A telomerase and its use in anti-aging

Abstract

The present invention provides a telomerase and its use in anti-aging. The telomerase is a site-directed modified telomerase variant with high activity for anti-aging. After being prepared into a composition, it can achieve good anti-aging effects and has a wide range of application fields.

Description

Technical Field

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

[0002] Cell senescence and carcinogenesis are important cell 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. During cell division, it consumes telomeres to prevent the gradual shortening and breakage of chromosome telomeres. With each cell division, the telomere shortens by a small segment. When the telomere shortens to a certain extent, the cell cannot maintain the normal chromosome structure, thus triggering the cell senescence mechanism. Telomerase can extend the lifespan of chromosomes by adding new telomere sequences to the ends of chromosomes, thereby maintaining the viability of cells.

[0003] Previous studies have shown that telomerase activity is affected by multiple regulatory factors. The most commonly studied factors are telomere length and telomerase expression levels. It has been 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 senescence and carcinogenesis. In addition, changes in chromosome structure and function, DNA damage and repair, and intracellular signaling pathways can also affect telomerase activity. Among existing treatment methods, telomerase inhibitors have become a research hotspot. These inhibitors can inhibit the growth and division of cancer cells by blocking telomerase activity. Some drugs have entered the clinical trial stage, showing potential anti-tumor activity. In addition, researchers are also exploring gene therapy methods for telomerase to restore or inhibit telomerase activity and further explore its role in cell senescence and carcinogenesis.

[0004] Also because telomerase has this amazing anti-aging effect, many telomerase series products have been developed in the prior art, and these products all have the effect of delaying aging. The products mainly achieve the effect of delaying aging by ingesting sufficient effective telomerase, which can enhance telomerase activity and promote cell division. There have also been scientific experiments showing that when 3% concentration of telomerase is applied to skin care products, it can gradually be seen that the basal layer of the skin begins to delay aging, achieving the so-called reverse aging and face-lifting effect. Similarly, in an experiment conducted by American scientists, after using skin care products containing telomerase for 4 weeks, obvious improvements were observed in areas prone to aging such as the corners of the eyes and mouth of the users. The wrinkles in these two areas were tightened by more than 20% and 50% respectively.

[0005] Although there are now many literatures proving that telomerase does have the above-mentioned various effects to a certain 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 telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC). Human telomerase reverse transcriptase (hTERT), as the catalytic subunit of telomerase, plays a decisive role in the activation of telomerase. The expression of hTERT can represent 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 amino acid sequence at position 252 from G to H and the amino acid sequence at position 594 from K to S on the basis of the amino acid sequence shown in SEQ ID NO: 1.

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

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

[0010] The composition used according to the present invention can be in various forms acceptable for topical application, especially in solid or liquid form, depending on the physiologically acceptable medium used.

[0011] The topical composition can be in the form of a water-in-oil (W / O) or oil-in-water (O / W) emulsion, suspension, oil, gel, paste, cream, lotion, solution, slurry, such as an aqueous, alcoholic, water-alcoholic or fatty solution, powder.

[0012] The polyol included as an embodiment may refer to a polyol, such as a fatty alcohol having two or more hydroxyl groups (-OH). An alcohol having two hydroxyl groups is called a diol or glycol, an alcohol having three hydroxyl groups is called a triol (e.g., glycerol), and an alcohol having four hydroxyl groups is called a tetrol (e.g., pentaerythritol). According to one 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, ethylhexanediol, 1,2-hexanediol, PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol, and pentylene glycol. The amount of the polyol included in the cosmetic composition of the present invention is not limited and may be appropriately adjusted by the user. According to one embodiment, the polyol may be 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 may be 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, relative to the total weight of the composition, the composition may include the polyol in an amount of 1 wt.% to 30 wt.%, such as 15 wt.% to 25 wt.%. According to one embodiment, relative to the total weight of the external phase, the composition may include the polyol in an amount of 5 wt.% to 35 wt.%, such as 20 wt.% to 30 wt.%.

[0013] Specifically, the stem cells are mesenchymal stem cells. Mesenchymal stem cells are a heterogeneous cell population derived from the stroma, 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. After cell resuscitation, subculture, and cell lysis of adipose tissue-derived seed mesenchymal stem cells, the cell lysate can be obtained. After lipid embedding, sterilization, and freeze-drying of the cell lysate, the active preservation period of the growth factors in the cell lysate can be increased. In a vacuum-sterile environment, the water in the cell lysate sublimes, causing less damage to the structure and characteristics of biological tissues and cells, enabling them to quickly enter a dormant state, thereby effectively extending 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 part by weight of glycerol, 0.2-0.6 part by weight of propylene glycol, 0.1-0.3 part by weight of sodium hyaluronate, and 0.05-0.2 part by weight of hydroxyacetophenone, 0.05-0.2 part by weight of olive fruit oil, 0.05-0.01 part by weight of vitamin E, 0.05-0.1 part by weight of dicaprylyl carbonate, and 0.01-0.02 part by weight of hydrogenated lecithin, 0.05-0.2 part by weight of adipose mesenchymal stem cell lysate and 0.05-0.2 part by weight of hTERT252 / 594 protein.

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

[0016] Beneficial effects:

[0017] (1) The stem cell lysate is rich in various growth factors and signaling molecules such as cytokines, small nucleic acids, DNA, etc. produced by stem cells, which can activate cells or tissues to achieve the purpose of anti-aging.

[0018] (2) The latest medical research has found that telomerase can repair damaged chromosome telomeres and enhance the regenerative capacity of collagen, elastic fibers, and hyaluronic acid, achieving the effects of wrinkle removal, firming, and enhancing skin elasticity. On this basis, the present invention provides an improved telomerase variant, which has further enhanced activity compared to the original telomerase. This 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 and wrinkle-removing cosmetic composition. It is composed of highly active telomerase, stem cell lysates, and specific adjuvants. The composition can overcome the problem of high storage costs due to the need for low-temperature preservation of conventional telomerase cosmetics. The composition of the present invention can maintain its activity for a long time at room temperature, with low storage costs and is suitable for marketization. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Western-Blot identification result diagram of the expressed hTERT protein DETAILED DESCRIPTION OF THE INVENTION

[0021] The following description is essentially exemplary only and is not intended to limit the present disclosure, application, and uses. It should be understood that in all these drawings, the same or similar reference numerals indicate the same or similar parts and features. Each drawing only schematically shows the concept and principle of the embodiments of the present disclosure, and does not necessarily show the specific dimensions and their ratios of the embodiments of the present disclosure. Specific parts in a specific drawing may be exaggerated to illustrate the relevant details or structures of the embodiments of the present disclosure. The disclosures of 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 a 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) and aiming at the amino acid sequence structure of hTERT, through computer simulation and preliminary three-dimensional modeling analysis, the inventors found that the telomerase mutant obtained by mutating the 252nd amino acid sequence of SEQ ID NO: 1 from G to H and the 594th K to S has improved activity.

[0024] Based on the NM_198253.2 sequence, the amino acid sequence at position 252 was changed to H and the amino acid sequence at position 594 was changed to S, and the corresponding codons were modified. Shanghai Yubo Biotechnology Co., Ltd. was commissioned to perform artificial synthesis and construct the corresponding pFastBacHTA-hTERT252 / 594. At the same time, 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 identified by IFA to obtain positive wells respectively. The positive wells were subjected to large-scale expression, affinity chromatography purification was carried out using Akata, and concentration was carried out using an ultrafiltration tube, and the protein concentration was adjusted to 50 mg / mL for standby.

[0025] Western-Blot identification: Take 5 μl of the two purified proteins and the control empty vector sf9 lysate, perform SDS-PAGE electrophoresis, and carry out Western-Blot experiment identification. The specific method is as follows: Activate the PVDF membrane with formaldehyde for 1 min and soak it in Bio-Red reagent for 1 min; Loading: Mix the samples in a 1:1 ratio, boil them in a microwave oven for 3 min, and the loading volume is 20 μl; Blotting: Transfer the protein from the gel to the PVDF membrane using a Bio-Red electroblotter, and the blotting time is 7 min; Blocking: After blotting, block the PVDF membrane with 5% skim milk for 1 hour; Wash the PVDF membrane with PBST (0.05% Tween-20), 5 min each time, wash 3 times, incubate with 6*His monoclonal antibody diluted 1:3000 times with 5% skim milk at 4°C on a shaker overnight; The next day, wash the PVDF membrane with PBST (0.05% Tween-20), 5 min each time, wash 3 times, incubate with HRP goat anti-mouse antibody diluted 1:8000 times with 5% skim milk at room temperature for 1 hour, wash the PVDF membrane with PBST (0.05% Tween-20) 3 times, 5 min each time; Develop the color with TMB-Blotting Solution and observe the results. From Figure 1 It can be seen that no band was produced in the control empty vector sf9 lysate, while relatively pure bands were shown in both the purified hTERT and hTERT252 / 594 proteins, indicating that relatively purified recombinant proteins were obtained by purification.

[0026] Example 2 Activity Identification of hTERT252 / 594

[0027] Take HaCaT cells in the logarithmic growth phase, digest them with 0.25% trypsin, wash them several times with PBS (pH 7.2), count them, and then adjust the cell concentration to 2x10 8 / L. Add 4 ml of the above cell suspension to each culture flask, place them in an incubator with 5% CO2, 37 °C, and saturated humidity for culture. After 24 h, randomly divide the culture flasks into groups. Add 4 ml of hTERT252 / 594 protein with final concentrations of 0.5% (m / v) and 1% (m / v) to the experimental groups respectively, add 4 ml of hTERT protein with the same concentration to the control group, and add an equal volume of 1640 culture medium to the blank group. Continue to culture for 48 h respectively, then take them out, digest and wash, and adjust the cell concentration to 1x10 9 / L. Aspirate 1 ml, quickly thaw it, add 50 ml of pre-cooled lysis buffer, suspend and mix well, incubate on ice for 30 min, centrifuge at 14000 r / min for 20 min in a 4 °C environment. Take 2 μl of the supernatant and add it to a TRAP reaction tube containing 45 μl of reaction mixture, mix well, add 30 ml of liquid paraffin, incubate in a 25 °C water bath for 30 min, inactivate it at 94 °C for 2 min on a PCR instrument, and then perform 35 cycles of amplification at 94 °C for 30 s, 48 °C for 30 s, and 72 °C for 90 s respectively. After extension at 72 °C for 5 min, take 25 μl of the amplified product and add it to each well of a microplate containing hybridization reaction solution, mix well, incubate at 37 °C for 60 min, add a color developer, develop color in the dark at 37 °C for 10 min, add a termination solution to terminate the reaction, and read the A value at a wavelength of 450 nm. The A value reflects the level of telomerase activity. Each sample in this experiment is set with duplicate tubes, and the results are shown in Table 1.

[0028] Table 1 Results of telomerase activity in each group

[0029] Group Value A 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 in the present invention is significantly higher than that of hTERT. Under the condition of 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 mesenchymal stem cells (product number SNP-M088, Shangen Biotech) were washed with 2 mL of 1×PBS buffer to remove residual proliferation medium, and then 2 mL of 1×trypsin was added. After digestion for 3 min until the cells could be completely detached by gentle pipetting, an equal volume of proliferation medium was added and mixed well. The mixture was centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of proliferation medium, pipetted and mixed well. Then, they were transferred into the mesenchymal stem cell system of Shangen Biotech (product number: SNPM-M088) and cultured at 37°C and 5% CO2 for 2 days to obtain passaged adipose mesenchymal stem cells; the cell suspension was centrifuged and the supernatant was discarded, and the cell pellet was collected; normal saline was added to the cell pellet to resuspend the cell pellet, and normal saline was added to obtain a cell suspension; the cell suspension was placed at -80°C and frozen for 30 min; this was repeated twice; then it was centrifuged and the supernatant was taken to obtain a cell lysate; the cell lysate was lipid-embedded, sterilized, and freeze-dried to obtain an adipose mesenchymal stem cell lysate.

[0033] Example 4 Preparation of a Composition Containing Telomerase

[0034] Take 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 hydroxyacetophenone, heat to 82°C, and keep stirring for 30 min to obtain a one-phase premix; subsequently, take 0.01 kg of olive fruit oil, 0.008 kg of vitamin E, 0.005 kg of dicaprylyl carbonate, and 0.001 kg of hydrogenated lecithin, heat to 82°C, and keep stirring for 30 min to obtain a two-phase premix; add the two-phase premix to the one-phase premix, keep stirring at 82°C at a speed of 500 r / min for 10 min, then cool to 37°C, add 0.01 kg of the adipose mesenchymal stem cell lysate prepared in Example 3 and 0.01 kg of hTERT252 / 594 protein, and keep stirring at a speed of 300 r / min for 20 min to obtain experimental composition A;

[0035] Take 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, heat to 82 °C, and keep stirring for 30 min to obtain a one-phase premix; Subsequently, take 0.01 kg of olive fruit oil, 0.008 kg of vitamin E, 0.005 kg of dicaprylyl carbonate, and 0.001 kg of hydrogenated lecithin, heat to 82 °C, and keep stirring for 30 min to obtain a two-phase premix; Add the two-phase premix to the one-phase premix, keep stirring at a temperature of 82 °C at a speed of 500 r / min for 10 min, then cool down to 37 °C, add 0.01 kg of the adipose mesenchymal stem cell lysate prepared in Example 3 and 0.01 kg of hTERT protein, and keep stirring at a speed of 300 r / min for 20 min to obtain Experimental Composition B;

[0036] Take 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, heat to 82 °C, and keep stirring for 30 min to obtain a one-phase premix; Subsequently, take 0.01 kg of olive fruit oil, 0.008 kg of vitamin E, 0.005 kg of dicaprylyl carbonate, and 0.001 kg of hydrogenated lecithin, heat to 82 °C, and keep stirring for 30 min to obtain a two-phase premix; Add the two-phase premix to the one-phase premix, keep stirring at a temperature of 82 °C at a speed of 500 r / min for 10 min, then cool down to 37 °C, add 0.01 kg of the adipose mesenchymal stem cell lysate prepared in Example 3, and keep stirring at a speed of 300 r / min for 20 min to obtain Experimental Composition C;

[0037] Take 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, heat to 82 °C, and keep stirring for 30 min to obtain a one-phase premix; Subsequently, take 0.01 kg of olive fruit oil, 0.008 kg of vitamin E, 0.005 kg of dicaprylyl carbonate, and 0.001 kg of hydrogenated lecithin, heat to 82 °C, and keep stirring for 30 min to obtain a two-phase premix; Add the two-phase premix to the one-phase premix, keep stirring at a temperature of 82 °C at a speed of 500 r / min for 10 min to obtain Experimental Composition D;

[0038] Select 100 volunteers and randomly divide them into 5 groups, with 20 people in each group, and conduct anti-aging tests with deionized water and the above 4 composition groups respectively. No cosmetics or topical medications can be used on the test areas for the first 3 days. Before the experiment, the volunteers need to clean their faces, and then each group of volunteers applies the corresponding sample 2 times every morning and evening, 1.0 mg / cm each time 2Apply it evenly. At the start of use on day 0 and after 9 consecutive weeks of use, use the American VISIA facial image analyzer to statistically compare the skin conditions of each subject, calculate the measured average value, and the results are shown in Table 2.

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

[0040] Group Change rate of skin wrinkles (%) 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, compared with the deionized water control group, the skin wrinkles in each experimental group were significantly reduced, with a significant difference (P < 0.05); in particular, the combination of telomerase hTERT252 / 594 and stem cell lysate had an extremely significant effect on reducing skin wrinkles.

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

[0043] Table 3 Results of skin water content

[0044] Group Change rate of skin moisture content (%) 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] As can be seen from the results in Table 3, compared with the deionized water control group, the skin water content in each experimental group was significantly increased, with a significant difference (P < 0.05); in particular, the combination of telomerase hTERT252 / 594 and stem cell lysate had an extremely significant effect on increasing skin water content.

[0046] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means 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 expressions 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. Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0047] Although the embodiments of the present invention have been described above in conjunction with the accompanying drawings, the present invention is not limited to the above specific embodiments and application fields. The above specific embodiments are merely illustrative and guiding, rather than restrictive. Those of ordinary skill in the art can also make many forms under the inspiration of this specification and without departing from the scope protected by the claims of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. An improved telomere reverse transcriptase, 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 and telomerase RNA components, 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 amino acid sequence at position 594 is mutated from K to S.

3. 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.

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