MRNA (messenger ribonucleic acid) molecule and application thereof in preparation of beauty and skin care products

Abstract

The invention relates to an mRNA (messenger Ribonucleic Acid) molecule and application thereof in preparation of beauty and skin care products, and particularly, the mRNA molecule comprises a nucleic acid sequence for coding collagen. After the mRNA provided by the invention is applied to the skin, the skin cells can generate collagen, and the aged skin is normalized by increasing the collagen content in the skin tissue, so that the effect of repairing the skin is achieved.

Description

Technical Field

[0001] The present invention belongs to the fields of medical beauty and cosmetics, and relates to an mRNA molecule and its application in the preparation of beauty and skin care products. Background Art

[0002] The skin is the largest organ of the human body, accounting for about 16% of the body weight. The skin covers the entire body surface and is the only organ with functions of protection, body temperature regulation, secretion and excretion of sweat and oil, absorption, sensation, participation in the body's metabolism and immunity against external invasions. The skin is mainly divided into three major layers, from the outside to the inside, namely the epidermis, the dermis, and the subcutaneous tissue.

[0003] Skin aging is a process in which the body functions develop naturally, either quickly or slowly, under the combined influence of internal and external factors. It is mainly caused by endogenous aging and exogenous aging. The main manifestations are that cell metabolism gradually slows down, collagen gradually loses, collagen is damaged, the skin thickness becomes thinner, elastic fibers break, resulting in skin aging problems such as dryness, roughness, wrinkles, and relaxation. With skin aging, protein synthesis decreases, proteolysis increases, humidity generally decreases, and there is general damage to the skin barrier, connective tissue, and cohesion.

[0004] Collagen plays a very important supporting role in the skin, and can maintain the firmness and elasticity of the skin. In particular, type I collagen is the most abundant collagen in the skin, accounting for about 90% of the total collagen content. Collagen is the main structural protein in the extracellular matrix (ECM). The reticular structure composed of collagen fibers (mainly composed of collagen) and elastic fibers (mainly composed of elastin) helps to enhance the structural scaffold and tensile strength of tissues. A large amount of water, extracellular matrix, and functional cells are distributed between the fibers, which is an important biochemical reaction site of the skin. In the ECM of the skin, the ECM interacts with dermal fibroblasts, and the collagen fiber scaffold is continuously remodeled.

[0005] mRNA (messenger RNA) is a nucleic acid molecule that plays a role in information transfer in cells. It is transcribed from genomic DNA and participates in the process of protein synthesis in cells. Compared with DNA, mRNA can mediate better transfection efficiency and longer protein expression time. mRNA can direct skin cells to produce specific proteins, such as collagen, thereby enhancing the elasticity and moisturizing ability of the skin and reducing the appearance of fine lines and wrinkles.

[0006] After investigation, at present, there is no in-depth research on the related technology of delivering mRNA into the skin to promote collagen growth. Summary of the Invention

[0007] To address the deficiencies in the existing technology, the objective of the present invention is to provide a method for delivering mRNA into the skin to promote collagen growth, enabling efficient expression of collagen after the mRNA penetrates the skin and is absorbed by skin cells, reversing the loss of collagen in damaged areas of aging skin, re-improving the cellular microenvironment of damaged skin, and regulating the skin state to achieve skin repair.

[0008] To achieve this objective, the present invention adopts the following technical solutions:

[0009] In the first aspect of the present invention, there is provided an mRNA molecule for introducing into the bottom layer of the skin for beauty purposes, which contains a nucleic acid sequence encoding collagen.

[0010] According to a specific embodiment of the present invention, the mRNA molecule contains a nucleic acid sequence encoding collagen, and the nucleic acid sequence encoding collagen contains a sequence shown in any one of SEQ ID NOs: 1 to 18.

[0011] In the present invention, SEQ ID NOs: 1 to 18 are only characteristic display sequences. According to the technical characteristics of mRNA technology, theoretically, the nucleotide sequence of the collagen can also be the sequences of other types of collagen. Under the condition of keeping other conditions unchanged, expressing other types of collagen, the relevant technical mechanisms are the same, and the objective of the present invention can also be achieved.

[0012] In the second aspect of the present invention, there is provided an mRNA molecule, wherein the mRNA molecule contains a nucleic acid sequence encoding collagen, and the nucleic acid sequence encoding collagen contains a sequence shown in any one of SEQ ID NOs: 1 to 8, SEQ ID NOs: 10 to 17.

[0013] According to a specific implementation scheme of the present invention, the mRNA molecule contains at least one mRNA sequence encoding the α1 chain of type I collagen and at least one mRNA sequence encoding the α2 chain of type I collagen. The mRNA sequence encoding the α1 chain of type I collagen has a sequence shown in any one of SEQ ID NOs: 1 to 9, and the mRNA sequence encoding the α2 chain of type I collagen has a sequence shown in any one of SEQ ID NOs: 10 to 18.

[0014] As a preferred implementation manner, the mRNA sequence encoding the α1 chain of type I collagen has the sequence shown in SEQ ID NO: 3, and the mRNA sequence encoding the α2 chain of type I collagen has the sequence shown in SEQ ID NO: 12.

[0015] As a preferred embodiment, the mRNA sequence encoding the α1 chain of type I collagen has the sequence shown in SEQ ID NO: 5, and the mRNA sequence encoding the α2 chain of type I collagen has the sequence shown in SEQ ID NO: 14.

[0016] As a preferred embodiment, the mRNA sequence encoding the α1 chain of type I collagen has the sequence shown in SEQ ID NO: 3, and the mRNA sequence encoding the α2 chain of type I collagen has the sequence shown in SEQ ID NO: 16.

[0017] According to the specific embodiments of the present invention, some or all of the nucleosides of the mRNA molecule of the present invention may be chemically modified nucleosides.

[0018] In the present invention, the chemically modified nucleosides are selected from one or more of 2-fluoro-2'-deoxyadenosine, 2-fluoro-2'-deoxyuridine, 2-fluoro-2'-deoxycytidine, 2-fluoro-2'-deoxyguanosine, 2-fluoro-2'-deoxy-5-methylcytidine, 2-fluoro-2'-deoxy-pseudouridine, 2-fluoro-2'-deoxy-N1-methyl-pseudouridine, 2-fluoro-2'-deoxy-N7-methyl-guanosine, 2-fluoro-2'-deoxy-5-methoxyuridine, 2-fluoro-2'-deoxy-N4-acetylcytidine, 2-fluoro-2'-deoxy-N6-methyladenosine, 5-methylcytidine, pseudouridine, N1-methyl-pseudouridine, N7-methyl-guanosine, 5-methoxyuridine, N4-acetylcytidine, and N6-methyladenosine.

[0019] According to the specific embodiments of the present invention, the mRNA molecule further has a 5' cap structure.

[0020] In the present invention, the 5' cap structure is selected from one of m7G, Cap0, Cap1, Cap2, modified Cap0, and modified Cap1 structures. Specifically, it may be Cap0, Cap1, Cap2, ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, or a 5' cap structure mentioned in patent applications CN202310091020.3 or CN202310391954.9.

[0021] According to the specific embodiments of the present invention, the mRNA molecule further has PolyA.

[0022] In the present invention, the PolyA structure is selected from one of the PolyAs mentioned in CN202211032978.7 or CN202311309226.5.

[0023] The third aspect of the present invention provides a beauty or pharmaceutical composition, wherein the beauty or pharmaceutical composition comprises the mRNA molecule described in the first aspect or the second aspect of the present invention.

[0024] According to a specific embodiment of the present invention, the beauty or pharmaceutical composition further comprises a medically, cosmetically and cosmetically acceptable matrix material.

[0025] In the present invention, the matrix material includes currently common matrix materials that have good compatibility with ribonucleic acid and will not cause denaturation or degradation of ribonucleic acid components. It can be one or more of natural polymer materials, or one or more of synthetic polymer materials, or a mixed system of natural polymer materials and synthetic polymer materials. Common matrix materials include, but are not limited to, starch, plant gum, animal gelatin, sodium hyaluronate, hyaluronic acid, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxyethylcellulose, and guar gum and its derivatives, polyvinyl alcohol, polyvinylpyrrolidone, acrylic polymers.

[0026] According to a specific embodiment of the present invention, the beauty or pharmaceutical composition further comprises a cosmetically, foodologically or pharmaceutically acceptable carrier.

[0027] In the present invention, the carrier is selected from one or more of excipients, disintegrants, binders, and lubricants.

[0028] The excipients include, but are not limited to, microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof.

[0029] The disintegrants include, but are not limited to, sodium starch glycolate, anhydrous calcium hydrogen phosphate, or a combination thereof.

[0030] The binders include, but are not limited to, polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof.

[0031] The lubricants include, but are not limited to, magnesium stearate, silicon dioxide, talc, or a combination thereof.

[0032] In one embodiment of the present invention, the beauty or pharmaceutical composition further comprises water, glycerin, propylene glycol, sodium hyaluronate, astaxanthin, citric acid, arbutin, PEG-40, mineral oil, 1,4-butanediol, isopropyl palmitate, caprylic / capric triglyceride, polydimethylsiloxane, PEG-60 sorbitan stearate, cetearyl alcohol, polysorbate-60, glyceryl stearate, phenoxyethanol, methylparaben.

[0033] According to a specific embodiment of the present invention, the cosmetic or pharmaceutical composition further comprises a preservative, a stabilizer, a surfactant, a solvent, a humectant, an emollient, an ultraviolet absorber, an antiseptic, a bactericide, an antioxidant, a pH regulator, organic and inorganic pigments, a fragrance, a cooling agent or an antiperspirant. Those skilled in the art can easily select the mixing amounts of additional components such as the above-mentioned humectants within the scope that does not impair the purpose and effects of the present invention.

[0034] According to a specific embodiment of the present invention, the cosmetic or pharmaceutical composition is sterile.

[0035] According to a specific embodiment of the present invention, the cosmetic or pharmaceutical composition is suitable for topical, transdermal, subcutaneous, intradermal, oral, intranasal, intratracheal, sublingual, buccal, rectal, vaginal, urethral, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intra-articular, periaricular, topical or epicutaneous administration.

[0036] As a preferred embodiment, the cosmetic or pharmaceutical composition is suitable for intradermal administration and superficial injection.

[0037] According to a specific embodiment of the present invention, the cosmetic or pharmaceutical composition can be made into a non-oral dosage form. The non-oral dosage form can be an injection or a topical skin agent. The topical skin agent can be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a medicated bandage, a lotion or a combination thereof.

[0038] The topical skin agent can be appropriately mixed with components commonly used in topical skin agents for cosmetics or drugs, etc., for example, aqueous components, oily components, powder components, alcohols, humectants, thickeners, ultraviolet absorbers, whitening agents, antiseptics, antioxidants, surfactants, fragrances, colorants, and various skin nutrients, etc.

[0039] The topical skin agent can be appropriately mixed with metal chelating agents such as disodium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc., agents such as caffeine, tannin, verapamil, glycyrrhizic acid, tranexamic acid and its derivatives or salts thereof, and sugars such as vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, glucose, fructose, trehalose, etc.

[0040] As a preferred embodiment, the cosmetic or pharmaceutical composition is a skin care product.

[0041] According to specific embodiments of the present invention, the cosmetic or pharmaceutical composition further includes the following dosage forms: lotion (moisturizing lotion), softening lotion, toner, astringent, emulsion, milk lotion, moisturizing emulsion, nutrient solution, massage cream, nutrient cream, moisturizing cream, hand cream, foundation, essence, nutrient essence, film, soap, facial cleansing foam, facial cleanser, facial cleansing cream, body lotion, body cleansing liquid, suspension, gel, powder, paste, facial mask or sheet mask or spray composition.

[0042] As a preferred embodiment, the cosmetic or pharmaceutical composition of the present invention is a preparation for use with a radiofrequency introducer, negative pressure introducer, microcrystalline introducer, microcurrent introducer, microneedle introducer, or syringe for introducing into the skin at the bottom layer.

[0043] The fourth aspect of the present invention provides a DNA molecule that can be used for transcription to obtain the mRNA molecule described in the first or second aspect of the present invention.

[0044] According to specific embodiments of the present invention, the DNA molecule further includes a promoter, a UTR sequence and a PolyA fragment are connected to the 3' end, and a UTR sequence and a Cap are connected to the 5' end.

[0045] The fifth aspect of the present invention provides a recombinant plasmid or recombinant cell that contains the DNA molecule described in the fourth aspect of the present invention.

[0046] According to specific embodiments of the present invention, the vector of the recombinant plasmid is an expression vector.

[0047] According to specific embodiments of the present invention, the recombinant plasmid or recombinant cell can be used for transcription to obtain the mRNA described in the first or second aspect of the present invention.

[0048] The sixth aspect of the present invention provides a kit that contains the mRNA molecule described in the first or second aspect of the present invention, the cosmetic or pharmaceutical composition described in the third aspect of the present invention, the DNA molecule described in the fourth aspect of the present invention, or the recombinant plasmid or recombinant cell described in the fifth aspect of the present invention.

[0049] According to specific embodiments of the present invention, the kit is a kit for preparing the mRNA molecule described in the first or second aspect of the present invention. The kit for preparing the mRNA molecule described in the first or second aspect of the present invention contains the DNA molecule described in the third aspect of the present invention, or the recombinant plasmid or recombinant cell described in the fourth aspect of the present invention.

[0050] According to the specific embodiments of the present invention, the kit is a kit for skin care or improving skin condition. The kit for skin care or improving skin condition contains the mRNA molecule described in the first or second aspect of the present invention or the beauty or pharmaceutical composition described in the third aspect of the present invention.

[0051] As a preferred embodiment, the kit for skin care or improving skin condition described in the present invention further comprises an auxiliary introduction device.

[0052] In the present invention, the auxiliary introduction device promotes the entry of the mRNA molecule through the skin into the underlying skin layer to achieve the purpose of effective delivery; among them, the auxiliary introduction device is mainly divided into a radiofrequency introduction instrument, a negative pressure introduction instrument, a microcrystal introduction instrument, a microcurrent introduction instrument, a microneedle introduction instrument, and a syringe according to different action mechanisms.

[0053] As a preferred embodiment, the auxiliary introduction device includes a negative pressure introduction instrument, a microcurrent introduction instrument, and a microneedle introduction instrument. More preferably, the auxiliary introduction device is a microneedle introduction instrument.

[0054] As a preferred embodiment, the auxiliary introduction device is a syringe.

[0055] The seventh aspect of the present invention provides a method for preparing the mRNA molecule described in the first or second aspect of the present invention, which includes:

[0056] (1) Cloning the DNA molecule described in the fourth aspect of the present invention into an expression plasmid to obtain a recombinant plasmid;

[0057] (2) Transferring the recombinant plasmid into a host cell to obtain a recombinant cell, extracting the plasmid from the amplified recombinant cell, and performing PCR amplification using the extracted plasmid as a template to obtain a DNA template for in vitro expression of mRNA;

[0058] (3) Constructing an RNA in vitro synthesis system including the DNA template to perform in vitro synthesis of mRNA to obtain the mRNA molecule.

[0059] In the present invention, when the mRNA molecule is a mixture of an mRNA molecule containing an mRNA sequence encoding the α1 chain of type I collagen and an mRNA molecule containing an mRNA sequence encoding the α2 chain of type I collagen, the above-obtained mRNA molecules can be mixed in proportion to obtain the mixture.

[0060] In the present invention, a DNA fragment for synthesizing and transcribing the mRNA is synthesized, and the DNA fragment is cloned into an expression plasmid to obtain a recombinant plasmid. The present invention has no special limitation on the method for synthesizing the DNA fragment corresponding to the mRNA, and conventional DNA synthesis methods in the art can be used. In the specific implementation process of the present invention, it is preferably entrusted to a biotechnology company for synthesis.

[0061] In the present invention, the DNA fragment is preferably cloned into the expression plasmid by a method of enzymatic digestion and ligation; in the present invention, the DNA fragment is preferably digested with BamHI and NheI enzymes to obtain a digested DNA fragment; the expression plasmid is preferably digested with BamHI and NheI enzymes to obtain a digested plasmid; then the digested DNA fragment and the digested plasmid are ligated to obtain a recombinant plasmid.

[0062] After obtaining the recombinant plasmid in the present invention, the recombinant plasmid is transferred into a host cell to obtain a recombinant cell, and the plasmid is extracted from the expanded recombinant cells, and a DNA template for in vitro expressing mRNA is obtained by PCR amplification using the extracted plasmid as a template. In the present invention, the host cell is preferably an Escherichia coli competent cell;

[0063] After obtaining the recombinant cells in the present invention, screening of positive recombinant cells and colony sequencing are preferably carried out. In the present invention, the screening of the positive recombinant cells is preferably carried out on a solid medium resistant to amp. In the present invention, single colonies on the solid medium resistant to amp are selected for colony PCR, and colonies with a target band in the colony PCR result are selected for sequencing. The present invention has no special limitation on the specific steps of the colony PCR, and conventional colony PCR steps in the art can be used.

[0064] In the present invention, the plasmid of the recombinant cells with correct sequencing is extracted; the present invention has no special limitation on the method for extracting the plasmid, and it is preferably carried out using a plasmid extraction kit. In the present invention, a DNA template for in vitro expressing mRNA is obtained by PCR amplification using the extracted plasmid as a template. The concentration of the DNA template is preferably 1 ng / μl. In the present invention, the amplification program of the PCR is preferably as follows: pre-denaturation at 98 °C for 3 minutes; denaturation at 98 °C for 10 seconds, annealing at 60 °C for 5 seconds, extension at 72 °C for 2 minutes, 34 cycles; and finally extension at 72 °C for 10 minutes.

[0065] In the present invention, after the PCR amplification reaction is completed, the amplification product is preferably detected by agarose gel electrophoresis to determine whether the reaction is successful; the parameters of the agarose gel electrophoresis detection are preferably as follows: 1.5% agarose, 5 V / min, 40 minutes. In the present invention, the appearance of a band of the target size in the agarose gel electrophoresis is considered a successful reaction.

[0066] After the PCR amplification reaction of the present invention is completed, preferably, the amplification product is concentrated and purified. In the present invention, the concentration is preferably carried out using a Millipore 30Kd ultrafiltration tube; the purification is preferably carried out using FPLC; after the purification in the present invention, preferably, the concentration of the purified template is detected by Nano Drop, as well as the ratios of 260 / 280 and 260 / 230. Preferably, the range of 260 / 280 is 1.8 - 2.1, and the range of 260 / 230 is greater than 2.0.

[0067] After obtaining the DNA template of the present invention, an in vitro RNA synthesis system including the DNA template is constructed to carry out in vitro synthesis of mRNA to obtain the active ingredient mRNA. In the present invention, based on 1600 μl, the in vitro RNA synthesis system includes the following components:

[0068]

[0069] In the present invention, the conditions for the in vitro RNA synthesis are preferably 36 - 38 °C for 8 - 12 hours, more preferably 37 °C for 10 hours. In the present invention, the in vitro RNA synthesis is preferably carried out in a constant temperature reactor; the in vitro RNA synthesis system is preferably placed in a 2 ml RNase-free Tube, and multiple tubes are reacted simultaneously at one time; the reaction reagents in the in vitro RNA synthesis system are added in the above order.

[0070] After the in vitro RNA synthesis of the present invention is completed, preferably, it further includes the steps of removing the DNA template, recovering the mRNA, and purifying the mRNA. In the present invention, the removal of the DNA template is preferably achieved by digestion with DNase I; the digestion preferably includes mixing DNase I with the solution after the in vitro RNA synthesis reaction; the volume ratio of DNase I to the solution after the in vitro RNA synthesis reaction is preferably 3:40; the mixing is preferably achieved by inverting the RNase-free Tube up and down, and the number of times of inverting is preferably 8 - 12 times, more preferably 10 times; after the mixing in the present invention, preferably, centrifugation is carried out to collect the solution at the bottom of the RNase-free Tube. In the present invention, the rotation speed of the centrifugation is preferably 800 - 1200 rpm, more preferably 1000 rpm; the time of the centrifugation is preferably 8 - 12 seconds, more preferably 10 seconds. The temperature of the digestion is preferably 37 °C; the time of the digestion is preferably 1 hour.

[0071] After the digestion is completed, it is preferred to detect the remaining DNA fragments in the present invention. In the present invention, the recovery of mRNA is preferably achieved by precipitating with the ammonium acetate solution; for the specific implementation method, refer to the description in the examples; after the mRNA is recovered in the present invention, the quality of the mRNA is detected; the quality detection includes the concentration of mRNA, the ratios of 260 / 280 and 260 / 230 of mRNA, and the value of A260 / A280 for pure mRNA is 2.0 - 2.1, and the range of A260 / A230 is 1.8 - 2.2. In the present invention, the purification of mRNA is achieved by HPLC purification. After the mRNA is purified in the present invention, it is preferred to aliquot the purified mRNA.

[0072] The eighth aspect of the present invention provides a beauty method, which includes applying the mRNA molecule described in the first aspect or the second aspect of the present invention, or the beauty or pharmaceutical composition described in the third aspect of the present invention to the skin.

[0073] According to the specific implementation scheme of the present invention, the method further includes promoting the entry of the mRNA molecule through the skin into the underlying layer of the skin by means of an auxiliary introduction device to achieve the purpose of effective delivery; among them, the auxiliary introduction device is mainly divided into a radio frequency introduction instrument, a negative pressure introduction instrument, a microcrystalline introduction instrument, a microneedle introduction instrument, and a syringe according to its different action mechanisms.

[0074] As a preferred implementation manner, the auxiliary introduction device includes a negative pressure introduction instrument, a microcurrent introduction instrument, and a microneedle introduction instrument. More preferably, the auxiliary introduction device is a microneedle introduction instrument.

[0075] As a preferred implementation manner, the auxiliary introduction device is a syringe.

[0076] According to the specific implementation manner of the present invention, the working concentration of the mRNA molecule is 0.001 ng / ml - 10 mg / ml.

[0077] As a preferred implementation manner, the working concentration of the mRNA molecule is 0.01 μg / ml - 1.0 mg / ml.

[0078] As a more preferred implementation manner, the working concentration of the mRNA molecule is 0.01 - 1.0 μg / ml.

[0079] As a preferred implementation manner, the working concentration of the mRNA molecule is 1.0 mg / ml.

[0080] As a more preferred implementation manner, the working concentration of the mRNA molecule is 0.01 μg / ml.

[0081] According to a specific embodiment of the present invention, the beauty method helps to improve skin wrinkles, and the skin wrinkles targeted refer to nasolabial folds, crow's feet, frown lines, worry lines, scars, glabellar lines, brow ptosis, tear troughs, nasolabial lines, bunny lines, cheek / mid-face sagging, marionette lines, poppy pits, smile lines, laugh lines, chin wrinkles, neck wrinkles, platysmal bands, and any combination thereof formed by superficial depressions.

[0082] The ninth aspect of the present invention provides the use of the mRNA molecule described in the first or second aspect of the present invention, the beauty or pharmaceutical composition described in the third aspect of the present invention, the DNA molecule described in the fourth aspect of the present invention, the recombinant plasmid or recombinant cell described in the fifth aspect of the present invention, or the kit described in the sixth aspect of the present invention in the preparation of a product for skin care or improving skin condition.

[0083] According to a specific embodiment of the present invention, the product is a drug, a cosmetic, or a kit.

[0084] According to a specific embodiment of the present invention, the improvement of the skin condition is to promote the expression of collagen.

[0085] According to a specific embodiment of the present invention, the improvement of the skin condition is to stimulate the proliferation of collagen fibers.

[0086] Beneficial effects:

[0087] The present invention provides an mRNA molecule and its use. The mRNA molecule contains a nucleic acid sequence encoding collagen. The mRNA sequence constructed in the present invention largely avoids sequence-independent apoptosis in mammalian cells, shows better serum stability and enhanced in vivo activity. The present invention also relates to the application of medical, beauty, and cosmetic compositions. By using liposomes or other gene delivery techniques, the composition is introduced into skin cells or absorbed by skin cells. Through the biological mechanism of mRNA translating proteins, skin cells produce collagen, and by upregulating the collagen content in the skin tissue, the aging skin is normalized. The preparation method of the beauty composition preparation provided by the present invention is simple, rapid, and has a high expression level of active ingredients. Combining the drug delivery method in the present invention can enhance the efficiency of mRNA preparation introduced into the skin, improve the utilization rate of the effective preparation, and thus achieve a more effective purpose of transdermal delivery of the skin and achieve the effect of skin repair.

[0088] The cosmetic preparation of the mRNA dosage form provided by the present invention includes mRNA that stimulates collagen regeneration. After introducing the preparation into skin cells, it can promote the expression of collagen in fibroblasts, making up for skin wrinkles caused by aging, light exposure, water loss, etc. The data in specific examples show the effectiveness data. According to the description of the examples, the mRNA with the ability to stimulate collagen growth factor provided by the present invention can be specifically and highly expressed intracellularly. During the use of the present invention, auxiliary devices such as radiofrequency introducers, negative pressure introducers, microcrystal introducers, and microcurrent introducers are combined to break through the skin absorption barrier and activate the expression of autologous collagen, solving the phenomenon that other skin care products are not easily permeable through the epidermis, stimulating the proliferation of collagen fibers, ensuring the integrity of skin tissue, making the skin firm, reducing fine lines, whitening and moisturizing, fading pigments, making the skin fair, bright, and producing collagen from autologous cells, without irritating the skin, so it can be safely used. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 It is a schematic structural diagram for encoding mRNA of the collagen growth factor stimulator;

[0090] Figure 2 It is the comparison result of the collagen content after separate transfection of cells with different sequences of SEQ ID No.1 - 18 and combined transfection of cells;

[0091] Figure 3 It is the quantitative result of the collagen content at the cellular level after cell transfection with mRNA;

[0092] Figure 4 It is the comparison result of the up - regulation rate of the cellular collagen content after cell transfection with mRNA;

[0093] Figure 5 It is the change in the content of type I collagen in the skin after mRNA enters the skin in the 3D skin test measured by the ELISA detection method;

[0094] Figure 6 It is the comparison result of the up - regulation rate of type I collagen in the skin after mRNA enters the skin in the 3D skin test measured by the ELISA detection method;

[0095] Figure 7 It is the immunofluorescence result of type I collagen in the skin section after mRNA enters the skin in the 3D skin test measured by the immunofluorescence method;

[0096] Figure 8 It is the comparison result of the up - regulation of the immunofluorescence intensity of type I collagen in the skin section after mRNA enters the skin in the 3D skin test measured by the immunofluorescence method;

[0097] Figure 9Results of observing the expression of fluorescent protein GFP mRNA in the skin by combining skin sections of mice in each experimental group with an auxiliary introduction instrument;

[0098] Figure 10 Rat skin sections and Masson staining results for observing the expression of type I collagen;

[0099] Figure 11 Mapping the thickness of the dermis layer of rat skin sections in each group to reflect the promoting effect of the mRNA preparation on the production of type I collagen. Detailed implementation manner

[0100] Before further describing the specific implementation manners of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific implementation manners; it should also be understood that the terms used in the embodiments of the present invention are for describing specific implementation manners and not for limiting the protection scope of the present invention.

[0101] When an embodiment gives a numerical range, it should be understood that unless otherwise specified in the present invention, either of the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials of the prior art similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0102] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional techniques in this technical field.

[0103] Example 1. Preparation of mRNA molecules

[0104] This example provides an mRNA molecule formed by connecting an mRNA molecule with PolyA at the 3' end. The sequence of the mRNA molecule includes a 5' cap structure, 5'UTR, target gene sequence, 3'UTR, and PolyA.

[0105] As Figure 1 shown, the mRNA molecule encoding collagen is prepared by the following steps:

[0106] Step S1, designing and synthesizing a plasmid vector with a promoter sequence and a target gene sequence. The plasmid vector is from a company, and the connection order of each part is 5'UTR - nucleotide sequence of the collagen gene - 3'UTR;

[0107] Step S2: Using the plasmid vector obtained in Step S1 as a template, perform in vitro transcription to obtain mRNA molecules. The sequence of the mRNA molecules includes a 5' cap structure, 5' UTR, target gene sequence, 3' UTR, and polyA.

[0108] The mRNA molecules are synthesized by the one-step method. Except for the cap structure, the other elements are integrated into the DNA template during plasmid construction.

[0109] In this example, the nucleotide sequence of the collagen gene is as shown in SEQ ID No. 1-18.

[0110] The 5' cap structure is m7G(5')ppp(5')(2'OMeA)pGp.

[0111] The 5' UTR sequence is as shown in SEQ ID No. 19.

[0112] The 3' UTR sequence is as shown in SEQ ID No. 20.

[0113] The polyA sequence is as shown in SEQ ID No. 21.

[0114] In this example, the specific synthesis method of the mRNA molecules in Step S2 is as follows:

[0115] 1. Amplify the DNA template from the constructed expression plasmid according to the following reaction system:

[0116] Reaction volume: 50 μl (the reaction volume for a single tube, multiple tubes can be reacted simultaneously at one time); the PCR amplification system (50 μl): PrimeSTAR Max Premix (2×) 25 μl, primer F 10 μmol / L 1.2 μl, primer R 10 μmol / L 1.2 μl, DNA template (1 ng / μl) 1 μl, and water 21.6 μl. The PCR amplification program is as follows: pre-denaturation at 98 °C for 3 min; denaturation at 98 °C for 10 s, annealing at 60 °C for 5 s, extension at 72 °C for 2 min, for 34 cycles; finally, extension at 72 °C for 10 min. After the reaction, combine the reaction solutions in a 1.5 ml Tube. Take 10 μl for DNA agarose gel electrophoresis (1.5% agarose, 5 V / min, 40 min). Confirm the success of the reaction according to the size of the target band in the electrophoresis.

[0117] Qualified standard: A single band appears in the electrophoresis detection and the size is correct.

[0118] 2. Ultrafiltration of the DNA template

[0119] Concentrate the obtained DNA template using a Millipore 30Kd ultrafiltration tube.

[0120] 3. FPLC Purification of DNA Template

[0121] Add the DNA obtained by ultrafiltration above to an equal volume of phenol / chloroform / isoamyl alcohol mixture (phenol / chloroform / isoamyl alcohol = 25 / 24 / 1). After thorough shaking, centrifuge at 12,000 g for 15 min. Discard the precipitate, transfer the supernatant to a new centrifuge tube, add 1 / 10 volume of 3 M NaAc (pH 5.2) of the supernatant volume, mix well, then add 2 volumes of absolute ethanol, mix well, and let stand at -20 °C for 30 min. Centrifuge at 4 °C and 12,000 g for 10 min, discard the supernatant. Wash the precipitate with 70% ethanol, centrifuge at 12,000 g for 5 min, take the supernatant, and air-dry it in a laminar flow hood for 5 min. Dissolve the purified DNA template with appropriate RNase-free water. Detect the concentration of the purified template, as well as the ratios of 260 / 280 and 260 / 230 using NanoDrop. Take samples for DNA agarose gel electrophoresis detection (1.5% agarose, 5 V / min, 40 min). Passing criteria: 260 / 280 is between 1.8 and 2.1, and 260 / 230 is between 1.6 and 2.2.

[0122] 4. Ultrafiltration of the Template after FPLC Purification

[0123] Concentrate the DNA template purified by FPLC using a Millipore 30Kd ultrafiltration tube, and elute and dissolve it with RNase-free water. Detect the concentration of the template after ultrafiltration, as well as the ratios of 260 / 280 and 260 / 230 using NanoDrop. Finally, dilute it to 150 ng / μl with RNase-free water.

[0124] 5. In Vitro Synthesis of mRNA

[0125] In a thermostatic reactor, perform the in vitro synthesis of mRNA. Carry out according to the following synthesis system (add reaction reagents from top to bottom):

[0126] Reaction volume, 1600 μl (placed in a 2 ml RNase-free Tube, which is the reaction volume for a single tube, and multiple tubes can be reacted simultaneously at one time): 440 μl of RNA-free water, 160 μl of 7.5 mM ATP, 160 μl of 7.5 mM UTP, 160 μl of 7.5 mM CTP, 160 μl of 7.5 mM GTP, 160 μl of 7.5 mM M7G(2’OMeA)pG, 40 μl of 150 ng / μl DNA template, 160 μl of 10× Buffer, and 160 μl of Enzyme Mix. The program for the in vitro synthesis of RNA is 37 °C for 10 h.

[0127] 6. Removal of DNA template by DNase I digestion

[0128] Add 120 μl of DNase I to each tube after in vitro synthesis of mRNA. Invert the tube 10 times to mix well, and centrifuge at 1000 rpm for 10 s. Place it back into the thermostatic reactor at 37 °C for 1 h.

[0129] 7. Precipitation and recovery of mRNA

[0130] Add an equal volume of ammonium acetate solution to each 50-ml tube in the previous step. Invert the tube 10 times to mix well. Place it at -20 °C for 2 h to precipitate. Centrifuge at 17000 g at 4 °C for 30 min. Discard the supernatant, and wash the precipitate with 70% ethanol. Centrifuge at 17000 g at 4 °C for 10 min. Discard the 70% ethanol, and dry it in the laminar flow hood. Add 20 μl of RNase-free water to each tube. Let it stand for 10 min, and then gently pipette to mix well. Use NanoDrop to detect that the concentration of the recovered mRNA is 5 μg / μl, A260 / A280 is 1.90, and A260 / A230 is 2.0. Take 1 μl and dilute it 10 times, and perform RNA ScreenTape assay and agarose gel electrophoresis to detect its fragment integrity.

[0131] 8. Purification of mRNA by LiCl precipitation

[0132] Add Rnase-free water with a volume 1.5 times that of the recovered mRNA in the previous step, and mix well. Add a LiCl solution pre-cooled to -20 °C with a volume 1.5 times that of the original mRNA, and mix well. Then let it stand at -20 °C for 2 h. Centrifuge at 16000 g for 20 min. Discard the supernatant, and wash the precipitate with 70% ethanol. Centrifuge at 16000 g for 15 min. Take the supernatant, and dry it in the laminar flow hood for 5 min. Dissolve the purified mRNA with an appropriate amount of RNase-free water.

[0133] 9. Obtaining of mRNA reagents

[0134] The mRNAs SEQ ID No.1 - No.18 obtained respectively through the above steps, SEQ ID No.1 - 9 express COL1A1, that is, the α1 chain of type I collagen, and SEQ ID No.10 - 18 express COL1A2, that is, the α2 chain of type I collagen. Currently, there are research reports that increasing the expression of COL1A1 protein alone can increase the expression of collagen in the skin. However, mRNAs SEQ ID No.1 - No.18 have the ability to increase collagen expression whether used alone or in combination. The cell transfection results of each mRNA group are shown in Figure 2 . In the following examples of this article, two mRNA sequences are used in combination in all mRNA test groups.

[0135] Example 2, mRNA cell experiment

[0136] S1 Cell culture and inoculation: Digest the cells and inoculate fibroblasts. Observe under an inverted microscope. When most cells become round and are in a suspended state, add DMEM medium containing serum at about 2 - 3 times the volume of trypsin to terminate digestion, and collect the cells into a centrifuge tube. Centrifuge at 1200 r / min for 5 min. After centrifugation, discard the supernatant. Add a certain volume of cell culture medium to the centrifuge tube, and pipette the cells with a bent pipette to mix evenly. Count the cells using a cell counter. Dilute the cells to the inoculation density and then inoculate them into a 96 - well plate, with 200 μL of liquid in each well. After inoculation, place it in a CO 2 incubator and culture for 24 h ± 2 h.

[0137] S2 Administration: Discard the medium in the 96 - well plate and carry out the administration operation. Add medium containing 1.0 μg / mL, 0.1 μg / mL, 0.01 μg / mL collagen mRNA to the sample wells. Since during the AI algorithm screening process, among the preferred 8 groups of sequences, all are effective relative to the wild - type, in this example, randomly select the compositions of SEQ ID No.3 and SEQ ID No.12, and mix them in a 1:1 volume ratio; add medium containing 250 ng / mL TGF to the positive control wells, and add normal cell culture medium to the blank control wells, 200 μL in each well. After administration, place the 96 - well plate in a CO 2 incubator and culture for 24 h ± 2 h. Cell supernatant collection: After the incubation culture ends, centrifuge to collect the culture solution and use an ELISA kit to measure the content of type I collagen (COL - 1). One - way ANOVA is performed on the test data of each group using SPSS. The statistical method uses a two - tailed test, and the test level = 0.05; calculate the up - regulation rate (%) of COL - 1 in each group according to the following formula:

[0138] COL - 1 up - regulation rate %=(content of the test group - content of the control group) / content of the control group×100%

[0139] From Figure 3 and Figure 4 the data, it can be concluded that the efficiency of the mRNA test group in promoting cell expression of collagen is much higher than that of the blank control group. The positive control group serves to prove the correctness of the cell experiment and the effectiveness of the positive substance. Compared with the positive control group, the up - regulation rate of the mRNA group is significantly higher. In the cell experiment, compared with the up - regulation efficiency of the positive control group, the mRNA group has a higher up - regulation efficiency. The content of collagen expressed by cells in the mRNA group is increased by 2 times compared with the blank control group.

[0140] Example 3, 3D skin experiment

[0141] Test system: T-Skin in vitro artificial full-thickness skin model detection kit, purchased from Shanghai Sianfuno Biotechnology Co., Ltd.; Culture environment: 37.0 ± 1 °C, 5 ± 1% CO2 (v / v), saturated humidity; Cell culture medium: Special medium for T-Skin in vitro artificial full-thickness skin model;

[0142] Main reagents: Vitamin C (Shanghai Macklin Biochemical Technology Co., Ltd.), COL-1 ELISA detection kit (Wuhan Boster Biological Engineering Co., Ltd.), DAPI nuclear staining solution (1 mg / mL), Primary antibody: Anti-Collagen1 antibody (Abcam, USA), Secondary antibody: Dylight-488 (Goat anti-rabbit IgG, Wuhan Boster Biological Engineering Co., Ltd.);

[0143] Main equipment: CO2 incubator, multifunctional microplate reader, biological safety cabinet, TS-8 type transfer decolorizing shaker, confocal microscope;

[0144] Test steps:

[0145] 1. Preparation of test substance: During the AI algorithm screening process, since the 8 selected sequences are all effective relative to the wild type, in this example, the combination of SEQ ID No.5 and SEQ ID No.14 is randomly selected from the preferred group, and the mixing ratio is 1:1 by the same volume as the test substance.

[0146] 2. The T-Skin model enters the laboratory on the 18th day of culture and is maintained in fresh maintenance medium at 37 °C and 5% CO2 for another 48 hours.

[0147] 3. Add the test substance (final concentration 1 mg / mL), positive control (200 μM vitamin C), and blank control (PBS) to the culture medium and incubate for 5 days. The test substance / control is directly added to the surface of the T-Skin model every other day. The culture medium is changed once a day for 5 consecutive days of treatment.

[0148] 4. Immunofluorescence semi-quantification of type I collagen in tissue sections of the T-Skin model: After 5 days of treatment, fix the T-Skin with 4% paraformaldehyde. TMTissues (at 4°C) were paraffin-embedded after dehydration treatment and sectioned. Subsequently, the tissue sections were dewaxed and hydrated, and antigen microwave heat repair was performed using a citric acid repair solution. A normal goat serum blocking solution was added and incubated at room temperature for 1 h. The excess liquid was discarded without washing. The primary antibody (diluted 15-fold) was added and incubated overnight at 4°C, and then washed 3 times with PBS for 5 min each time. The fluorescent secondary antibody (diluted 100-fold) was added and incubated at 37°C for 1 h, and then washed once with PBS for 5 min. DAPI nuclear staining solution (1:1000) was added and incubated at room temperature for 2 min, and then washed 4 times with PBS for 5 min each time. The slides were sealed with an anti-fluorescence quenching agent and observed under a microscope (DAPI: Ex = 358 nm, Em = 461 nm; type I collagen: Ex = 493 nm, Em = 518 nm). Images were collected and the fluorescence intensity was analyzed using Image J.

[0149] 5. Determination of type I collagen (COL-1) content using an ELISA kit: After 5 days of treatment, the culture medium under the model was collected and stored at -80°C, and an ELISA kit was used for determination.

[0150] Result analysis: Calculate the upregulation rate of COL-1 (%) in each group.

[0151] Upregulation rate of COL-1 (%) = (content in the experimental group - content in the control group) / content in the control group × 100%

[0152] The test data of each group were analyzed using an independent samples T-test with SPSS. The statistical method used a two-tailed test, and the significance level = 0.05; Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 The data showed the test results: When the type I collagen content in the sample group > the control group and there was a significant difference after statistical analysis (P < 0.05), it was considered that the sample had the effect of promoting the secretion of type I collagen by fibroblasts in the dermis layer of the T-Skin model tissue.

[0153] Example 4. Verification of the effect of auxiliary instruments through animal experiments

[0154] Preparation of instruments before the experiment: Purchase daily skin care instruments in a shopping mall. The skin care claims to have the function of increasing the skin permeability of cosmetics. For easy understanding and differentiation from the mechanism, they are respectively a microcurrent introduction instrument, a microneedle introduction instrument, and a negative pressure introduction instrument; the above three introduction instruments can be directly purchased from the mall and belong to daily use instruments. In this patent, the brand and type of the introduction instrument are not restricted and protected by rights.

[0155] Preparation of main reagents: In this experiment, the coding mRNA that can express green fluorescent protein was selected as the marker. Green fluorescent protein (GFP) is a protein composed of about 238 amino acids. It can be excited by light from blue to ultraviolet and emit green fluorescence. It is a commonly used indicator protein in biomolecular experiments and can quickly locate the position of expressing cells, facilitating the detection of the distribution of mRNA in the skin after it is introduced into the skin.

[0156] Preparation of experimental animals: Twenty-five 10-week-old healthy female Balb / C mice with qualified quarantine were selected and randomly divided into a blank control group, an eGFP mRNA group, an eGFP mRNA + microcurrent applicator test group (hereinafter referred to as the mRNA microcurrent group), an eGFP mRNA + microneedle applicator test group (hereinafter referred to as the mRNA microneedle group), and an eGFP mRNA + negative pressure applicator test group (hereinafter referred to as the mRNA negative pressure group), with 5 mice in each group.

[0157] Experimental procedures: Four to 24 hours before the experiment, the hair on both sides of the spine on the back of the mice was removed (an area of about 3 cm x 3 cm), which served as the test and observation site. In order to facilitate observation and repeated experiments, hair removal may be required repeatedly.

[0158] After hair removal on the back of the mice, the mice were anesthetized with 1.5% isoflurane, and the back of the mice was wiped with disinfected alcohol. After the alcohol volatilized, the eGFP mRNA solution was evenly applied to the back of the mice at a dose of 5 μg per mouse, and the application range was controlled within a 1 cm x 1 cm square. Subsequently, according to the operation manual, the auxiliary introduction instrument was used to perform a 15-minute liquid introduction treatment on the back of the mice; the blank control group used physiological saline as the control reagent to apply to the back of the mice.

[0159] After the experiment, the remaining excess liquid on the back of the mice was wiped clean, the anesthesia was lifted, and after observing for 24 hours, the skin was excised and placed in 4% formaldehyde for fixation. Subsequently, frozen sections were made and observed under a fluorescence microscope.

[0160] Compared with the blank control group, different degrees of green fluorescent clusters were observed in the mRNA groups under the fluorescence microscope (as shown in Figure 9 ), which proved that the mRNA penetrated the skin and was taken up by skin cells and expressed fluorescent proteins; compared with the group that only applied mRNA, the expression levels of fluorescent proteins in the experimental groups using auxiliary introduction devices all increased to varying degrees. Among them, more obvious green fluorescence could be observed in the epidermis layer of the mRNA microcurrent group and the mRNA negative pressure group.

[0161] Example 5. Verification of collagen expression effect by rat skin test

[0162] Preparation of instruments before the experiment: Purchase daily skincare instruments in a shopping mall. The skincare is claimed to have the function of increasing the skin permeability of cosmetics. For the convenience of understanding and differentiation from the mechanism, they are respectively a microcurrent introducer, a microneedle introducer, and a negative pressure introducer. The above three types of introducers can be directly purchased from the mall and belong to daily-use instruments. Therefore, in this patent, the brands and types of the introducers are not restricted and protected by rights.

[0163] Preparation of main reagents: In this experiment, the coding mRNA that can express collagen is selected as the marker. Because during the AI algorithm screening process, among the preferably selected 8 groups of sequences, they are all effective relative to the wild type. Therefore, in this embodiment, a composition of SEQ ID No.7 and SEQ ID No.16 is randomly selected from the preferred groups, and the mixing ratio is 1:1 by the same volume.

[0164] Preparation of experimental animals: Select 25 healthy 4-week-old female SD rats with qualified quarantine, and randomly divide them into a blank control group, a collagen mRNA + microcurrent introducer test group (hereinafter referred to as the mRNA microcurrent group), a collagen mRNA + microneedle introducer test group (hereinafter referred to as the mRNA microneedle group), a collagen mRNA + negative pressure introducer test group (hereinafter referred to as the mRNA negative pressure group), and a collagen mRNA intradermal injection as the positive control group, with 5 rats in each group.

[0165] Experimental procedure: 4 to 24 hours before the experiment, remove the hair on both sides of the rat's back spine (about a 3 cm x 3 cm area) as the test and observation site. For the convenience of observation and repeated experiments, hair removal may be required repeatedly. After the rat's back is depilated, anesthetize the rat with 1.5% isoflurane, wipe the rat's back with disinfected alcohol, and after the alcohol has evaporated, apply the eGFP mRNA solution at a dose of 10 μg per rat evenly on the rat's back, controlling the application range within a 1 cm x 1 cm square. Then, according to the operation manual, use an auxiliary introduction instrument to perform a 15-minute liquid introduction treatment on the rat's back; the blank control group uses physiological saline as the control reagent and applies it to the rat's back in the same way. After the experiment, wipe off the remaining excess liquid on the rat's back, relieve the anesthesia, observe for 48 hours, then take the skin at the experimental site, fix it overnight with 4% paraformaldehyde, make paraffin sections, and stain the sections with masson and take pictures for analysis.

[0166] At Figure 10 It is possible to visually compare the changes in collagen fibers in the dermis layer among different groups. The dermis layer is mainly composed of collagen fibers and elastic fibers, which directly determine the elasticity of the skin. Figure 11 The data results show that the positive control group has a promoting effect on the upregulation of collagen through intradermal injection, and the expression of collagen in the skin can also be increased by microneedle introduction, showing a significant difference compared with the control group.

[0167] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. An mRNA molecule for importing into the underlying layer of the skin for beauty treatment, which contains a nucleic acid sequence encoding collagen.

2. An mRNA molecule, wherein, the mRNA molecule contains a nucleic acid sequence encoding collagen, and the nucleic acid sequence encoding collagen contains a sequence shown in any one of SEQ ID NO: 1-8, SEQ ID NO: 10-17; preferably, the mRNA molecule contains at least one mRNA sequence encoding the α1 chain of type I collagen and at least one mRNA sequence encoding the α2 chain of type I collagen. The mRNA sequence encoding the α1 chain of type I collagen has a sequence shown in any one of SEQ ID NO: 1-9, and the mRNA sequence encoding the α2 chain of type I collagen has a sequence shown in any one of SEQ ID NO: 10-18.

3. The mRNA molecule according to claim 2, wherein, the mRNA molecule also has a 5' cap structure, 5' UTR, 3' UTR, and / or PolyA.

4. A beauty or medicinal composition, wherein, the beauty or medicinal composition contains the mRNA molecule according to any one of claims 1-3; preferably, the beauty or medicinal composition further includes a medically, cosmetically, and pharmaceutically acceptable matrix material; preferably, the beauty or medicinal composition further includes a cosmetically or pharmaceutically acceptable carrier; preferably, the beauty or medicinal composition is a preparation for importing into the underlying layer of the skin using a radiofrequency import device, a negative pressure import device, a microcrystalline import device, a microcurrent import device, a microneedle import device, or a syringe.

5. A DNA molecule that can be used for transcription to obtain the mRNA molecule according to any one of claims 1-3.

6. A recombinant plasmid or recombinant cell that contains the DNA molecule according to claim 5, preferably, the recombinant plasmid or recombinant cell can be used for transcription to obtain the mRNA molecule according to any one of claims 1-3.

7. A kit that contains the mRNA molecule according to any one of claims 1-3, the beauty or medicinal composition according to claim 4, the DNA molecule according to claim 5, or the recombinant plasmid or recombinant cell according to claim 6.

8. The kit according to claim 7, wherein, the kit further includes an auxiliary import device, preferably, the auxiliary import device includes a radiofrequency import device, a negative pressure import device, a microcrystalline import device, a microcurrent import device, a microneedle import device, or a syringe.

9. A method for preparing the mRNA molecule according to any one of claims 1-3, which comprises: (1) cloning the DNA molecule according to claim 5 into an expression vector to obtain a recombinant plasmid; (2) transferring the recombinant plasmid into a host cell to obtain a recombinant cell, extracting the plasmid from the amplified recombinant cell, and using the extracted plasmid as a template for PCR amplification to obtain a DNA template for in vitro expression of mRNA; (3) constructing an in vitro RNA synthesis system including the DNA template to perform in vitro synthesis of mRNA to obtain the mRNA molecule.

10. Use of the mRNA molecule according to any one of claims 1-3, the cosmetic or pharmaceutical composition according to claim 4, the DNA molecule according to claim 5, the recombinant plasmid or recombinant cell according to claim 6, or the kit according to claim 7 or 8 in the preparation of a product for skin care or improving skin condition, Preferably, the product is a drug, a cosmetic or a kit, Preferably, the improvement of skin condition includes promoting the expression of collagen and stimulating the proliferation of collagen fibers.

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