mRNA molecule and use thereof in preparation of beauty and skin care products

Abstract

Provided are an mRNA molecule and a use thereof in preparation of beauty and skin care products. Specifically, the mRNA molecule comprises a nucleic acid sequence encoding collagen. After the mRNA is applied to skin, skin cells can generate collagen, and thus aged skin is normalized by up-regulating the content of the collagen in skin tissue, achieving the repairing effect of skin.

Description

An mRNA molecule and its application in preparing beauty and skin care products Technical Field

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

[0002] Skin is the largest organ in the human body, accounting for approximately 16% of body weight. Covering the entire body, it is the only organ that protects, regulates body temperature, secretes and excretes sweat and oil, absorbs, senses, participates in the body's metabolism, and protects against external invasions. Skin is divided into three main layers: the epidermis, dermis, and subcutaneous tissue.

[0003] Skin aging is a natural process in which human functions evolve at varying speeds, influenced by both internal and external factors. It is primarily caused by both intrinsic and extrinsic factors. Key manifestations include a gradual slowing of cellular metabolism, the loss and destruction of collagen, thinning of the skin, and the breakdown of elastic fibers, leading to dryness, roughness, wrinkles, and sagging skin. As skin ages, protein synthesis decreases, proteolysis increases, humidity decreases, and the skin barrier, connective tissue, and cohesion become generally damaged.

[0004] Collagen plays a very important supporting role in the skin, maintaining its firmness and elasticity, especially type I collagen, which 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 network structure composed of collagen fibers (mainly composed of collagen) and elastic fibers (mainly composed of elastin) helps to enhance the structural support and tensile strength of the tissue. A large amount of water, extracellular matrix and functional cells are distributed between the fibers, which is an important site for biochemical reactions in the skin. In the ECM of the skin, the ECM interacts with dermal fibroblasts, and the collagen fiber scaffold is constantly reshaped.

[0005] Messenger RNA (mRNA) is a nucleic acid molecule that transmits information within cells. It is transcribed from genomic DNA and participates in protein synthesis within cells. Compared to DNA, mRNA can mediate better transfection efficiency and longer-lasting protein expression. mRNA can instruct skin cells to produce specific proteins, such as collagen, thereby enhancing skin's elasticity and moisturizing ability, and reducing the appearance of fine lines and wrinkles.

[0006] Upon investigation, it was found that there is no in-depth research on the technology of delivering mRNA into the skin to promote collagen growth. Summary of the Invention

[0007] In order to address the deficiencies in the prior art, the purpose of the present invention is to provide a method for delivering mRNA into the skin to promote collagen growth, so that mRNA can penetrate the skin and be absorbed by skin cells to promote efficient expression of collagen, reverse the loss of collagen in damaged areas of aging skin, improve the cellular microenvironment of damaged skin, and regulate the skin condition to achieve skin repair.

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

[0009] In a first aspect, the present invention provides an mRNA molecule for introduction into the underlying skin for cosmetic purposes, comprising a nucleic acid sequence encoding collagen.

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

[0011] In the present invention, SEQ ID NOs: 1-18 are merely representative sequences. Based on the characteristics of mRNA technology, the nucleotide sequences of the collagen proteins described herein can theoretically also represent sequences of other types of collagen. While maintaining other conditions unchanged, the related technical mechanisms for expressing other types of collagen are the same and can still achieve the objectives of the present invention.

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

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

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

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

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

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

[0018] In the present invention, the chemically modified nucleoside is 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 a specific embodiment 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 can 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 one of the 5' cap structures mentioned in patent application CN202310091020.3 or CN202310391954.9.

[0021] According to a specific embodiment of the present invention, the mRNA molecule further comprises 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 cosmetic or pharmaceutical composition, wherein the cosmetic or pharmaceutical composition comprises the mRNA molecule according to the first or second aspect of the present invention.

[0024] According to a specific embodiment of the present invention, the cosmetic or pharmaceutical composition further comprises a base material acceptable for medical, cosmetic and cosmetic use.

[0025] In the present invention, the matrix material includes commonly available matrix materials that have good compatibility with RNA and do not cause denaturation or degradation of RNA components. The matrix material may be one or more natural polymer materials, one or more synthetic polymer materials, or a mixture of natural and synthetic polymer materials. Common matrix materials include, but are not limited to, starch, plant gums, animal gelatin, sodium hyaluronate, hyaluronic acid, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, guar gum and its derivatives, polyvinyl alcohol, polyvinyl pyrrolidone, and acrylic polymers.

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

[0027] In the present invention, the carrier is selected from one or more of an excipient, a disintegrant, a binder, and a lubricant.

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

[0029] The disintegrant includes but is not limited to sodium starch glycolate, anhydrous calcium hydrogen phosphate or a combination thereof.

[0030] The binder includes, but is not limited to, polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, or a combination thereof.

[0031] The lubricant includes, but is not limited to, magnesium stearate, silicon dioxide, talc, or a combination thereof.

[0032] In one embodiment of the present invention, the cosmetic or pharmaceutical composition further comprises water, glycerin, propylene glycol, sodium hyaluronate, astaxanthin, citric acid, arbutin, PEG-40, mineral oil, 1.4-butylene glycol, isopropyl palmitate, caprylic / capric triglyceride, dimethicone, PEG-60 sorbitan stearate, cetearyl alcohol, polysorbate 60, glyceryl stearate, phenoxyethanol, and methylparaben.

[0033] According to a specific embodiment of the present invention, the cosmetic or pharmaceutical composition may further include a preservative, a stabilizer, a surfactant, a solvent, a humectant, an emollient, a UV absorber, a preservative, a bactericide, an antioxidant, a pH adjuster, an organic or inorganic pigment, a fragrance, a cooling agent, or an antiperspirant. The amount of additional ingredients such as the humectant can be readily selected by those skilled in the art without impairing the purpose and effects of the present invention.

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

[0035] According to a particular embodiment of the present invention, the cosmetic or pharmaceutical composition is suitable for topical, transdermal, subcutaneous, intradermal, oral, intranasal, intratracheal, sublingual, buccal, rectal, vaginal, transurethral, ​​inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, local or epidermal 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 prepared into a non-oral dosage form. The non-oral dosage form can be an injection or a skin topical preparation. The skin topical preparation can be a cream, a gel, an ointment, a skin emulsion, a skin suspension, a transdermal patch, a medicated bandage, an emulsion, or a combination thereof.

[0038] The skin external preparation can be appropriately mixed with ingredients commonly used in skin external preparations such as cosmetics or medicines, for example, aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants and various skin nutrients, as needed.

[0039] The skin external preparation can be appropriately mixed with metal chelating agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc., caffeine, tannin, verapamil, glycyrrhizic acid, tranexamic acid and its derivatives or salts thereof, 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 a specific embodiment of the present invention, the cosmetic or pharmaceutical composition further comprises the following dosage forms: lotion (skin lotion), softener, toner, astringent, emulsion, milk lotion, moisturizing lotion, nutrient solution, massage cream, nutrient cream, moisturizing cream, hand cream, foundation, essence, nutrient essence, film, soap, cleansing foam, cleansing milk, 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 introduction into the underlying skin via a radiofrequency introduction device, a negative pressure introduction device, a microcrystal introduction device, a microcurrent introduction device, a microneedle introduction device, or a syringe.

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

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

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

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

[0047] According to a specific embodiment of the present invention, the recombinant plasmid or recombinant cell can be used to transcribe and obtain the mRNA described in the first aspect or the second aspect of the present invention.

[0048] The sixth aspect of the present invention provides a kit comprising 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 a specific embodiment 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 comprises 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 a specific embodiment of the present invention, the kit is a kit for skin care or skin improvement. The kit for skin care or skin improvement comprises the mRNA molecule of the first or second aspect of the present invention or the cosmetic or pharmaceutical composition of the third aspect of the present invention.

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

[0052] In the present invention, the auxiliary introduction device promotes the mRNA molecules to pass through the skin into the underlying skin layer, thereby achieving the purpose of effective delivery; wherein the auxiliary introduction device is mainly divided into radio frequency introduction device, negative pressure introduction device, microcrystal introduction device, microcurrent introduction device, microneedle introduction device, and syringe according to its different mechanism of action.

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

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

[0055] A seventh aspect of the present invention provides a method for preparing the mRNA molecule according to the first or second aspect of the present invention, comprising:

[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 comprising an mRNA sequence encoding type I collagen α1 chain and an mRNA molecule comprising an mRNA sequence encoding type I collagen α2 chain, the mixture can be obtained by mixing the mRNA molecules obtained above in proportion.

[0060] In the present invention, a DNA fragment that transcribes the mRNA is synthesized and cloned into an expression plasmid to obtain a recombinant plasmid. The present invention does not particularly limit the method for synthesizing the DNA fragment corresponding to the mRNA; conventional DNA synthesis methods in the art can be used. In the specific implementation of the present invention, the synthesis is preferably commissioned to a biotechnology company.

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

[0062] After obtaining the recombinant plasmid, the present invention transfers the recombinant plasmid into host cells to obtain recombinant cells. The plasmid is extracted from the expanded recombinant cells, and PCR amplification is performed using the extracted plasmid as a template to obtain a DNA template for in vitro expression of mRNA. In the present invention, the host cells are preferably competent Escherichia coli cells;

[0063] After obtaining recombinant cells, the present invention preferably performs screening for positive recombinant cells and colony sequencing. In the present invention, screening for positive recombinant cells is preferably performed on an AMP-resistant solid culture medium. In the present invention, single colonies on the AMP-resistant solid culture medium are selected for colony PCR, and colonies containing the target band in the colony PCR results are selected for sequencing. The present invention does not specifically limit the specific steps of the colony PCR; conventional colony PCR procedures in the art can be employed.

[0064] In the present invention, plasmids from recombinant cells with correct sequencing are extracted. The present invention does not specifically limit the method for extracting the plasmids, but preferably employs a plasmid extraction kit. In the present invention, PCR amplification is performed using the extracted plasmids as templates to obtain a DNA template for in vitro mRNA expression. The concentration of the DNA template is preferably 1 ng / μl. In the present invention, the PCR amplification procedure is preferably as follows: initial denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, and extension at 72°C for 2 minutes, for 34 cycles; and a final extension at 72°C for 10 minutes.

[0065] In the present invention, after the PCR amplification reaction is completed, the amplified product is preferably subjected to agarose gel electrophoresis to determine whether the reaction is successful. The parameters for the agarose gel electrophoresis are preferably as follows: 1.5% agarose, 5 V / min, 40 minutes. In the present invention, the appearance of a band of the desired size on agarose gel electrophoresis is considered a successful reaction.

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

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

[0068] 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 performed in a thermostatic reactor; the in vitro RNA synthesis system is preferably placed in a 2 ml RNase-free tube, with multiple tubes reacted simultaneously at one time; and the reaction reagents in the in vitro RNA synthesis system are added in the order described above.

[0069] After the in vitro synthesis of RNA is completed, the present invention preferably 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 DNase I digestion; the digestion preferably includes mixing DNase I with the solution after the RNA in vitro synthesis reaction; the volume ratio of the DNase I to the solution after the RNA in vitro synthesis reaction is preferably 3:40; the mixing is preferably achieved by turning the RNase-free Tube upside down, and the number of inversions is preferably 8 to 12 times, more preferably 10 times; after the mixing, the present invention preferably performs centrifugation to collect the solution to the bottom of the RNase-free Tube. In the present invention, the centrifugal speed is preferably 800 to 1200 rpm, more preferably 1000 rpm; the centrifugal time is preferably 8 to 12 seconds, more preferably 10 seconds. The digestion temperature is preferably 37°C; the digestion time is preferably 1 hour.

[0070] The present invention preferably performs a residual DNA fragment detection after the digestion is completed. In the present invention, the recovery of mRNA is preferably achieved by precipitation with the ammonium acetate solution; the specific implementation method is described in the examples; after the mRNA is recovered, the present invention performs a quality test on the mRNA; the quality test includes the concentration of the mRNA, the 260 / 280 and 260 / 230 ratios of the mRNA, and the A260 / A280 value of the pure mRNA is 2.0 to 2.1, and the A260 / A230 range is 1.8 to 2.2. In the present invention, the purification of mRNA is achieved by HPLC purification. After the purification of mRNA according to the present invention, the purified mRNA is preferably packaged.

[0071] The eighth aspect of the present invention provides a cosmetic method, comprising applying the mRNA molecule according to the first or second aspect of the present invention, or the cosmetic or pharmaceutical composition according to the third aspect of the present invention to the skin.

[0072] According to a specific embodiment of the present invention, the method further includes promoting the mRNA molecules to pass through the skin into the underlying skin layer by means of an auxiliary introduction device to achieve the purpose of effective delivery; wherein the auxiliary introduction device is mainly divided into a radio frequency introduction device, a negative pressure introduction device, a microcrystal introduction device, a microneedle introduction device, and a syringe according to its different mechanism of action.

[0073] As a preferred embodiment, the auxiliary introduction device includes a negative pressure introduction device, a microcurrent introduction device, and a microneedle introduction device. More preferably, the auxiliary introduction device is a microneedle introduction device.

[0074] As a preferred embodiment, the auxiliary introduction device is a syringe.

[0075] According to a specific embodiment of the present invention, the working concentration of the mRNA molecule is 0.001 ng / ml-10 mg / ml.

[0076] As a preferred embodiment, the working concentration of the mRNA molecule is 0.01 μg / ml-1.0 mg / ml.

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

[0078] As a preferred embodiment, the working concentration of the mRNA molecule is 1.0 mg / ml.

[0079] As a more preferred embodiment, the working concentration of the mRNA molecule is 0.01 μg / ml.

[0080] According to a specific embodiment of the present invention, the cosmetic method helps to improve skin wrinkles, and the targeted skin wrinkles are nasolabial folds, crow's feet, frown lines, worry lines, scars, glabellar lines, brow drooping, tear troughs, nasal cheek lines, bunny lines, cheek / mid-face drooping, marionette lines, poppy pits, smile lines, laugh lines, chin wrinkles, neck lines, platysma bands and any combination thereof formed by superficial depressions.

[0081] 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 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, 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.

[0082] According to a specific embodiment of the present invention, the product is a medicine, a cosmetic or a kit.

[0083] According to a specific embodiment of the present invention, the improvement of skin condition is to promote the expression of collagen.

[0084] According to a specific embodiment of the present invention, the improvement of skin condition is to stimulate collagen fiber proliferation. Beneficial effects:

[0085] The present invention provides an mRNA molecule and its use, wherein the mRNA molecule comprises a nucleic acid sequence encoding collagen. The mRNA sequence constructed by the present invention largely avoids sequence-independent apoptosis in mammalian cells, exhibiting better serum stability and enhanced in vivo activity. The present invention also relates to the use of medical, cosmetic, and cosmetic compositions. Utilizing liposomes or other gene delivery technologies, the composition is introduced into skin cells or absorbed by skin cells, whereupon, through the biological mechanism of mRNA translation of protein, the skin cells produce collagen. By increasing the collagen content in skin tissue, aging skin is normalized. The preparation method of the cosmetic composition provided by the present invention is simple, rapid, and has a high expression level of active ingredients. Combined with the drug delivery method of the present invention, the efficiency of mRNA delivery into the skin can be enhanced, and the utilization rate of the effective formulation can be improved, thereby achieving the purpose of more effective transdermal delivery and achieving the effect of skin repair.

[0086] The cosmetic preparation in mRNA dosage form provided by the present invention includes mRNA that stimulates collagen regeneration. After the preparation is introduced into skin cells, it can promote the expression of collagen in fibroblasts, compensating for skin wrinkles and other conditions caused by aging, light exposure, water loss, etc. The data in the specific examples show the effectiveness data; according to the records of the examples, the mRNA that stimulates collagen growth factor provided by the present invention can be expressed at a high level specifically in cells; during use, the present invention is combined with auxiliary equipment such as radio frequency introduction instruments, negative pressure introduction instruments, microcrystal introduction instruments, microcurrent introduction instruments, etc. to break through the skin absorption barrier and activate autologous collagen expression, solving the problem that other skin care products are difficult to penetrate the epidermis, stimulating collagen fiber proliferation, ensuring the integrity of skin tissue, making the skin firm, fine lines faded, whitening and moisturizing, and lightening pigmentation, making the skin white and translucent, and since autologous cells produce collagen, it will not irritate the skin, so it can be used safely. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIG1 is a schematic diagram of the structure of mRNA encoding collagen growth factor;

[0088] FIG2 is a comparison of the collagen content of cells transfected with different sequences of SEQ ID No. 1-18 alone and in combination;

[0089] Figure 3 is the quantitative results of collagen content at the cellular level after cells were transfected with mRNA;

[0090] Figure 4 is a comparison of the upregulation rates of cellular collagen content after cell transfection with mRNA;

[0091] FIG5 shows the changes in type I collagen content in the skin after 3D skin test mRNA enters the skin using the ELISA detection method;

[0092] FIG6 is a comparison of the upregulation rate of type I collagen in the skin after the 3D skin test mRNA enters the skin using the ELISA detection method;

[0093] FIG7 shows the immunofluorescence results of type I collagen in skin sections after 3D skin test mRNA entered the skin using immunofluorescence assay;

[0094] FIG8 is a comparison of the results of the upregulation of type I collagen immunofluorescence intensity in skin sections after 3D skin test mRNA enters the skin using immunofluorescence assay;

[0095] FIG9 shows the results of observing the expression of fluorescent protein GFP mRNA in the skin of mice in each experimental group using the assisted introduction instrument;

[0096] Figure 10 shows the rat skin sections and Masson staining results, observing the expression of type I collagen;

[0097] FIG11 shows the thickness of the dermis layer in skin sections of rats in each group, which reflects the promoting effect of the mRNA preparation on the production of type I collagen. DETAILED DESCRIPTION

[0098] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0099] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0100] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in this technical field.

[0101] Example 1. Preparation of mRNA molecules

[0102] This embodiment provides an mRNA molecule, which is formed by connecting an mRNA molecule to a 3' end Poly A. The sequence of the mRNA molecule includes a 5' cap structure, a 5' UTR, a target gene sequence, a 3' UTR and Poly A.

[0103] As shown in FIG1 , the mRNA molecule encoding collagen is prepared by the following steps:

[0104] Step S1, design and synthesize a plasmid vector with a promoter sequence and a target gene sequence. The plasmid vector is a company vector, and the connection order of each part is 5'UTR-collagen gene nucleotide sequence-3'UTR;

[0105] Step S2, performing in vitro transcription using the plasmid vector of step S1 as a template to obtain an mRNA molecule, wherein the sequence of the mRNA molecule includes a 5' cap structure, a 5' UTR, a target gene sequence, a 3' UTR and plolyA;

[0106] The mRNA molecule is synthesized in a one-step process. Except for the cap structure, all other elements are integrated into the DNA template during plasmid construction.

[0107] In this embodiment, the nucleotide sequences of the collagen genes are shown in SEQ ID Nos. 1 to 18.

[0108] The 5' cap structure is m7G(5')ppp(5')(2'OMeA)pGp.

[0109] The 5'UTR sequence is shown as SEQ ID No.19.

[0110] The 3'UTR sequence is shown as SEQ ID No. 20.

[0111] The polyA sequence is shown as SEQ ID No.21.

[0112] In this embodiment, the specific method for synthesizing the mRNA molecule in step S2 is as follows:

[0113] 1. Amplify the constructed expression plasmid using the following reaction system for DNA template amplification:

[0114] Reaction volume, 50 μl (representing the reaction volume of a single tube; multiple tubes can be reacted simultaneously); PCR amplification system (50 μl): Prime STAR 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 procedure was as follows: initial denaturation at 98°C for 3 min; 34 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 5 s, and extension at 72°C for 2 min; and final extension at 72°C for 10 min. After completion of the reaction, the reaction mixtures were combined in a 1.5 ml tube. 10 μl of the sample was subjected to DNA agarose gel electrophoresis (1.5% agarose, 5 V / min, 40 min). The success of the reaction was confirmed by the size of the target band on the electrophoresis.

[0115] Eligibility criteria: Electrophoresis detection shows a single band with the correct size.

[0116] 2. DNA template ultrafiltration

[0117] The DNA template obtained above was concentrated using Millipore 30Kd ultrafiltration tube.

[0118] 3. FPLC purification of DNA template

[0119] Add an equal volume of phenol / chloroform / isoamyl alcohol mixture (phenol / chloroform / isoamyl alcohol = 25 / 24 / 1) to the ultrafiltrated DNA, shake thoroughly, and centrifuge at 12,000 g for 15 minutes. Remove the precipitate, transfer the supernatant to a new centrifuge tube, add 1 / 10 the supernatant volume of 3M NaAc (pH 5.2), mix thoroughly, then add 2 volumes of anhydrous ethanol, mix thoroughly, and let stand at -20°C for 30 minutes. Centrifuge at 12,000 g for 10 minutes at 4°C, and discard the supernatant. Wash the precipitate with 70% ethanol, centrifuge at 12,000 g for 5 minutes, remove the supernatant, and air-dry in a clean hood for 5 minutes. Dissolve the purified DNA template in appropriate RNase-free water. Determine the concentration of the purified template and the 260 / 280 and 260 / 230 ratios using a NanoDrop. Samples are then analyzed by DNA agarose gel electrophoresis (1.5% agarose, 5 V / min, 40 minutes). Pass criteria: 260 / 280 between 1.8 and 2.1, 260 / 230 between 1.6 and 2.2.

[0120] 4. Template ultrafiltration after FPLC purification

[0121] Concentrate the FPLC-purified DNA template using Millipore 30Kd ultrafiltration tubes and elute with RNase-free water. Determine the ultrafiltration template concentration and the 260 / 280 and 260 / 230 ratios using a NanoDrop. Finally, dilute to 150 ng / μl with RNase-free water.

[0122] 5. In vitro synthesis of mRNA

[0123] In a thermostatic reactor, in vitro mRNA synthesis was performed according to the following synthesis system (reaction reagents were added from top to bottom):

[0124] The reaction volume was 1600 μl (placed in a 2 ml RNase-free tube, representing the reaction volume for a single tube; multiple tubes were reacted simultaneously): 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 in vitro RNA synthesis procedure was 37°C for 10 h.

[0125] 6. DNase I digestion to remove DNA template

[0126] Add 120 μl of DNase I to each tube after in vitro mRNA synthesis. Invert 10 times to mix thoroughly. Centrifuge at 1000 rpm for 10 seconds. Return to the thermostat at 37°C for 1 hour.

[0127] 7. mRNA precipitation and recovery

[0128] Add an equal volume of ammonium acetate solution to each 50ml Tube in the previous step. Mix by inverting 10 times. Place at -20℃ for 2h to precipitate. Centrifuge at 17000g, 4℃, 30min. Remove the supernatant and wash the precipitate with 70% ethanol. Centrifuge at 17000g, 4℃, 10min. Remove the 70% ethanol, evaporate to dryness in a clean bench, and add 20ml of RNase-free water to each tube. After standing for 10min, gently blow with a pipette to mix. The recovered mRNA concentration detected by NanoDrop was 5μg / μl, A260 / A280 was 1.90, and A260 / A230 was 2.0. Take 1μl, dilute 10 times, and perform RNA ScreenTape assay and agarose gel electrophoresis to detect the integrity of the fragment.

[0129] 8. Purification of mRNA by LiCl precipitation

[0130] Add 1.5 times the volume of mRNA recovered in the previous step to RNase-free water and mix thoroughly. Add 1.5 times the volume of pre-chilled LiCl solution at -20°C (1.5 times the volume of the original mRNA) and mix thoroughly. Incubate at -20°C for 2 hours. Centrifuge at 16,000g for 20 minutes. Discard the supernatant, wash the precipitate with 70% ethanol, and centrifuge at 16,000g for 15 minutes. Remove the supernatant and air-dry in a clean hood for 5 minutes. Dissolve the purified mRNA in appropriate RNase-free water.

[0131] 9. Obtaining mRNA reagents

[0132] The mRNA SEQ IDs 1-18 obtained through the above steps are: SEQ IDs 1-9 express COL1A1, i.e., type I collagen α1 chain; and SEQ IDs 10-18 express COL1A2, i.e., type I collagen α2 chain. Studies have reported that increasing COL1A1 protein expression alone can increase collagen expression in the skin. mRNA SEQ IDs 1-18, whether used alone or in combination, have the ability to increase collagen expression. The cell transfection results for each mRNA group are shown in Figure 2. In the examples hereinbelow, all mRNA test groups used a combination of two mRNA sequences.

[0133] Example 2, mRNA cell assay

[0134] S1 Cell Culture and Inoculation: Digest cells and inoculate fibroblasts. Observe under an inverted microscope. When most cells become round and suspended, add approximately 2-3 times the volume of trypsin-containing DMEM medium to terminate digestion. Collect the cells into a centrifuge tube and centrifuge at 1200 rpm for 5 minutes. After centrifugation, discard the supernatant and add a certain volume of cell culture medium to the centrifuge tube. Mix the cells by pipetting with an elbow pipette and count them using a cell counter. Dilute the cells to the inoculation density and inoculate them into a 96-well plate with 200 μL per well. After inoculation, place the cells in a CO2 incubator and incubate for 24 hours ± 2 hours.

[0135] S2 administration: discard the culture medium in the 96-well plate and carry out the administration operation. 1.0 μg / mL, 0.1 μg / mL, and 0.01 μg / mL collagen mRNA culture medium were added to the sample wells. Because the 8 preferred sequences were all effective compared to the wild type during the AI ​​algorithm screening process, the preferred group in this embodiment randomly selected a combination of SEQ ID No. 3 and SEQ ID No. 12, and the mixing ratio was 1:1 with equal volume; 250 ng / mL TGF culture medium was added to the positive control wells, and normal cell culture medium was added to the blank control wells, 200 μL per well. After the administration is completed, the 96-well plate is placed in a CO2 incubator and cultured for 24h±2h. Cell supernatant collection: After the incubation and culture, the culture medium was collected by centrifugation and the type I collagen (COL-1) content was determined using an ELISA kit. The test data of each group were subjected to one-way analysis of variance using SPSS, and the statistical method used was a two-tailed test with a test level of 0.05; the COL-1 upregulation rate (%) of each group was calculated according to the following formula:

[0136] COL-1 up-regulation rate % = (content in the experimental group - content in the control group) / content in the control group × 100%

[0137] From the data in Figures 3 and 4, it can be concluded that the mRNA test group is much more efficient in promoting cell collagen expression than the blank control group. The positive control group serves to verify the correctness of the cell experiment and reflect the effectiveness of the positive substance. Compared with the positive control group, the mRNA group has a significantly higher upregulation rate. In the cell experiment, the mRNA group has a higher upregulation efficiency than the positive control group. Compared with the blank control group, the cell expression of collagen content increased by 2 times.

[0138] Example 3: 3D skin test

[0139] Experimental system: T-Skin in vitro artificial full-thickness skin model test kit, purchased from Shanghai Si'anfuno Biotechnology Co., Ltd.; culture environment: 37.0±1°C, 5±1% CO2 (v / v), saturated humidity; cell culture medium: T-Skin in vitro artificial full-thickness skin model-specific culture medium;

[0140] Main reagents: Vitamin C (Shanghai MacLean Biochemical Technology Co., Ltd.), COL-1 ELISA detection kit (Wuhan Boster Bioengineering 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 Bioengineering Co., Ltd.);

[0141] Main equipment: CO2 incubator, multifunctional microplate reader, biological safety cabinet, TS-8 transfer and decolorization shaker, confocal microscope;

[0142] Test steps:

[0143] 1. Preparation of test substances: Because the eight preferred groups of sequences were all effective relative to the wild type during the AI ​​algorithm screening process, the preferred group in this example randomly selected a combination of SEQ ID No. 5 and SEQ ID No. 14, and the mixture ratio was 1:1 with the same volume as the test substance.

[0144] 2. The T-Skin model was brought into the laboratory on the 18th day of culture and maintained in fresh maintenance medium at 37°C and 5% CO2 for another 48 hours.

[0145] 3. Add the test substance (final concentration of 1 mg / mL), a positive control (200 μM vitamin C), and a blank control (PBS) to the culture medium and incubate for 5 days. Every other day, apply the test substance / control substance directly to the T-Skin model surface. Change the culture medium daily for 5 consecutive days.

[0146] 4. Semi-quantitative immunofluorescence analysis of type I collagen in T-skin model tissue sections: After 5 days of treatment, the T-skin was fixed with 4% paraformaldehyde. TMTissues were dehydrated (4°C), embedded in paraffin, and sectioned. Tissue sections were then dewaxed and hydrated, followed by microwave-assisted antigen retrieval with citrate retrieval solution. Normal goat serum blocking solution was added and incubated at room temperature for 1 hour. Excess liquid was removed without washing. Primary antibody (15-fold dilution) was added and incubated at 4°C overnight, followed by three 5-minute washes with PBS. Fluorescent secondary antibody (100-fold dilution) was added and incubated at 37°C for 1 hour, followed by one 5-minute wash with PBS. DAPI nuclear stain (1:1000) was added and incubated at room temperature for 2 minutes. Washes were performed four times with PBS for 5 minutes each. Slides were mounted with anti-quencher and observed under a microscope (DAPI: Ex = 358 nm, Em = 461 nm; type I collagen: Ex = 493 nm, Em = 518 nm). Images were acquired, and fluorescence intensity analyzed using Image J.

[0147] 5. Determination of type I collagen (COL-1) content using an ELISA kit: After 5 days of treatment, the culture medium below the model was collected, stored at -80°C, and determined using an ELISA kit.

[0148] Result analysis: The COL-1 up-regulation rate (%) of each group was calculated.

[0149] COL-1 up-regulation rate (%) = (content in experimental group - content in control group) / content in control group × 100%

[0150] The test data of each group were subjected to independent sample T test using SPSS, and the statistical method adopted was two-tailed test with a test level of 0.05; the data in Figures 5, 6, 7 and 8 show the test results: when the type I collagen content in the sample group was greater than that in 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 the dermal fibroblasts of the T-Skin model tissue.

[0151] Example 4: Animal Experiment Verification of the Effect of Auxiliary Instruments

[0152] Equipment preparation before the experiment: Purchase daily skin care devices in the shopping mall. Skin care devices claim to have the function of increasing the skin permeability of cosmetics. For the convenience of understanding, they are distinguished from each other based on the mechanism, namely microcurrent introduction device, microneedle introduction device, and negative pressure introduction device. The above three introduction devices can be purchased directly from the mall. They are daily-use instruments. This patent does not restrict or protect the brands and types of introduction devices.

[0153] Preparation of key reagents: This experiment uses mRNA encoding green fluorescent protein as a marker. Green fluorescent protein (GFP), a protein composed of approximately 238 amino acids, can be excited by light from blue to ultraviolet, emitting green fluorescence. It is a commonly used indicator protein in biomolecular experiments, allowing rapid localization of expressing cells and facilitating detection of mRNA distribution in the skin.

[0154] Experimental animal preparation: Twenty-five healthy Balb / C female mice aged 10 weeks that passed quarantine were selected and randomly divided into a blank control group, an eGFP mRNA group, an eGFP mRNA + microcurrent introduction instrument test group (hereinafter referred to as the mRNA microcurrent group), an eGFP mRNA + microneedle introduction instrument test group (hereinafter referred to as the mRNA microneedle group), and an eGFP mRNA + negative pressure introduction instrument test group (hereinafter referred to as the mRNA negative pressure group), with 5 mice in each group.

[0155] Experimental Procedure: 4-24 hours before the experiment, remove the hair on both sides of the mouse spine (approximately 3cm x 3cm area) on the back to serve as the testing and observation site. Repeated hair removal may be necessary to facilitate observation and repeat testing.

[0156] After depilation of the mouse back, the mice were anesthetized with 1.5% isoflurane and wiped with disinfectant alcohol. After the alcohol evaporated, the eGFP mRNA solution was evenly applied to the mouse back at a dose of 5 μg / mouse, controlling the application range within a 1 cm x 1 cm square. Subsequently, according to the instruction manual, the auxiliary introduction instrument was used to introduce liquid into the mouse back for 15 minutes; in the blank control group, normal saline was used as a control reagent to apply to the mouse back.

[0157] After the experiment, the excess fluid on the back of the mouse was wiped clean, and the anesthesia was released. After 24 hours of observation, the skin was excised and fixed in 4% formaldehyde. Frozen sections were then taken and observed under a fluorescence microscope.

[0158] Compared with the blank control group, the mRNA groups all showed varying degrees of green fluorescent groups under a fluorescence microscope (as shown in Figure 9), which proved that the mRNA passed through the skin and was taken up by skin cells to express fluorescent protein; compared with the group that applied mRNA alone, the expression levels of fluorescent proteins in the experimental groups using the auxiliary introduction device increased to varying degrees, among which the mRNA microcurrent group and the mRNA negative pressure group could observe more obvious green fluorescence in the epidermis.

[0159] Example 5: Rat skin test to verify collagen expression effect

[0160] Equipment preparation before the experiment: Purchase daily skin care devices in the shopping mall. Skin care devices claim to have the function of increasing the skin permeability of cosmetics. For the convenience of understanding, they are distinguished from each other based on the mechanism, namely microcurrent introduction device, microneedle introduction device, and negative pressure introduction device. The above three introduction devices can be purchased directly from the mall. They are daily-use instruments. This patent does not restrict or protect the brands and types of introduction devices.

[0161] Preparation of main reagents: In this experiment, mRNA encoding collagen was selected as the marker. During the AI ​​algorithm screening process, the eight preferred sequences were all effective compared to the wild type. Therefore, the preferred group in this example randomly selected a combination of SEQ ID No. 7 and SEQ ID No. 16, and the mixing ratio was 1:1 with equal volume.

[0162] Experimental animal preparation: 25 healthy 4-week-old SD female rats that passed quarantine were selected and randomly divided into a blank control group, a collagen mRNA + microcurrent introduction instrument test group (hereinafter referred to as the mRNA microcurrent group), a collagen mRNA + microneedle introduction instrument test group (hereinafter referred to as the mRNA microneedle group), and a collagen mRNA + negative pressure introduction instrument test group (hereinafter referred to as the mRNA negative pressure group). Collagen mRNA was injected intradermally as a positive control group, with 5 rats in each group.

[0163] Experimental Procedure: 4 to 24 hours before the experiment, the hair on both sides of the rat spine (approximately a 3 cm x 3 cm area) was removed from the dorsal spine to serve as the experimental and observation site. Repeated hair removal may be necessary to facilitate observation and repeat testing. After depilation, the rats were anesthetized with 1.5% isoflurane and wiped with disinfectant alcohol. After the alcohol evaporated, the eGFP mRNA solution was evenly applied to the rat's back at a dose of 10 μg / mouse, controlling the application area within a 1 cm x 1 cm square. Following the instructions, the liquid was introduced into the rat's back using an auxiliary infusion device for 15 minutes. A blank control group was treated with saline as a control reagent, and the same procedure was followed. After the experiment, any excess liquid on the rat's back was wiped clean, and anesthesia was released. After 48 hours of observation, the skin at the experimental site was fixed overnight with 4% paraformaldehyde and paraffin sections were prepared. The sections were then Masson-stained and photographed for analysis.

[0164] Figure 10 provides a visual comparison of the changes in collagen fibers in the dermis after treatment between the groups. The dermis is primarily composed of collagen and elastic fibers, which directly determine skin elasticity. Figure 11 shows that intradermal injection in the positive control group promoted collagen upregulation. Microneedle injection also significantly increased collagen expression in the skin, with significant differences compared to the control group.

[0165] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. An mRNA molecule for introduction into the bottom layer of the skin for beauty treatment, comprising a nucleic acid sequence encoding collagen.

2. An mRNA molecule, wherein The mRNA molecule comprises a nucleic acid sequence encoding collagen, wherein the nucleic acid sequence encoding collagen comprises a sequence shown in any one of SEQ ID NOs: 1 to 8 and SEQ ID NOs: 10 to 17, Preferably, the mRNA molecule comprises at least one mRNA sequence encoding type I collagen α1 chain and at least one mRNA sequence encoding type I collagen α2 chain, the mRNA sequence encoding type I collagen α1 chain has a sequence shown in any one of SEQ ID NOs: 1 to 9, and the mRNA sequence encoding type I collagen α2 chain has a sequence shown in any one of SEQ ID NOs: 10 to 18.

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

4. A cosmetic or pharmaceutical composition, wherein: The cosmetic or pharmaceutical composition comprises the mRNA molecule according to any one of claims 1 to 3; Preferably, the cosmetic or pharmaceutical composition further comprises a base material acceptable for medical, cosmetic and cosmetic use; Preferably, the cosmetic or pharmaceutical composition further comprises a cosmetically or pharmaceutically acceptable carrier; Preferably, the cosmetic or medicinal composition is a preparation for introduction into the underlying layer of the skin via a radio frequency introduction device, a negative pressure introduction device, a microcrystal introduction device, a microcurrent introduction device, a microneedle introduction device, or a syringe.

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

6. A recombinant plasmid or recombinant cell comprising the DNA molecule according to claim 5, Preferably, the recombinant plasmid or recombinant cell can be used to transcribe the mRNA molecule according to any one of claims 1 to 3.

7. A kit comprising the mRNA molecule according to any one of claims 1 to 3, the cosmetic or pharmaceutical 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 comprises an auxiliary introduction device; preferably, the auxiliary introduction device comprises a radio frequency introduction device, a negative pressure introduction device, a microcrystal introduction device, a microcurrent introduction device, a microneedle introduction device, or a syringe.

9. A method for preparing the mRNA molecule according to any one of claims 1 to 3, comprising: (1) cloning the DNA molecule described in 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 performing PCR amplification using the extracted plasmid as a template to obtain a DNA template for in vitro expression of mRNA; (3) constructing an RNA in vitro 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 to 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 improvement of skin condition, Preferably, the product is a medicine, 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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