml of the above cell suspension to the culture flasks, place them in an incubator with 5% CO2, 37°C and saturated humidity, and randomly divide the culture flasks into groups after 24 hours. Add 4 ml of hTERT252 / 594 protein with a final concentration of 0.5% (m / v) and 1% (m / v) to the experimental group, add 4 ml of hTERT protein with the same concentration to the control group, and add the same volume of 1640 culture medium to the blank group. Continue to culture for 48 hours, then take out, digest and wash, and adjust the cell concentration to 1x10 9 / L. 1ml was aspirated, thawed quickly, added to 50ml pre-cooled lysis buffer, suspended, mixed, ice bathed for 30min, centrifuged at 14000r / min for 20min in a 4℃ environment, 2μl supernatant was taken, added to the TRAP reaction tube containing 45μl reaction mixture, mixed, added to 30ml liquid paraffin, kept warm in a 25℃ water bath for 30min, inactivated at 94℃ for 2min on the PCR instrument, and then amplified for 35 times at 94℃, 30s, 48℃, 30s, 72℃, 90s, respectively, and extended at 72℃ for 5min, 25μl of the amplified product was taken and added to each well of the microplate containing the hybridization reaction solution, mixed, kept warm at 37℃ for 60min, added the color developer, developed the color at 37℃ in the dark for 10min, added the stop solution to stop the reaction, and read the A value at a wavelength of 450nm. The A value reflects the level of telomerase activity. In this experiment, each sample was tested in duplicate, and the results are shown in Table 1.

[0028] Table 1 Telomerase activity results of each group

[0029] Group A Value Control group 0.615±0.021 Control group (0.5%) 0.768±0.034 Control group (1%) 0.825±0.028 Experimental group (0.5%) 0.846±0.037 Experimental group (1%) 0.979±0.041

[0030] As can be seen from Table 1, the activity of the mutated hTERT252 / 594 protein of the present invention is significantly improved compared with that of hTERT. At a concentration of 0.5%, the activity of the hTERT252 / 594 protein is even higher than that of 1% hTERT.

[0031] Example 3 Preparation of stem cell lysate

[0032] Primary mouse adipose-derived mesenchymal stem cells (Cat. No. SNP-M088, Sean Biotech) were washed with 2 mL of 1× PBS buffer to remove the residual proliferation medium, and then 2 mL of 1× trypsin was added to digest for 3 min until the cells were completely detached by gently blowing. Then, an equal volume of proliferation medium was added to mix, and the mixture was centrifuged at 800 rpm for 5 min. The supernatant was discarded, and 1 mL of proliferation medium was added to resuspend the cells, and the mixture was mixed by blowing. Then, the cells were transferred to the Sean Biotech mesenchymal stem cell system (Cat. No. SNPM-M088). , cultured at 37°C and 5% CO2 for 2 days to obtain adipose-derived mesenchymal stem cells for passage culture; the cell suspension was centrifuged and the supernatant was discarded to collect the cell pellet; physiological saline was added to the cell pellet, the cell pellet was resuspended, and physiological saline was added to obtain a cell suspension; the cell suspension was placed at -80°C and frozen for 30 minutes; repeated twice; then centrifuged, the supernatant was taken to obtain a cell lysate; the cell lysate was lipid-embedded, sterilized, and freeze-dried to obtain adipose-derived mesenchymal stem cell lysate.

[0033] Example 4 Preparation of a composition containing telomerase

[0034] 0.7 kg of water, 0.07 kg of glycerol, 0.035 kg of propylene glycol, 0.015 kg of sodium hyaluronate, and 0.01 kg of parahydroxyacetophenone were taken, heated to 82° C., and stirred for 30 min to obtain a one-phase premix; then, 0.01 kg of olive oil, 0.008 kg of vitamin E, 0.005 kg of dicaprylyl carbonate, and 0.001 kg of hydrogenated lecithin were taken, heated to 82° C., and stirred for 30 min to obtain a two-phase premix; the two-phase premix was added to the one-phase premix, and stirred at 82° C. at a speed of 500 r / min for 10 min, then cooled to 37° C., 0.01 kg of the adipose mesenchymal stem cell lysate prepared in Example 3 and 0.01 kg of hTERT252 / 594 protein were added, and stirred at a speed of 300 r / min for 20 min to obtain the experimental composition A;

[0035] 0.7 kg of water, 0.07 kg of glycerol, 0.035 kg of propylene glycol, 0.015 kg of sodium hyaluronate, and 0.01 kg of parahydroxyacetophenone were taken, heated to 82° C., and stirred for 30 min to obtain a one-phase premix; then, 0.01 kg of olive oil, 0.008 kg of vitamin E, 0.005 kg of dicaprylyl carbonate, and 0.001 kg of hydrogenated lecithin were taken, heated to 82° C., and stirred for 30 min to obtain a two-phase premix; the two-phase premix was added to the one-phase premix, and stirred at 82° C. at a speed of 500 r / min for 10 min, then cooled to 37° C., 0.01 kg of the adipose mesenchymal stem cell lysate prepared in Example 3 and 0.01 kg of hTERT protein were added, and stirred at a speed of 300 r / min for 20 min to obtain the experimental composition B;

[0036] 0.7 kg of water, 0.07 kg of glycerol, 0.035 kg of propylene glycol, 0.015 kg of sodium hyaluronate, and 0.01 kg of p-hydroxyacetophenone were taken, heated to 82° C., and stirred for 30 minutes to obtain a one-phase premix; then, 0.01 kg of olive oil, 0.008 kg of vitamin E, 0.005 kg of dicaprylyl carbonate, and 0.001 kg of hydrogenated lecithin were taken, heated to 82° C., and stirred for 30 minutes to obtain a two-phase premix; the two-phase premix was added to the one-phase premix, and stirred at 82° C. at a speed of 500 r / min for 10 minutes, then cooled to 37° C., 0.01 kg of the adipose mesenchymal stem cell lysate prepared in Example 3 was added, and stirred at a speed of 300 r / min for 20 minutes to obtain the experimental composition C;

[0037] 0.7 kg of water, 0.07 kg of glycerol, 0.035 kg of propylene glycol, 0.015 kg of sodium hyaluronate, and 0.01 kg of p-hydroxyacetophenone were taken, heated to 82° C., and stirred for 30 minutes to obtain a one-phase premix; then, 0.01 kg of olive oil, 0.008 kg of vitamin E, 0.005 kg of dicaprylyl carbonate, and 0.001 kg of hydrogenated lecithin were taken, heated to 82° C., and stirred for 30 minutes to obtain a two-phase premix; the two-phase premix was added to the one-phase premix, and stirred at 82° C. and 500 r / min for 10 minutes to obtain experimental composition D;

[0038] 100 volunteers were randomly divided into 5 groups, 20 people in each group, and deionized water and the above 4 composition groups were used for anti-aging tests. No cosmetics or topical drugs were used on the test area for 3 days before the experiment. Before the experiment, volunteers were required to clean their faces, and then each group of volunteers applied the corresponding samples twice a day, morning and evening, at 1.0 mg / cm 2The amount was evenly applied. On the 0th day of use and after 9 weeks of continuous use, the American VISIA facial image analyzer was used to perform statistical comparisons on the skin conditions of each subject, and the measured average values ​​were calculated. The results are shown in Table 2.

[0039] Table 2 Comparison of skin anti-wrinkle effects

[0040] Group Skin wrinkle change rate (%) Deionized water control group 1.25±0.08 Experimental composition A -47.53±0.57# Experimental composition B -38.65±0.34# Experimental composition C -28.56±0.29# Experimental composition D -15.27±0.21#

[0041] As can be seen from Table 2, the skin wrinkles of each experimental group were significantly reduced compared with the deionized water control group, with significant differences (P<0.05); in particular, the combination of telomerase hTERT252 / 594 and stem cell lysate had an extremely significant effect in reducing skin wrinkles.

[0042] At the same time, the skin moisture content of each group of subjects was tested. Each data was tested three times, and the average value of each group was calculated. The skin moisture content change rate was calculated according to the following formula: Skin moisture content change rate = (skin moisture content after use - skin moisture content before use) / skin moisture content before use × 100%; the test results are shown in Table 3:

[0043] Table 3 Skin moisture content results

[0044] Group Skin moisture content change rate (%) Deionized water control group 4.91±0.24 Experimental composition A 46.54±2.52# Experimental composition B 40.35±1.48# Experimental composition C 32.43±2.67# Experimental composition D 28.64±1.54#

[0045] From the results in Table 3, it can be seen that the skin water content of each experimental group was significantly increased compared with the deionized water control group, and the difference was significant (P<0.05); in particular, the combination of telomerase hTERT252 / 594 and stem cell lysate had an extremely significant effect of increasing skin water content.

[0046] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction 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 may be combined in any one or more embodiments or examples in a suitable manner. Although embodiments of the present invention have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

[0047] Although the embodiments of the present invention are described above in conjunction with the accompanying drawings, the present invention is not limited to the above specific embodiments and application fields, and the above specific embodiments are only illustrative and instructive, rather than restrictive. A person of ordinary skill in the art can also make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present invention, all of which belong to the protection of the present invention.

Claims

1. An improved telomere reverse transcriptase (TERT), characterized in that The telomere reverse transcriptase is based on the amino acid sequence shown in SEQ ID NO: 1, with only the 252nd amino acid sequence mutated from G to H and the 594th K mutated to S.

2. An improved telomerase, characterized in that The invention is composed of telomere reverse transcriptase (TERT) and telomerase RNA component (TERC), wherein the telomere reverse transcriptase is based on the amino acid sequence as shown in SEQ ID NO: 1, and only the amino acid sequence at position 252 is mutated from G to H, and the K at position 594 is mutated to S.

3. An anti-aging composition, characterized in that The invention comprises the improved telomere reverse transcriptase as claimed in claim 1.

4. The composition according to claim 3, characterized in that The composition also contains a pharmaceutically acceptable carrier.

5. An anti-aging cosmetic, characterized in that The cosmetic composition comprises 5-10 parts by weight of water, 0.5-1 parts by weight of glycerol, 0.2-0.6 parts by weight of propylene glycol, 0.1-0.3 parts by weight of sodium hyaluronate, 0.05-0.2 parts by weight of p-hydroxyacetophenone, 0.05-0.2 parts by weight of olive oil, 0.05-0.01 parts by weight of vitamin E, 0.05-0.1 parts by weight of dicaprylyl carbonate, 0.01-0.02 parts by weight of hydrogenated lecithin, 0.05-0.2 parts by weight of adipose mesenchymal stem cell lysate and 0.05-0.2 parts by weight of the telomere reverse transcriptase described in claim 1.

Images