Small molecule nucleic acid RNA composition and application thereof in preparation of skin beautifying and repairing products

Abstract

The invention relates to a micromolecule RNA (Ribonucleic Acid) composition and an application thereof in preparing a skin beautifying and repairing product. Comprising tiRNA-1: 34-Glu-CTC-1-M2 and tRF-1: 30-Glu-CTC-1-M2, and the nucleotide sequences of the tiRNA-1: 34-Glu-CTC-1-M2 and the tRF-1: 30-Glu-CTC-1-M2 are as shown in SEQ ID The invention further provides application of the small molecule RNA composition in preparation of skin beautifying and repairing products. It is found for the first time that the composition of tiRNA-1: 34-Glu-CTC-1-M2 and tRF-1: 30-Glu-CTC-1-M2 can promote proliferation and migration of human skin cells, collagen synthesis and secretion in the human skin cells and expression of genes related to angiogenesis are improved, and meanwhile aging of the human skin cells is delayed. Besides, the small molecule nucleic acid RNA composition reduces expression of inflammation-related factors so as to exert an anti-inflammatory effect, exerts the effects of promoting skin regeneration and repair, improving skin aging, resisting inflammation, promoting skin collagen and blood vessel regeneration and the like, can be used for preparing skin beautifying and repairing products, provides a new method and a new thought for the field of skin beauty treatment, and has wide application prospects. Good commercial application prospects are realized.

Description

Technical Field

[0001] The invention relates to a small molecule nucleic acid RNA composition and application thereof in preparing a skin beauty and repair product, belonging to the technical field of biomedicine. Background Art

[0002] The skin is not only the largest organ in the human body, but also the "first line of defense" when we come into contact with the outside world. It can effectively protect the body from harmful external factors, such as bacteria, viruses, ultraviolet rays, chemicals, and physical injuries (such as abrasions, collisions, etc.). The surface of the skin has a stratum corneum and sebum, which can prevent water loss and the entry of harmful substances. In addition, the skin has many other important physiological functions, such as protection, sensory function, temperature regulation, secretion, synthesis, excretion, immune defense, and storage. These functions enable the skin to play a vital role in maintaining human health, protecting internal organs, and regulating physiological states.

[0003] The health and beauty of the skin has a profound impact on one's health, overall image and self-confidence. Skin beauty repair is not only for existing skin problems, but also a preventive maintenance and long-term care. Through skin repair, the overall health of the skin can be effectively improved, the aging process can be delayed, and the natural beauty of the skin can be enhanced. For modern people, maintaining good skin repair and care can not only achieve a better appearance, but also enhance self-confidence and mental health. Therefore, the importance of skin repair in the beauty and health industry is self-evident.

[0004] Although skin beauty and repair products have achieved remarkable results in improving skin health, delaying aging, and repairing damaged skin, these products on the market still have some shortcomings and limitations, mainly including: (1) Many side effects: The main functional ingredients of most skin beauty products are chemical synthetic preparations. Although they have a certain degree of skin beauty function, they contain a variety of chemical additives that may irritate the skin and cause side effects such as skin irritation, redness, dryness or peeling; (2) Dependence: Although many skin repair products can provide certain improvement effects in the short term, these effects are often temporary and require long-term use to maintain. Some products (such as moisturizing, anti-aging essences, etc.) may make the skin dependent on external nourishment. After discontinuation of use, the skin may rebound and even feel drier, duller or age faster; (3) There are problems with ingredients and safety: Many skin repair products contain high concentrations of active ingredients, such as vitamin C, A acid, peptides, etc. Although these ingredients have a repairing effect on the skin, they may also cause irritation or adverse reactions to certain skin. Some skin care products contain chemical ingredients such as preservatives and fragrances. Long-term use may burden the skin and cause allergies or other adverse reactions. (4) Long repair process: The self-repair process of the skin is a long and complex process. Many repair products require long-term use to see obvious results. For example, improving acne scars, pigmentation, fine lines and other problems may require months or even longer of continuous use to see results. For problems such as pigmentation, wrinkles and acne scars, especially deep skin damage, many skin care products may not show results quickly. Professional treatment or stronger treatment methods (such as laser, microneedle therapy) are required to accelerate the effect. (5) Formula complexity and mixing problems: With the increasing diversity of beauty repair products on the market, different brands and types of products have differences in ingredients. Consumers may encounter formula complexity problems when choosing. Stacking too many products may also cause excessive burden on the skin and even adverse reactions. Some repair products may contain interacting ingredients. For example, acid ingredients and vitamin C may cause irritation when used at the same time. Conflicts between products may affect the repair effect and may even cause allergies or irritation.

[0005] In recent years, small molecule nucleic acid RNA drugs have shown great application potential in the field of skin repair. Small molecule nucleic acid RNA drugs can inhibit the expression of specific genes by binding to specific mRNA, thereby accurately regulating the skin repair process. This targeting effect makes small molecule nucleic acid RNA drugs more selective than traditional drugs and treatments, and can reduce interference with non-target genes, thereby avoiding common side effects in traditional drug treatments. Small molecule nucleic acid RNA drugs can regulate the expression of key regeneration and repair genes in the skin and promote the healing process of damaged skin. For example, by regulating cell proliferation, migration, differentiation and other processes, small molecule nucleic acid RNA can accelerate wound healing, promote skin regeneration, and even repair long-term skin damage such as acne marks and scars. During the aging process, the synthesis of collagen and elastin in the skin decreases, resulting in sagging skin, wrinkles and fine lines. Studies have shown that certain small RNA drugs can regulate the expression of aging-related genes and promote the synthesis of collagen and elastin, thereby effectively resisting aging. In the process of skin damage or aging, inflammatory response is an important factor in aggravating skin damage and aging. Small molecule nucleic acid RNA drugs can reduce the negative effects of inflammation and promote skin recovery by regulating inflammatory signaling pathways and inhibiting excessive immune responses. Small molecule nucleic acid RNA drugs usually produce a long-term therapeutic effect by targeting and regulating gene expression, unlike some topical drugs or traditional treatments that require frequent use. A relatively long-lasting therapeutic effect can be achieved simply through proper administration or local application.

[0006] At present, the research on small molecule nucleic acid RNA drugs, especially tRFs, is still in its early stages, and the research on the role of tRFs in skin beauty and repair is still very lacking. Therefore, it is urgent to explore the role of tRFs in skin beauty and repair, and at the same time provide new, effective and safe small molecule RNA drugs for skin beauty and repair. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a small molecule nucleic acid RNA composition and its application in the preparation of skin beauty and repair products.

[0008] The technical solution of the present invention is as follows:

[0009] A small molecule nucleic acid RNA composition, comprising tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2;

[0010] The nucleotide sequence of the tiRNA-1:34-Glu-CTC-1-M2 is shown in SEQ ID NO.1;

[0011] The nucleotide sequence of the tRF-1:30-Glu-CTC-1-M2 is shown in SEQ ID NO.2.

[0012] Preferably according to the present invention, the molar ratio of the tiRNA-1:34-Glu-CTC-1-M2 to tRF-1:30-Glu-CTC-1-M2 is (1-5):(1-5).

[0013] Application of the above small molecule nucleic acid RNA composition in the preparation of skin beauty and repair products.

[0014] Preferably, according to the present invention, the small molecule nucleic acid RNA composition in the skin beauty and repair product has a proliferation promoting effect on human umbilical vein endothelial cells and human immortalized epidermal cells, and resists skin aging and promotes skin renewal by promoting the proliferation, regeneration and renewal of human skin cells. For example, it improves wrinkles, pigmentation, enlarged pores, rough skin, dry skin, uneven skin tone and other problems caused by skin aging.

[0015] Preferably, the small molecule nucleic acid RNA composition in the skin beauty and repair product has a migration promoting effect on human umbilical vein endothelial cells, alleviates the decline in skin repair ability during skin aging by promoting angiogenesis, and promotes skin repair and wound healing. For example, it improves the dull skin color, poor skin elasticity, slow skin wound healing, skin aging and other problems caused by abnormal skin angiogenesis.

[0016] Preferably, according to the present invention, the small molecule nucleic acid RNA composition in the skin beauty and repair product achieves the effect of repairing the skin barrier by upregulating the expression levels of angiogenesis-related markers vascular endothelial growth factor, transforming growth factor β, collagen 1 and collagen 3 in human umbilical vein endothelial cells. For example, it improves wrinkles, fine lines, sagging skin, thinning skin, reduced skin elasticity, dry skin, rough skin, slow skin wound healing and other problems caused by the loss of skin collagen.

[0017] Preferably, according to the present invention, the small molecule nucleic acid RNA composition in the skin beauty and repair product has an anti-aging effect on human umbilical vein endothelial cells, and improves skin aging, improves skin elasticity and firmness, hydration, improves pigmentation and uneven skin color, and enhances skin immune function by delaying the aging of human umbilical vein endothelial cells. The cell aging is natural aging, stress aging, or skin cell aging caused by large-scale replicative passage expansion in vitro. The natural aging refers to the aging of skin cells caused by intrinsic factors of the cells; the stress aging refers to the aging of skin cells caused by light stimulation (ultraviolet rays), electrical stimulation (ionizing radiation) or drug stimulation (doxorubicin, hydrogen peroxide, RO3306, Nutlin-3a) and other risk factors.

[0018] Preferably, according to the present invention, the small molecule nucleic acid RNA composition in the skin beauty and repair product significantly inhibits the differentiation of macrophages M0 to M1 to achieve anti-inflammatory effects, such as improving redness, swelling, pain, itching, dryness, infection, allergic reactions and chronic inflammation (eczema, acne, psoriasis, etc.) caused by inflammation, and enhancing the skin barrier function.

[0019] Preferably according to the present invention, the skin comprises sensitive skin.

[0020] Preferably according to the present invention, the product includes medicine, medical device, health care product, skin care product or cosmetics.

[0021] Further preferably, the medicine, medical device, health product, skin care product or cosmetic can be facial care products (lotion, toner, emulsion, essence, cream, facial mask, facial spray, etc.), body care products (body lotion, body oil, essential oil, etc.), lip care products (lipstick, lip mask, lip balm, lip mask, etc.), eye care products (eye cream, eye mask, eye essence, etc.), skin anti-inflammatory products, skin anti-aging products, skin injection beauty and skin care products (water light injection, etc.), oral beauty and skin care products (oral liquid, etc.), etc.

[0022] A skin beauty and repair product comprises the above-mentioned small molecule nucleic acid RNA composition.

[0023] Preferably according to the present invention, the skin beauty and repair product further comprises a pharmaceutically acceptable carrier, adjuvant, additive, supplement, dispersant or stabilizer.

[0024] Preferably according to the present invention, the skin beauty and repair product is an injection, capsule, tablet, oral preparation or microcapsule preparation.

[0025] Beneficial effects:

[0026] 1. The present invention first discovered that the composition of tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2 can promote the proliferation and migration of human skin cells, and increase the expression of genes related to collagen synthesis and secretion and angiogenesis in human skin cells, while delaying the aging of human skin cells. In addition, the small molecule nucleic acid RNA composition of tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2 reduces the expression of inflammation-related factors and thus exerts an anti-inflammatory effect. The small molecule nucleic acid RNA composition of tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2 has the effects of promoting skin regeneration and repair, improving skin aging, anti-inflammatory, promoting skin collagen and angiogenesis, etc., and can be used to prepare skin beauty and repair products, providing new methods and new ideas for the field of skin medical beauty, and has good commercial application prospects.

[0027] 2. The use of the small molecule nucleic acid RNA composition provided by the present invention in the preparation of skin beauty and repair products is also disclosed for the first time. The small molecule nucleic acid RNA composition has the advantages of simple preparation method, easy access to raw materials, engineering, large-scale and standardized production, and small RNA dosage required for skin beauty and repair. Compared with traditional skin beauty products, the small molecule nucleic acid RNA composition has low cost, sufficient control strategies such as sterility / microorganisms / impurities, perfect preparation process and quality system, and long duration of skin beauty and repair effect. In addition, the main functional ingredients of most skin beauty products are chemical synthetic preparations. Although they have a certain degree of skin beauty function, they contain a variety of chemical additives, which may irritate the skin and cause allergic symptoms. Therefore, compared with other skin beauty products, the small molecule nucleic acid RNA composition of the present invention plays a role at the level of gene or protein expression, has higher specificity and targeting, and the small molecule nucleic acid RNA has high activity, short half-life, small side effects, high safety, and is not easy to produce immune rejection. In addition, the design method of the small molecule nucleic acid RNA composition is simple, and it is easy to be made into various dosage forms, which can be widely used, providing a new choice for the improvement of skin repair and skin beauty efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a result diagram showing that a small molecule nucleic acid RNA composition (tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2) significantly promotes the proliferation of human umbilical vein endothelial cells and human immortalized epidermal cells;

[0029] In the figure: A is a graph showing the result of CCK-8 experiment to detect the proliferation ability of human umbilical vein endothelial cells; B is a graph showing the result of CCK-8 experiment to detect the proliferation ability of human immortalized epidermal cells.

[0030] Figure 2 The graph shows the result that the small molecule nucleic acid RNA composition (tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2) significantly promotes the migration of human umbilical vein endothelial cells.

[0031] Figure 3 It is a result diagram showing that a small molecule nucleic acid RNA composition (tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2) significantly promotes the expression of angiogenesis-related markers in human umbilical vein endothelial cells;

[0032] In the figure: A is the result of qRT-PCR experiment to detect the expression of vascular endothelial growth factor (pDGFA) related to angiogenesis in human umbilical vein endothelial cells; B is the result of qRT-PCR experiment to detect the expression of transforming growth factor β (TGF-β) related to angiogenesis in human umbilical vein endothelial cells.

[0033] Figure 4 It is a result diagram showing that a small molecule nucleic acid RNA composition (tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2) significantly promotes the expression of markers related to collagen synthesis and secretion in human umbilical vein endothelial cells;

[0034] In the figure: A is the result of qRT-PCR experiment to detect the expression of type I collagen (COL1), a marker related to collagen synthesis and secretion, in human umbilical vein endothelial cells; B is the result of qRT-PCR experiment to detect the expression of type III collagen (COL3), a marker related to collagen synthesis and secretion, in human umbilical vein endothelial cells.

[0035] Figure 5 This is a result diagram showing that a small molecule nucleic acid RNA composition (tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2) significantly delays the senescence of human umbilical vein endothelial cells;

[0036] In the figure: A is a staining image of the aging of human umbilical vein endothelial cells detected by β-galactosidase staining experiment; B is a statistical result of the aging of human umbilical vein endothelial cells detected by β-galactosidase staining experiment.

[0037] Figure 6 It is a diagram showing the verification results of the anti-inflammatory function of the small molecule nucleic acid RNA composition (tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2);

[0038] In the figure: A is a qRT-PCR experiment to detect the expression of IL-1β, a marker of macrophage M1; B is a qRT-PCR experiment to detect the expression of IL-6, a marker of macrophage M1; C is a qRT-PCR experiment to detect the expression of TNF-α, a marker of macrophage M1. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the embodiments are all common commercially available products unless otherwise specified.

[0040] Human umbilical vein endothelial cells (HUVEC) and human immortalized epidermal cells (HaCaT) were purchased from Wuhan Saiweier Biotechnology Co., Ltd., China.

[0041] The nucleotide sequence of tiRNA-1:34-Glu-CTC-1-M2 in the small molecule nucleic acid RNA composition of the present invention is:

[0042] 5'-TCCCTGGTGGTCTAGTGGTTAGGATTCGGCGCTC-3' (SEQ ID NO. 1).

[0043] The nucleotide sequence of tRF-1:30-Glu-CTC-1-M2 is:

[0044] 5'-TCCCTGGTGGTCTAGTGGTTAGGATTCGGC-3' (SEQ ID NO. 2).

[0045] Then, Qingke Biotechnology Co., Ltd. was commissioned to artificially synthesize and mix the above-mentioned specific nucleotide sequences by chemical methods to obtain a small molecule nucleic acid RNA composition, wherein the molar ratio of tiRNA-1:34-Glu-CTC-1-M2 to tRF-1:30-Glu-CTC-1-M2 was 1:1.

[0046] siRNA-NC is a conventional public sequence and is available from Qingke Biotechnology Co., Ltd.

[0047] Example 1

[0048] Human umbilical vein endothelial cells (HUVEC) and human immortalized epidermal cells (HaCaT) were treated with small molecule nucleic acid RNA compositions (tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2) by cell transfection method, and the effects of the small molecule nucleic acid RNA compositions on the proliferation of human umbilical vein endothelial cells (HUVEC) and human immortalized epidermal cells (HaCaT) were detected.

[0049] The specific implementation process is as follows:

[0050] (1) Cell transfection

[0051] Human umbilical vein endothelial cells (HUVEC) and human immortalized epithelial cells (HaCaT) were cultured at 2×10 3 / mL was inoculated in DMEM (Yuanpei Biotechnology, China) culture medium in a 96-well plate (NEST, China), and the RNA-specific transfection reagent RNAFit (Hanheng Biotechnology) and serum-free Opti-MEM medium (Thermo) were used for cell transfection, and the small molecule nucleic acid RNA composition was transfected into human umbilical vein endothelial cells (HUVEC) and human immortalized epidermal cells (HaCaT) respectively, so that the final concentration of the small molecule nucleic acid RNA composition was 100nM, and the experimental group of human skin cells transfected with the small molecule nucleic acid RNA composition was obtained; at the same time, the NC group was the control group, and the same dose of siRNA-NC was given to human umbilical vein endothelial cells (HUVEC) according to the same method; after 6 hours of transfection and culture, the fresh culture medium containing 10% serum was replaced, and the CCK-8 experiment was performed 48 hours after transfection;

[0052] (2) CCK-8 experiment

[0053] The control group and the experimental group in step (1) were taken, and CCK-8 reagent was added at 48 hours after transfection, and incubated at 37°C in the dark for 2 hours; then the absorbance (OD) value of the cells in the control group and the experimental group at a wavelength of 450nm was detected by using a microplate reader (TECAN, Switzerland), and the effect of the small molecule nucleic acid RNA composition on the proliferation ability of human skin cells was analyzed. The results are as follows: Figure 1 shown.

[0054] Depend on Figure 1 The CCK-8 test results of A show that, compared with the human umbilical vein endothelial cells (HUVEC) in the control group, treatment with the small molecule nucleic acid RNA composition can significantly improve the proliferation ability of human umbilical vein endothelial cells (HUVEC).

[0055] Depend on Figure 1 The CCK-8 experimental results of B show that, compared with the human immortalized epidermal cells HaCaT in the control group, treatment with the small molecule nucleic acid RNA composition can significantly improve the proliferation ability of human immortalized epidermal cells HaCaT.

[0056] The results of this example show that the small molecule nucleic acid RNA composition of the present invention has the effect of significantly improving the proliferation ability of human umbilical vein endothelial cells (HUVEC) and human immortalized epidermal cells (HaCaT), promoting the proliferation, regeneration and renewal of human skin cells, and promoting skin renewal.

[0057] Example 2

[0058] Human umbilical vein endothelial cells (HUVEC) were treated with the small molecule nucleic acid RNA composition by cell transfection method, and the effect of the small molecule nucleic acid RNA composition on the migration of human umbilical vein endothelial cells (HUVEC) was detected.

[0059] The specific implementation process is as follows:

[0060] (1) Cell transfection

[0061] Human umbilical vein endothelial cells (HUVEC) were cultured at 1×10 6 / mL was inoculated in a six-well plate (NEST, China) in DMEM (Yuanpei Biotechnology, China) culture medium, and RNA-specific transfection reagent RNAFit (Hanheng Biotechnology) and serum-free Opti-MEM medium (Thermo) were used for cell transfection, and the small molecule nucleic acid RNA composition was transfected into human umbilical vein endothelial cells (HUVEC) to make the final concentration of the small molecule nucleic acid RNA composition 100nM, thereby obtaining the experimental group of human umbilical vein endothelial cells (HUVEC) transfected with the small molecule nucleic acid RNA composition; meanwhile, the NC group was the negative control group, in which the same dose of siRNA-NC was given to human umbilical vein endothelial cells (HUVEC) according to the same method; after transfection and culture for 6h, the fresh culture medium containing 10% serum was replaced;

[0062] (2) Cell scratch test

[0063] Human umbilical vein endothelial cells (HUVEC) of the control group and the experimental group in step (1) were streaked along a straight line on the cells using a sterile pipette tip, washed three times with PBS buffer to remove floating cells, and then replaced with fresh serum-free culture medium; and at 0h, 12h, and 24h, the scratches were observed and photographed using an inverted optical microscope to analyze the effect of the small molecule nucleic acid RNA composition on the migration ability of human umbilical vein endothelial cells (HUVEC). The results are as follows: Figure 2 shown.

[0064] Depend on Figure 2 The results of the cell scratch experiment showed that compared with the human umbilical vein endothelial cells (HUVEC) in the negative control (NC) group, treatment with the small molecule nucleic acid RNA composition can significantly promote the migration of human umbilical vein endothelial cells (HUVEC).

[0065] The results of this example show that the small molecule nucleic acid RNA composition of the present invention can significantly improve the migration ability of human umbilical vein endothelial cells (HUVEC), promote skin cell migration, help promote angiogenesis, maintain skin barrier function, alleviate the decline in skin repair ability during skin aging, and promote skin repair and wound healing.

[0066] Example 3

[0067] Human umbilical vein endothelial cells (HUVEC) were treated with the small molecule nucleic acid RNA composition by cell transfection method, and the effect of the small molecule nucleic acid RNA composition on the expression of angiogenesis-related markers in human umbilical vein endothelial cells (HUVEC) was detected.

[0068] The specific implementation process is as follows:

[0069] (1) Cell transfection

[0070] Human umbilical vein endothelial cells (HUVEC) were cultured at 1×10 6 / mL was inoculated in a six-well plate (NEST, China) in DMEM (Yuanpei Biotechnology, China) culture medium, and RNA-specific transfection reagent RNAFit (Hanheng Biotechnology) and serum-free Opti-MEM medium (Thermo) were used for cell transfection, and the small molecule nucleic acid RNA composition was transfected into human umbilical vein endothelial cells (HUVEC) to make the final concentration of the small molecule nucleic acid RNA composition 100nM, thereby obtaining the experimental group of human umbilical vein endothelial cells (HUVEC) transfected with the small molecule nucleic acid RNA composition; meanwhile, the NC group was the negative control group, in which the same dose of siRNA-NC was given to human umbilical vein endothelial cells (HUVEC) according to the same method; after transfection and culture for 6h, the fresh culture medium containing 10% serum was replaced;

[0071] (2) qRT-PCR experiment

[0072] Human skin cells from the control group and the experimental group in step (1) were taken, and cell samples were collected 48 hours after transfection, RNA was extracted, and qRT-PCR experiments were performed to detect the expression of angiogenesis-related markers vascular endothelial growth factor and transforming growth factor β, and the effect of the small molecule nucleic acid RNA composition on the expression of angiogenesis-related markers vascular endothelial growth factor (pDGFA) and transforming growth factor β (TGF-β) in human umbilical vein endothelial cells HUVEC was analyzed. The results are as follows: Figure 3 shown.

[0073] Depend on Figure 3 The qRT-PCR results showed that compared with the human umbilical vein endothelial cells (HUVEC) in the negative control (NC) group, the expression of vascular endothelial growth factor (pDGFA) and transforming growth factor β (TGF-β) in the human umbilical vein endothelial cells treated with the small molecule nucleic acid RNA composition was significantly increased, that is, the small molecule nucleic acid RNA composition can promote the expression of angiogenesis-related markers in HUVEC.

[0074] The results of this example show that the small molecule nucleic acid RNA composition of the present invention can significantly increase the expression of angiogenesis-related markers in human umbilical vein endothelial cells (HUVEC), promote skin angiogenesis, help promote wound healing, promote oxygen and nutrient supply to the skin, improve skin barrier function, accelerate the repair and regeneration of aged skin, promote inflammatory response and immune repair, anti-aging, and improve pigmentation.

[0075] Example 4

[0076] Human umbilical vein endothelial cells (HUVEC) were treated with the small molecule nucleic acid RNA composition by cell transfection method, and the effect of the small molecule nucleic acid RNA composition on the expression of markers related to collagen synthesis and secretion in human umbilical vein endothelial cells (HUVEC) was detected.

[0077] The specific implementation process is as follows:

[0078] (1) Cell transfection

[0079] Human skin cells (human umbilical vein endothelial cells HUVEC) were cultured at 1×10 6 / mL was inoculated in a six-well plate (NEST, China) in DMEM (Yuanpei Biotechnology, China) culture medium, and RNA-specific transfection reagent RNAFit (Hanheng Biotechnology) and serum-free Opti-MEM medium (Thermo) were used for cell transfection, and the small molecule nucleic acid RNA composition was transfected into human umbilical vein endothelial cells (HUVEC) to make the final concentration of the small molecule nucleic acid RNA composition 100nM, thereby obtaining the experimental group of human umbilical vein endothelial cells (HUVEC) transfected with the small molecule nucleic acid RNA composition; meanwhile, the NC group was the negative control group, in which the same dose of siRNA-NC was given to human umbilical vein endothelial cells (HUVEC) according to the same method; after transfection and culture for 6h, the fresh culture medium containing 10% serum was replaced;

[0080] (2) qRT-PCR experiment

[0081] HUVECs from the control group and the experimental group in step (1) were taken, and cell samples were collected 48 hours after transfection, RNA was extracted, and qRT-PCR experiments were performed to detect the expression of collagen formation-related markers collagen 1 and collagen 3, and the effect of the small molecule nucleic acid RNA composition on the expression of collagen synthesis and secretion-related markers collagen 1 (COL1) and collagen 3 (COL3) in human umbilical vein endothelial cells (HUVEC) was analyzed. The results are as follows: Figure 4 shown.

[0082] Depend on Figure 4The qRT-PCR results showed that compared with the human umbilical vein endothelial cells (HUVEC) in the negative control (NC) group, the expression levels of collagen 1 (COL1) and collagen 3 (COL3) in the human umbilical vein endothelial cells treated with the small molecule nucleic acid RNA composition were significantly increased, that is, the small molecule nucleic acid RNA composition can promote the expression of markers related to collagen synthesis and secretion in human umbilical vein endothelial cells (HUVEC).

[0083] The results of this example show that the small molecule nucleic acid RNA composition of the present invention can significantly increase the expression of markers related to collagen synthesis and secretion in human umbilical vein endothelial cells (HUVEC), promote the synthesis and secretion of skin collagen, help maintain the structure and elasticity of the skin, promote skin repair and healing, anti-aging, anti-oxidation, enhance skin barrier function, promote hydration, anti-wrinkle, whitening, etc.

[0084] Example 5

[0085] 1. Cell transfection

[0086] Human umbilical vein endothelial cells (HUVEC) were cultured at 1×10 5 / mL was inoculated in a 24-well plate (NEST, China) in DMEM (Yuanpei Biotechnology, China) culture medium, and RNA-specific transfection reagent RNAFit (Hanheng Biotechnology) and serum-free Opti-MEM medium (Thermo) were used for cell transfection, and the small molecule nucleic acid RNA composition was transfected into human umbilical vein endothelial cells (HUVEC) to make the final concentration of the small molecule nucleic acid RNA composition 100nM, thereby obtaining the experimental group of human umbilical vein endothelial cells HUVEC transfected with the small molecule nucleic acid RNA composition; meanwhile, the NC group was the negative control group, in which the same dose of siRNA-NC was given to human umbilical vein endothelial cells (HUVEC) according to the same method; after transfection and culture for 6h, the fresh culture medium containing 10% serum was replaced;

[0087] 2. Construction of human umbilical vein endothelial cell aging model

[0088] Human umbilical vein endothelial cells (HUVEC) were treated with doxorubicin (100 nM) for 24 hours to obtain a drug-stimulated HUVEC aging model.

[0089] 3. β-Galactosidase staining

[0090] Human umbilical vein endothelial cells (HUVEC) in the control group and the experimental group of step 1 were treated with 100 nM doxorubicin for 24 h, and then a β-galactosidase staining experiment was performed; specifically, a β-galactosidase kit (Biyuntian; C0602) was used for staining according to its instructions to detect the senescence of human umbilical vein endothelial cells (HUVEC), and photos were taken under a microscope to count the proportion of SA-β-gal positive cells. The results are as follows: Figure 5 Shown, scale bar = 50 μm.

[0091] Depend on Figure 5 The β-galactosidase staining results show that compared with the human umbilical vein endothelial cells (HUVEC) in the negative control (NC) group, the β-galactosidase positivity rate in the human umbilical vein endothelial cells was reduced after treatment with the small molecule nucleic acid RNA composition, that is, the small molecule nucleic acid RNA composition can improve the aging of human umbilical vein endothelial cells.

[0092] The results of this example show that the small molecule nucleic acid RNA composition of the present invention can significantly delay the aging of human umbilical vein endothelial cells (HUVEC), improve skin aging, help improve skin elasticity and firmness, hydration, improve pigmentation and uneven skin color, enhance skin immune function, antioxidant capacity, skin barrier function, etc.

[0093] Example 6

[0094] 1. Primary culture and induction of macrophages

[0095] The mice were euthanized, and the femur and tibia of the mice were peeled off with tweezers, surgical scissors and other instruments, and the ends of the femur and tibia were cut off with surgical scissors. PBS was sucked out of the femur and tibia using a 10mL syringe. Blow the bone marrow out of the femur and tibia. Use a 1mL pipette to repeatedly blow the suspension to fully disperse the cells. Then filter with a 70μm cell filter and transfer the cells to a new 15mL centrifuge tube. Place the centrifuge tube in a centrifuge, centrifuge at 1500rpm for 5min, discard the supernatant, add 1mL ACK lysing buffer to resuspend the cells, and place on ice for 10 minutes to fully lyse the red blood cells. Then add 2mL of culture medium to terminate the lysis. Place the centrifuge tube in a centrifuge, centrifuge at 1500rpm for 5min, discard the supernatant, resuspend the cells with macrophage induction medium, place them in a cell culture dish, and culture them in an incubator. The medium was completely replaced on the 4th day of culture, and macrophage M0 was obtained on the 7th day of culture.

[0096] 2. Macrophage differentiation induction

[0097] Macrophages M0 were digested and placed in a cell culture dish. After the cells attached to the wall and began to grow normally, IFN-γ at a concentration of 20 ng / mL and LPS at a concentration of 100 ng / mL were added and treated for 12 hours to induce macrophages M0 to polarize to M1 type.

[0098] 3. Cell transfection

[0099] The RNA-specific transfection reagent RNAFit (Hanheng Bio) and serum-free Opti-MEM medium (Thermo) were used for cell transfection. The small molecule nucleic acid RNA composition was transfected into the M1 macrophages, and the final concentration of the small molecule nucleic acid RNA composition was 100 nM, thereby obtaining the experimental group macrophages transfected with the small molecule nucleic acid RNA composition; meanwhile, the NC group was the negative control group, in which the macrophages were given the same dose of siRNA-NC according to the same method; after transfection and culture for 6 hours, the fresh culture medium containing 10% serum was replaced.

[0100] 4. qRT-PCR experiment

[0101] Take the macrophages of the control group and the experimental group in step 3, collect cell samples 24 hours after transfection, extract RNA, and perform qRT-PCR experiments to detect the expression of macrophage M1-related indicators and analyze the effect of the small molecule nucleic acid RNA composition on the expression of macrophage M1 markers. The results are as follows: Figure 6 shown.

[0102] Depend on Figure 6 The qRT-PCR results of A showed that compared with the macrophages in the negative control (NC) group, the expression levels of macrophage M1 markers (IL-1β, IL-6, TNF-α) in the macrophages treated with the small molecule nucleic acid RNA composition were significantly reduced, that is, the small molecule nucleic acid RNA composition inhibited the differentiation of macrophages M0 to M1.

[0103] The results of this example show that the small molecule nucleic acid RNA composition of the present invention can significantly inhibit the differentiation of macrophages M0 to M1, exert an anti-inflammatory effect, and help treat tissue damage, pigmentation and scar formation, impaired skin barrier function, accelerated aging, acne, eczema, psoriasis, skin allergies, etc. caused by chronic skin inflammation.

[0104] The embodiments described in the present invention are only preferred specific implementation schemes of the present invention and are not used to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A small molecule nucleic acid RNA composition, characterized in that: The small molecule nucleic acid RNA composition includes tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2; The nucleotide sequence of the tiRNA-1:34-Glu-CTC-1-M2 is shown in SEQ ID NO.1; The nucleotide sequence of the tRF-1:30-Glu-CTC-1-M2 is shown in SEQ ID NO.

2.

2. The small molecule nucleic acid RNA composition according to claim 1, characterized in that: The molar ratio of the tiRNA-1:34-Glu-CTC-1-M2 and tRF-1:30-Glu-CTC-1-M2 is (1-5):(1-5).

3. Use of the small molecule nucleic acid RNA composition according to claim 1 in the preparation of skin beauty and repair products.

4. The use according to claim 3, characterized in that The small molecule nucleic acid RNA composition in the skin beauty and repair product has a proliferation-promoting effect on human umbilical vein endothelial cells and human immortalized epidermal cells, and resists skin aging and promotes skin renewal by promoting the proliferation, regeneration and renewal of human skin cells; The small molecule nucleic acid RNA composition in the skin beauty and repair product has a migration-promoting effect on human umbilical vein endothelial cells, alleviates the decline in skin repair ability during skin aging by promoting angiogenesis, and promotes skin repair and wound healing.

5. The use according to claim 3, characterized in that The small molecule nucleic acid RNA composition in the skin beauty repair product achieves the effect of repairing the skin barrier by upregulating the expression levels of angiogenesis-related markers vascular endothelial growth factor, transforming growth factor β, collagen 1 and collagen 3 in human umbilical vein endothelial cells; The small molecule nucleic acid RNA composition in the skin beauty and repair product has an anti-aging effect on human umbilical vein endothelial cells. By delaying the aging of human umbilical vein endothelial cells, it improves skin aging, improves skin elasticity and firmness, hydration, improves pigmentation and uneven skin color, and enhances skin immune function.

6. The use according to claim 3, characterized in that The small molecule nucleic acid RNA composition in the skin beauty and repair product significantly inhibits the differentiation of macrophages M0 to M1, thereby achieving an anti-inflammatory effect.

7. The use according to claim 3, characterized in that The skin includes sensitive skin; the product includes medicine, medical device, health care product, skin care product or cosmetics.

8. A skin beauty and repair product, characterized in that: Comprising the small molecule nucleic acid RNA composition according to claim 1.

9. The skin beauty and repair product according to claim 8, characterized in that: The skin beauty and repair product also includes pharmaceutically acceptable carriers, adjuvants, additives, supplements, dispersants or stabilizers.

10. The skin beauty and repair product according to claim 8, characterized in that: The skin beauty and repair product is an injection, capsule, tablet or oral preparation.

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