SiRNA (small interfering Ribonucleic Acid) of targeted skin aging promoting gene and application of siRNA

Abstract

The invention relates to siRNA of a targeted skin aging promoting gene and application of the siRNA, and belongs to the technical field of biology. The invention provides siRNA (small interfering Ribonucleic Acid) of a targeted skin aging promoting gene. A nucleotide sequence of a positive-sense strand of the siRNA is shown as any one of SEQ ID NO. 1 to SEQ ID NO. 4. The siRNA can target a skin aging promoting gene, is high in interference efficiency, and has high inhibitory activity on the skin aging promoting gene; moreover, the research shows that the exosome loaded with the siRNA has the capability of resisting skin aging (including improving wrinkles and improving the smooth degree of the skin). Therefore, the siRNA has great application prospects in preparation of products for resisting skin aging, diseases caused by skin aging promoting genes and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to siRNA targeting genes promoting skin aging and application thereof. Background Art

[0002] Ultraviolet radiation is the main factor causing exogenous skin aging, which is also called photoaging. Compared with natural aging, skin aging caused by photoaging is more serious. The most obvious difference between it and natural aging in terms of symptoms is that wrinkles caused by natural aging are thinner and shallower, while wrinkles caused by photoaging are thicker and deeper. In addition, the speed of skin aging caused by photoaging is significantly faster than natural aging. Therefore, photoaging in exogenous aging can be used as a good starting point for skin anti-aging treatment.

[0003] The extracellular matrix (ECM) is a complex network composed of macromolecules, the main components of which include collagen, elastin, non-collagen glycoproteins and proteoglycans. Studies have shown that photoaging can cause changes in the components of the extracellular matrix of the skin, leading to skin aging (mainly manifested as: the cleavage of mucopolysaccharide components such as hyaluronic acid in the matrix, increased solubility, affecting its structure and function, and ultimately leading to dry, loose, and inelastic skin). At the same time, studies have confirmed that some genes that promote skin aging can have a destructive effect on maintaining the normal structure of the extracellular matrix of the skin. Therefore, knocking down the expression of these genes that promote skin aging in vivo, and then maintaining the normal structure of the extracellular matrix of the skin, is the key to resisting skin aging caused by photoaging.

[0004] Small interfering RNA (siRNA) is a double-stranded RNA molecule composed of 20 to 25 nucleotides. In the RNA interference (RNAi) pathway, siRNA interferes with gene expression by hybridizing with complementary mRNA molecules. This interference triggers mRNA degradation and inhibits the gene expression of specific genes. If the expression of skin-promoting aging genes in vivo can be effectively knocked down by siRNA, it will be very helpful for anti-aging treatment of the skin. In addition, the gene knockdown effect achieved by direct transfection of exogenous siRNA is usually short-lived. Therefore, on the basis of developing siRNA that can effectively knock down the expression of skin-promoting aging genes in vivo, long-term maintenance of the stability and biological activity of siRNA in vivo is also critical for anti-aging treatment of the skin. Summary of the invention

[0005] To solve the above problems, the present invention provides a siRNA targeting a gene that promotes skin aging, wherein the siRNA comprises a sense strand and an antisense strand with a length of 20 to 25 nucleotides; the sense strand of the siRNA can at least partially reverse complement the antisense strand to form a double-stranded region; the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in any one of SEQ ID NOs. 1 to 4.

[0006] In one embodiment of the present invention, the antisense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in any one of SEQ ID NOs. 5 to 8.

[0007] In one embodiment of the present invention, the nucleotide sequence of the sense strand of the siRNA is shown in any one of SEQ ID NOs. 1 to 4; the nucleotide sequence of the antisense strand of the siRNA is shown in any one of SEQ ID NOs. 5 to 8.

[0008] In one embodiment of the present invention, the skin aging promoting genes include a gene encoding an inflammatory factor α (TNF-α gene), a gene encoding a 15-hydroxyprostaglandin dehydrogenase (15-PGDH gene), a gene encoding a hyaluronic acid glucosaminidase 2 (HYAL2 gene) and / or a gene encoding a matrix metalloproteinase 1 (MMP-1 gene);

[0009] When the skin aging-promoting gene is a gene encoding an inflammatory factor α, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.1, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.5;

[0010] When the skin aging promoting gene is a gene encoding 15-hydroxyprostaglandin dehydrogenase, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.2, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.6;

[0011] When the skin aging-promoting gene is a gene encoding hyaluronic acid glucosidase 2, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.3, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.7;

[0012] When the skin aging promoting gene is a gene encoding matrix metalloproteinase 1, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.4, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.8.

[0013] In one embodiment of the present invention, when the gene promoting skin aging is a gene encoding an inflammatory factor α, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.5;

[0014] When the skin aging promoting gene is a gene encoding 15-hydroxyprostaglandin dehydrogenase, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.6;

[0015] When the skin aging promoting gene is a gene encoding hyaluronic acid glucosaminidase 2, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.7;

[0016] When the skin aging promoting gene is a gene encoding matrix metalloproteinase 1, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO.8.

[0017] In one embodiment of the present invention, the skin aging promoting gene is a gene encoding hyaluronic acid glucosidase 2 or a gene encoding matrix metalloproteinase 1.

[0018] In one embodiment of the present invention, the skin aging-promoting gene is a gene encoding matrix metalloproteinase-1.

[0019] The present invention also provides a recombinant plasmid, wherein the recombinant plasmid carries the above siRNA.

[0020] In one embodiment of the present invention, the vector of the recombinant plasmid includes at least one of a viral vector or a non-viral vector; the viral vector includes at least one of a flavivirus vector, a retrovirus vector, a phage vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a hybrid virus vector, a baculovirus vector, a herpes simplex virus vector or a lentivirus vector; the non-viral vector includes a plasmid vector.

[0021] In one embodiment of the present invention, the plasmid vector comprises at least one of pLKO.1 plasmid, pcDNA6.2 plasmid, PGEM-3zf plasmid, PUC19 plasmid or PUC57 plasmid.

[0022] In one embodiment of the present invention, the preparation method of the recombinant plasmid is: designing shRNA according to siRNA; connecting shRNA with a linearized vector to obtain the recombinant plasmid.

[0023] In one embodiment of the present invention, when the gene promoting skin aging is a gene encoding an inflammatory factor α, the nucleotide sequence of the siRNA target sequence (sense) of the shRNA is shown as SEQ ID NO.9, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO.39; when the gene promoting skin aging is a gene encoding 15-hydroxyprostaglandin dehydrogenase, the nucleotide sequence of the siRNA target sequence of the shRNA is shown as SEQ ID NO.10; when the gene promoting skin aging is a gene encoding hyaluronic acid glucosaminidase 2, the nucleotide sequence of the siRNA target sequence of the shRNA is shown as SEQ ID NO.11; when the gene promoting skin aging is a gene encoding matrix metalloproteinase 1, the nucleotide sequence of the siRNA target sequence of the shRNA is shown as SEQ ID NO.12.

[0024] The present invention also provides an engineered cell, wherein the genome of the engineered cell is integrated with the above siRNA; or the engineered cell carries the above recombinant plasmid.

[0025] In one embodiment of the present invention, the method for preparing the engineered cells is: transfecting the above-mentioned recombinant plasmid and the virus packaging plasmid into host cells simultaneously to obtain the engineered cells.

[0026] In one embodiment of the present invention, the host cell includes at least one of an immortalized cell or a stem cell; the stem cell includes at least one of an embryonic stem cell or an adult stem cell; the immortalized cell includes at least one of a 293T cell, a 293FT cell, a NIH / 3T3 cell or a L929 cell; the adult stem cell includes at least one of a hematopoietic stem cell, a bone marrow mesenchymal stem cell, a neural stem cell, a liver stem cell, a muscle satellite cell, a skin mesenchymal stem cell, an intestinal epithelial stem cell, a retinal stem cell or a pancreatic stem cell.

[0027] The present invention also provides an exosome, wherein the exosome is loaded with the above-mentioned siRNA; or, the exosome is loaded with the above-mentioned recombinant plasmid.

[0028] In one embodiment of the present invention, the exosomes are secreted by the above-mentioned engineered cells.

[0029] In one embodiment of the present invention, the method for preparing the exosomes is: inoculating the above-mentioned engineered cells into a cell culture medium for culturing to obtain a culture product; and isolating the exosomes loaded with the above-mentioned siRNA from the culture product.

[0030] In one embodiment of the present invention, when the gene promoting skin aging is a gene encoding an inflammatory factor α, the exosomes are loaded with siRNA having a nucleotide sequence of a positive chain as shown in SEQ ID NO.1 and a nucleotide sequence of an antisense chain as shown in SEQ ID NO.5;

[0031] When the skin aging promoting gene is a gene encoding 15-hydroxyprostaglandin dehydrogenase, the exosomes are loaded with siRNA having a nucleotide sequence of a positive chain as shown in SEQ ID NO.2 and a nucleotide sequence of an antisense chain as shown in SEQ ID NO.6;

[0032] When the skin aging-promoting gene is a gene encoding hyaluronic acid glucosidase 2, the exosomes are loaded with siRNA having a nucleotide sequence of a positive chain as shown in SEQ ID NO.3 and a nucleotide sequence of an antisense chain as shown in SEQ ID NO.7;

[0033] When the gene promoting skin aging is a gene encoding matrix metalloproteinase 1, the exosomes are loaded with siRNA having a nucleotide sequence of a positive chain as shown in SEQ ID NO.4 and a nucleotide sequence of an antisense chain as shown in SEQ ID NO.8.

[0034] In one embodiment of the present invention, the skin aging promoting gene is a gene encoding hyaluronic acid glucosidase 2 or a gene encoding matrix metalloproteinase 1.

[0035] In one embodiment of the present invention, the skin aging-promoting gene is a gene encoding matrix metalloproteinase-1.

[0036] The present invention also provides the use of the above siRNA or the above recombinant plasmid or the above engineered cell or the above exosome in preparing a product for resisting skin aging or treating diseases caused by genes promoting skin aging.

[0037] In one embodiment of the present invention, the genes promoting skin aging include a gene encoding inflammatory factor α, a gene encoding 15-hydroxyprostaglandin dehydrogenase, a gene encoding hyaluronic acid glucosidase 2 and / or a gene encoding matrix metalloproteinase 1.

[0038] In one embodiment of the present invention, the skin aging promoting gene is a gene encoding hyaluronic acid glucosidase 2 or a gene encoding matrix metalloproteinase 1.

[0039] In one embodiment of the present invention, the skin aging-promoting gene is a gene encoding matrix metalloproteinase-1.

[0040] In one embodiment of the present invention, the product is a medicine.

[0041] In one embodiment of the present invention, the drug further contains a pharmaceutically acceptable carrier.

[0042] In one embodiment of the present invention, the pharmaceutically acceptable carrier comprises at least one of liposomes, magnetic nanoparticles, exosomes or solid lipid nanoparticles.

[0043] The present invention also provides a product for resisting skin aging or treating diseases caused by genes that promote skin aging, wherein the ingredients of the product include the above-mentioned siRNA or the above-mentioned recombinant plasmid or the above-mentioned engineered cells or the above-mentioned exosomes.

[0044] In one embodiment of the present invention, the genes promoting skin aging include a gene encoding inflammatory factor α, a gene encoding 15-hydroxyprostaglandin dehydrogenase, a gene encoding hyaluronic acid glucosidase 2 and / or a gene encoding matrix metalloproteinase 1.

[0045] In one embodiment of the present invention, the skin aging promoting gene is a gene encoding hyaluronic acid glucosidase 2 or a gene encoding matrix metalloproteinase 1.

[0046] In one embodiment of the present invention, the skin aging-promoting gene is a gene encoding matrix metalloproteinase-1.

[0047] In one embodiment of the present invention, the product is a medicine.

[0048] In one embodiment of the present invention, the drug further contains a pharmaceutically acceptable carrier.

[0049] In one embodiment of the present invention, the pharmaceutically acceptable carrier comprises at least one of liposomes, magnetic nanoparticles, exosomes or solid lipid nanoparticles.

[0050] The technical solution of the present invention has the following advantages:

[0051] 1. The present invention provides a siRNA targeting a gene that promotes skin aging, wherein the nucleotide sequence of the sense strand of the siRNA is shown in any one of SEQ ID NO.1 to 4; the antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO.5 to 8. The siRNA can target the gene that promotes skin aging, and has high interference efficiency and high inhibitory activity on the gene that promotes skin aging; and studies have shown that exosomes loaded with the siRNA have the ability to resist skin aging (including improving wrinkles and improving skin smoothness). Therefore, the siRNA has great application prospects in the preparation of products for resisting skin aging and diseases caused by genes that promote skin aging.

[0052] Further, the skin aging promoting gene includes a gene encoding an inflammatory factor α (TNF-α gene), a gene encoding 15-hydroxyprostaglandin dehydrogenase (15-PGDH gene), a gene encoding hyaluronic acid glucosidase 2 (HYAL2 gene) and / or a gene encoding matrix metalloproteinase 1 (MMP-1 gene); when the skin aging promoting gene is a gene encoding an inflammatory factor α, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.1, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.5; when the skin aging promoting gene is a gene encoding 15-hydroxyprostaglandin dehydrogenase, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.2, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.6; when the skin aging promoting gene is a gene encoding hyaluronic acid glucosidase 2, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.3, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.7; when the skin aging promoting gene is a gene encoding matrix metalloproteinase 1, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.4, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.8. Wherein:

[0053] Collagen is the main component of the extracellular matrix, and inflammatory factor α (TNF-α) can increase the level of intracellular ROS, causing abnormal expression of collagen content in skin cells, disrupting the redox system and affecting human skin. Therefore, knocking down the expression of the gene encoding inflammatory factor α through siRNA can resist skin aging.

[0054] Prostaglandin E2 (PGE2) is a lipid hormone-like signaling molecule. Inhibiting the action of PGE2 will inhibit tissue regeneration, and 15-hydroxyprostaglandin dehydrogenase is the main degradation enzyme of PGE2. Therefore, knocking down the expression of the gene encoding 15-hydroxyprostaglandin dehydrogenase through siRNA can promote the accumulation of PGE2, thereby improving skin tissue repair and regeneration and anti-aging.

[0055] Hyaluronic acid (HA) is the main structure for absorbing and maintaining moisture in the skin, and hyaluronan aminoglycosidase 2 (HYAL2) is the most important hyaluronan degrading enzyme in the skin. Therefore, knocking down the gene encoding hyaluronan aminoglycosidase 2 through siRNA can inhibit the degradation of the skin's own HA and promote the accumulation of HA in the skin, thereby increasing the skin's moisture content and slowing down the appearance of skin aging.

[0056] The collagen in adult skin is mainly type I collagen and type III collagen. Among them, type I collagen mainly enhances the skin's flexibility and makes the skin firm, and type III collagen makes the skin tender. Matrix metalloproteinase 1 is the main enzyme that degrades type I collagen and type III collagen. Therefore, knocking down the gene encoding matrix metalloproteinase 1 through siRNA can reduce the degradation of extracellular matrix components, maintain the normal structure of collagen fibers and elastic fibers, and thus prevent the appearance of wrinkles and other aging manifestations on the skin.

[0057] Further, the skin aging-promoting gene is a gene encoding hyaluronic acid glucosidase 2 or a gene encoding matrix metalloproteinase 1; when the skin aging-promoting gene is a gene encoding hyaluronic acid glucosidase 2, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.7; when the skin aging-promoting gene is a gene encoding matrix metalloproteinase 1, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.8. The exosomes loaded with the siRNA have the best effect of anti-skin aging (including improving wrinkles and improving skin smoothness).

[0058] 2. The present invention provides an exosome, wherein the exosome is loaded with siRNA targeting a skin-promoting aging gene; the nucleotide sequence of the sense strand of the siRNA is as shown in any one of SEQ ID NO.1 to 4; the antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO.5 to 8. The siRNA loaded with the exosome can target the skin-promoting aging gene, and has high interference efficiency and high inhibitory activity on the skin-promoting aging gene; and studies have shown that the exosome has the ability to resist skin aging (including improving wrinkles and improving skin smoothness). Therefore, the exosome has great application prospects in the preparation of products for resisting skin aging and diseases caused by skin-promoting aging genes.

[0059] In addition, the exosomes have the following advantages: using exosomes for skin anti-aging can avoid immune rejection reactions and greatly improve safety; exosomes can be stored for a long time at -80°C and maintain biological activity, and are easy to transport for a long time; exosomes are small in size and can be directly taken up by cells, affecting the function of target cells and improving the efficiency of action; as a subcellular structure secreted by cells, exosomes have no relevant ethical restrictions when used; exosomes can be extracted from various cell culture media by gradient centrifugation, which is easy to use.

[0060] Further, the skin aging-promoting gene is a gene encoding hyaluronic acid glucosidase 2 or a gene encoding matrix metalloproteinase 1; when the skin aging-promoting gene is a gene encoding hyaluronic acid glucosidase 2, the nucleotide sequence of the sense chain of the siRNA loaded by the exosomes is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.7; when the skin aging-promoting gene is a gene encoding matrix metalloproteinase 1, the nucleotide sequence of the sense chain of the siRNA loaded by the exosomes is shown in SEQ ID NO.4, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.8. The exosomes have the best effect of anti-skin aging (including improving wrinkles and improving skin smoothness). BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 : Interference efficiency of siRNA targeting TNF-α gene.

[0062] Figure 2 : Interference efficiency of siRNA targeting 15-PGDH gene.

[0063] Figure 3 : Interference efficiency of siRNA targeting HYAL2 gene.

[0064] Figure 4 : Interference efficiency of siRNA targeting MMP-1 gene.

[0065] Figure 5 : shRNA design flow chart.

[0066] Figure 6 : Plasmid map of recombinant plasmid pLKO.1shRNA.

[0067] Figure 7 : Schematic diagram of the construction process of engineered exosomes.

[0068] Figure 8 : Expression level of siRNA in engineered exosomes (siRNA targeting TNF-α gene).

[0069] Fig. 9 : Expression level of siRNA in engineered exosomes (siRNA targeting 15-PGDH gene).

[0070] Fig.10 : Expression level of siRNA in engineered exosomes (siRNA targeting HYAL2 gene).

[0071] Fig.11 : Expression level of siRNA in engineered exosomes (siRNA targeting MMP-1 gene).

[0072] Fig.12 :Dermatoscopy results of different groups of photoaging models. DETAILED DESCRIPTION

[0073] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0074] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0075] Experimental Example 1: Acquisition of siRNA targeting genes that promote skin aging

[0076] The specific process is as follows:

[0077] 1. Design

[0078] According to the coding region sequences of TNF-α gene, 15-PGDH gene, HYAL2 gene and MMP-1 gene, siRNA targeting TNF-α gene, 15-PGDH gene, HYAL2 gene and MMP-1 gene was designed (see Table 1 for details), and the siRNA is as follows:

[0079] siRNA 1 targeting TNF-α gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.5;

[0080] siRNA 2 targeting TNF-α gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.13, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.14;

[0081] siRNA3 targeting TNF-α gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.15, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.16;

[0082] siRNA 1 targeting 15-PGDH gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.17, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.18;

[0083] siRNA 2 targeting 15-PGDH gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.6;

[0084] siRNA3 targeting 15-PGDH gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.19, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.20;

[0085] siRNA 1 targeting the HYAL2 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.21, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.22;

[0086] siRNA 2 targeting the HYAL2 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.7;

[0087] siRNA3 targeting HYAL2 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.23, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.24;

[0088] siRNA 1 targeting MMP-1 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.25, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.26;

[0089] siRNA 2 targeting MMP-1 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.27, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.28;

[0090] siRNA3 targeting MMP-1 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.8.

[0091] 2. Screening

[0092] 5 μL of transfection reagent lipo2000 (purchased from Thermo Fisher Scientific) was mixed with 100 μL of Opti-MEM (purchased from Thermo Fisher Scientific) and allowed to stand for 5 minutes to obtain mixture A; different siRNAs were diluted to a concentration of 100 pmol with 100 μL of Opti-MEM and allowed to stand for 5 minutes to obtain mixture B; mixture A and mixture B were mixed and allowed to stand for 20 minutes to obtain transfection solutions containing different siRNAs;

[0093] NIH / 3T3 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured at 1×10 5The cells were inoculated into 6-well plates with 2 mL of DMEM medium (purchased from Thermo Fisher Scientific) containing 10% (v / v) fetal bovine serum per well and incubated in 5% (v / v) CO 2 , and cultured in a 37°C cell culture incubator for 24 h; after culturing for 24 h, a blank control group, a negative control group (siNC), a TNF-α-siRNA1 experimental group, a TNF-α-siRNA2 experimental group, a TNF-α-siRNA3 experimental group, a 15-PGDH-siRNA1 experimental group, a 15-PGDH-siRNA2 experimental group, a 15-PGDH-siRNA3 experimental group, a HYAL2-siRNA1 experimental group, a HYAL2-siRNA2 experimental group, a HYAL2-siRNA3 experimental group, a MMP-1-siRNA1 experimental group, a MMP-1-siRNA2 experimental group, and a MMP-1-siRNA3 experimental group were set up in a 6-well plate, with 4 replicate wells in each group;

[0094] After the setup was completed, transfection solutions containing different siRNAs were added to the wells of the experimental groups (TNF-α-siRNA1 experimental group was given a transfection solution containing siRNA1 targeting the TNF-α gene, TNF-α-siRNA2 experimental group was given a transfection solution containing siRNA2 targeting the TNF-α gene, TNF-α-siRNA3 experimental group was given a transfection solution containing siRNA3 targeting the TNF-α gene, and so on), and transfection solutions containing siNC were added to the wells of the negative control group (the nucleotide sequence of the sense chain of siNC is shown in SEQ ID NO.29, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.30), and transfection solutions without siRNA were added to the blank control group. The cells were incubated at 5% (v / v) CO 2 , cultured in a 37°C cell culture incubator for 6 h for transfection; after 6 h of transfection, discard the old cell culture medium, add 2 mL of new DMEM containing 10% (v / v) fetal bovine serum to each well of the 6-well plate, and incubate at 5% (v / v) CO 2 , continue culturing in a 37°C cell culture incubator for 24 h for transfection;

[0095] 24 hours after transfection, the cell lysate (purchased from Sigma) was added to a 6-well plate at a volume of 500 μL per well, and chloroform was added to a 6-well plate at a volume of 100 μL per well, first vortexed vigorously, then placed on ice for 5 minutes, and finally centrifuged at 12000 rpm and 4°C for 10 minutes; after centrifugation, the supernatant was taken, and an equal volume of isopropanol was added to the supernatant, first placed at room temperature (25°C) for 16 hours, and then centrifuged at 12000 rpm and 4°C for 10 minutes; after centrifugation, the precipitate was taken, and the precipitate was resuspended to the original volume (i.e., the same volume as the supernatant) with 75% (v / v) ethanol, and centrifuged at 12000 rpm and 4°C for 10 minutes; after centrifugation, the precipitate was taken to obtain mRNA; the extracted mRNA was used as the test sample, and the expression of the target gene in the cells after different siRNA interference was detected by qPCR to verify the interference efficiency of the designed siRNA, and the verification results are shown in Figure 1 to Figure 4 ;

[0096] The specific steps of qPCR are as follows:

[0097] Reverse transcription: Add the reagents according to the reaction system in Table 2 to a 200 μL enzyme-free centrifuge tube, vortex to mix, and place in a PCR instrument to obtain cDNA (see Table 2 for the specific reverse transcription reaction procedure);

[0098] qPCR: Add the reagents to a 200 μL enzyme-free centrifuge tube according to the reaction system in Table 3, vortex and centrifuge, and then add to a 96-well plate (see Table 3 for the specific qPCR reaction procedure and Table 4 for the primers used for qPCR).

[0099] Depend on Figure 1 It can be seen that among the siRNAs targeting the TNF-α gene, siRNA1 has the best interference efficiency and significantly knocks down TNF-α expression, and it is named si-TNF-α.

[0100] Depend on Figure 2 It can be seen that among the siRNAs targeting the 15-PGDH gene, siRNA2 has the best interference efficiency and significantly knocks down the expression of 15-PGDH. It is named si-15-PGDH.

[0101] Depend on Figure 3 It can be seen that among the siRNAs targeting the HYAL2 gene, siRNA2 has the best interference efficiency and significantly knocks down the expression of HYAL2. It is named si-HYAL2.

[0102] Depend on Figure 4 It can be seen that among the siRNAs targeting the MMP-1 gene, siRNA3 has the best interference efficiency and significantly knocks down the expression of MMP-1. It is named si-MMP-1.

[0103] Table 1 siRNA and its sequence

[0104]

[0105] Table 2 Reverse transcription reaction system and reaction procedure

[0106]

[0107]

[0108] Table 3 qPCR reaction system and reaction procedure

[0109]

[0110] Table 4 qPCR primers and their sequences

[0111]

[0112] Experimental Example 2: The specific process of constructing an engineered polyclonal cell line secreting exosomes is as follows:

[0113] 1. Construction of recombinant plasmid pLKO.1shRNA

[0114] According to the nucleotide sequence of the sense strand as shown in SEQ ID NO.1 and the nucleotide sequence of the antisense strand as shown in SEQ ID NO.5, shRNA was designed (the design idea of ​​shRNA is shown in Figure 5 , shRNA consists of a restriction site, a siRNA target sequence sense, a stem-loop sequence loop, a reverse complementary sequence of the siRNA target sequence antisense, repeated base T and a restriction site), and a pLKO.1puro lentiviral vector (purchased from GenScript, Age1 (accggt), EcoR1 (gaattc) restriction sites, AMP resistance, promoter U6, starting with G) was used to construct a recombinant plasmid pLKO.1shRNA-1 inserted with shRNA (the nucleotide sequence of the siRNA target sequence of shRNA is shown in SEQ ID NO.9, and the plasmid map is shown in Figure 6 , the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.39).

[0115] 2. Construction of engineered cell lines

[0116] Lentivirus packaging: 60 μL of transfection reagent lipo2000 (purchased from Thermo Fisher Scientific) was mixed with 750 μL of Opti-MEM medium (purchased from Thermo Fisher Scientific) and allowed to stand for 5 minutes to obtain mixture A; 12 μg of recombinant plasmid pLKO.1shRNA-1, 6 μg of lentivirus packaging plasmid psPAX2 (purchased from GenScript), and 1.5 μg of lentivirus packaging plasmid pMD2G (purchased from GenScript) were mixed with 750 μL of Opti-MEM medium (purchased from GenScript) and allowed to stand for 5 minutes to obtain mixture B; mixture A and mixture B were mixed and allowed to stand for 20 minutes to obtain transfection solution; 293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured at 3×10 5 The inoculum was inoculated into each well of a 10 cm culture dish with 8 mL of cell culture medium (the cell culture medium was DMEM medium containing 10% fetal bovine serum, 10% refers to the volume ratio, and DMEM medium was purchased from Thermo Fisher Scientific) and incubated at 37°C and 5% (v / v) CO 2 Incubate for 24 hours in an incubator. After 24 hours, discard the cell culture medium in the 10 cm culture dish, add the transfection solution to the 10 cm culture dish, and incubate at 37°C, 5% (v / v) CO 2 The cells were cultured in an incubator for 6 h for transfection. After 6 h of transfection, the cell culture medium in the 10 cm culture dish was discarded and replaced with new cell culture medium. The cells were cultured in an incubator at 37 °C and 5% (v / v) CO 2 The cells were cultured in an incubator for 48 hours and then transfected. After 48 hours of transfection, the culture supernatant was collected in a biosafety cabinet. The culture supernatant was filtered with a 0.22 μm filter to obtain a cell culture fluid containing the lentivirus.

[0117] Lentivirus infection: 2 μL of infection aid Polybrene (1000×, purchased from Bio-Tech) was mixed with 2 mL of cell culture medium containing lentivirus to obtain infection medium; 293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured at 1×10 5 The inoculum size was inoculated into 6-well plates with 2 mL of cell culture medium added to each well (the cell culture medium was DMEM medium containing 10% fetal bovine serum, 10% refers to the volume ratio, and DMEM medium was purchased from Thermo Fisher Scientific) and incubated at 37°C and 5% (v / v) CO 2 Incubate for 24 h in an incubator (only one well was inoculated here); after 24 h of incubation, discard the cell culture medium in the 6-well plate, add the infection solution to the 6-well plate, and incubate at 37°C and 5% (v / v) CO 2 The cells were incubated in an incubator for 48 h for infection. After 48 h of infection, the infection solution in the 6-well plate was discarded, and 2 mL of drug screening medium (the drug screening medium is MSCM medium containing 1 μg / mL puromycin, purchased from Dakoway) was added to the 6-well plate at each well. The cells were incubated at 37°C and 5% (v / v) CO2 The cells were cultured in an incubator for 48 h for drug screening. After 48 h of drug screening, 200 μL of trypsin digestion solution (purchased from Thermo Fisher Scientific) was added to a 6-well plate at 37°C and 5% (v / v) CO 2 Incubate in an incubator for 30 seconds for digestion; after 30 seconds of digestion, first add an equal amount of drug-sieve medium to the cell digestion solution obtained by digestion to terminate digestion, then centrifuge the cell digestion solution obtained by digestion to collect the cell precipitate; resuspend the cell precipitate with 1 mL of drug-sieve medium to obtain a cell resuspension; take the cell resuspension obtained by digestion containing 450 (1.5×60×5=450) cells and dilute it to 90 mL (1.5×200×60×5=90 mL) with drug-sieve medium to obtain a cell dilution; spread the cell dilution on 5 96-well plates, spread the middle 60 wells of each 96-well plate, and spread the cell dilution in an amount of 200 μL / well (to ensure 1.5 cells per well), and add 200 μL / well PBS buffer to the remaining wells for blocking; after spreading, place the 96-well plate at 37°C and 5% (v / v) CO 2 The culture medium was cultured in an incubator. During the culture process, the medium was observed every two days to see if it was necessary to change the medium (if the medium turned yellow, a new drug-screened medium was replaced. If the medium was still pink, there was no need to replace the new drug-screened medium and wait for the next replacement cycle). After two weeks of culture, the number of cells in each well of the 96-well plate was observed under an inverted microscope, and the wells with 1 to 2 cells were marked. The marked positive cells were expanded step by step from 96-well plate to 48-well plate to 12-well plate until the required cell amount was obtained for monoclonal cell line verification. If the verification was correct, the engineered polyclonal cell line 293T / pLKO.1shRNA-1 secreting exosomes was obtained (the construction process of the engineered cell line is shown in Figure 7 );

[0118] Among them, the specific verification process of monoclonal cell lines is as follows:

[0119] 96-well plate culture: After two weeks of culture, select the wells with 1-2 cells, discard the cell culture medium in the 96-well plate, add 20 μL of trypsin digestion solution to each well of the 96-well plate, and incubate at 37°C, 5% (v / v) CO 2 Incubate in an incubator for 15 seconds for digestion; after 15 seconds of digestion, add an equal amount of drug screening medium to the trypsin digestion solution to the cell digestion solution obtained by digestion to terminate the digestion, then centrifuge the cell digestion solution obtained by digestion, collect the cell pellets from all wells, and resuspend them with 2.4 mL of cell culture medium (the cell culture medium is DMEM culture medium containing 10% fetal bovine serum, 10% refers to the volume ratio, and the DMEM culture medium is purchased from Thermo Fisher Scientific) to obtain a cell resuspension;

[0120] 24-well plate culture: Take the cell resuspension obtained from the 96-well plate culture and inoculate 100 μL per well into a 24-well plate and incubate at 37°C and 5% (v / v) CO 2 Incubate for 72 h in an incubator. After 72 h, discard the cell culture medium in the 24-well plate, add 80 μL of trypsin digestion solution to each well of the 24-well plate, and incubate at 37°C, 5% (v / v) CO 2 Incubate in an incubator for 15 seconds for digestion; after 15 seconds of digestion, add an equal amount of cell culture medium to the trypsin digestion solution to terminate the digestion, then centrifuge the cell digestion solution obtained by digestion, collect the cell pellets from all wells, and resuspend them in 1.2 mL of cell culture medium to obtain a cell resuspension;

[0121] 12-well plate culture: Take the cell resuspension obtained from the 24-well plate culture and inoculate 100 μL per well into a 12-well plate and incubate at 37°C, 5% (v / v) CO 2 The cells were cultured in an incubator for 72 h. After 72 h of culture, the cell culture fluid in the 12-well plate was collected.

[0122] Engineered exosome detection: The cell culture fluid obtained by expanding the culture of unengineered 293T cells was used as a control. The cell exosome extraction kit (purchased from Novazon) was used to extract the engineered exosomes loaded with si-TNF-α in the cell culture fluid obtained by expanding the culture of positive cells. The content of si-TNF-α in the engineered exosomes loaded with si-TNF-α was detected by qPCR. The test results are shown in Figure 8 ;

[0123] The specific steps of qPCR are as follows:

[0124] Reverse transcription: Add the reagents according to the reaction system in Table 5 to a 200 μL enzyme-free centrifuge tube, vortex mix, and place in a PCR instrument to obtain cDNA (see Table 5 for the specific reverse transcription reaction procedure);

[0125] qPCR: Add the reagents to a 200 μL enzyme-free centrifuge tube according to the reaction system in Table 6, vortex and centrifuge, and then add to a 96-well plate (see Table 6 for the specific qPCR reaction procedure, and the probe used for qPCR is synthesized by Yike Bio).

[0126] Depend on Figure 8 It can be seen that the absolute expression amount (expression level) of si-TNF-α in the engineered exosomes loaded with si-TNF-α is as high as 4 pmol / g total RNA. This result shows that positive cells can stably express si-TNF-α, and the engineered polyclonal cell line 293T / pLKO.1shRNA-1 that secretes exosomes is successfully constructed.

[0127] Table 5 Reverse transcription reaction system and reaction procedure

[0128]

[0129] Table 6 qPCR reaction system and reaction procedure

[0130]

[0131] Experimental Example 3: Construction of an engineered polyclonal cell line secreting exosomes

[0132] The specific process is as follows:

[0133] According to the nucleotide sequence of the sense strand as shown in SEQ ID NO.2 and the nucleotide sequence of the antisense strand as shown in SEQ ID NO.6, shRNA was designed (the design idea of ​​shRNA is shown in Figure 5 ), and the pLKO.1puro lentiviral vector was used to construct a recombinant plasmid pLKO.1shRNA-2 inserted with shRNA (the nucleotide sequence of the siRNA target sequence of shRNA is shown in SEQ ID NO.10, and the plasmid map is shown in Figure 6 , the nucleotide sequence of the recombinant plasmid is based on SEQ ID NO.39, SEQ ID NO.9 is replaced by SEQ ID NO.10); based on Experimental Example 2, the recombinant plasmid pLKO.1shRNA-1 is replaced by the recombinant plasmid pLKO.1shRNA-2 to obtain the engineered polyclonal cell line 293T / pLKO.1shRNA-2 that secretes exosomes. The content of si-15-PGDH in the engineered exosomes secreted by the engineered polyclonal cell line 293T / pLKO.1shRNA-2 is shown in Fig. 9 .Depend on Fig. 9 It can be seen that the absolute expression amount (expression level) of si-15-PGDH in the engineered exosomes loaded with si-15-PGDH is as high as 3.8 pmol / g total RNA. This result shows that positive cells can stably express si-15-PGDH, and the engineered polyclonal cell line 293T / pLKO.1shRNA-2 that secretes exosomes is successfully constructed.

[0134] Experimental Example 4: Construction of an engineered polyclonal cell line secreting exosomes

[0135] The specific process is as follows:

[0136] Based on the nucleotide sequence of the sense strand as shown in SEQ ID NO.3 and the nucleotide sequence of the antisense strand as shown in SEQ ID NO.7 of si-HYAL2, shRNA was designed (the design idea of ​​shRNA is shown in Figure 5), and the pLKO.1puro lentiviral vector was used to construct a recombinant plasmid pLKO.1shRNA-3 inserted with shRNA (the nucleotide sequence of the siRNA target sequence of shRNA is shown in SEQ ID NO.11, and the plasmid map is shown in Figure 6 , the nucleotide sequence of the recombinant plasmid is based on SEQ ID NO.39, SEQ ID NO.9 is replaced by SEQ ID NO.11); based on Experimental Example 2, the recombinant plasmid pLKO.1shRNA-1 is replaced by the recombinant plasmid pLKO.1shRNA-3 to obtain the engineered polyclonal cell line 293T / pLKO.1shRNA-3 that secretes exosomes. The content of si-HYAL2 in the engineered exosomes secreted by the engineered polyclonal cell line 293T / pLKO.1shRNA-3 is shown in Fig.10 .Depend on Fig.10 It can be seen that the absolute expression amount (expression level) of si-HYAL2 in the engineered exosomes loaded with si-HYAL2 was as high as 4.2 pmol / g total RNA. This result showed that positive cells can stably express si-HYAL2, and the engineered polyclonal cell line 293T / pLKO.1shRNA-3 that secretes exosomes was successfully constructed.

[0137] Experimental Example 5: Construction of an engineered polyclonal cell line secreting exosomes

[0138] The specific process is as follows:

[0139] Based on the nucleotide sequence of the sense strand as shown in SEQ ID NO.4 and the nucleotide sequence of the antisense strand as shown in SEQ ID NO.8 of si-MMP-1, shRNA was designed (the design idea of ​​shRNA is shown in Figure 5 ), and the pLKO.1puro lentiviral vector was used to construct a recombinant plasmid pLKO.1shRNA-4 inserted with shRNA (the nucleotide sequence of the siRNA target sequence of shRNA is shown in SEQ ID NO.12, and the plasmid map is shown in Figure 6 , the nucleotide sequence of the recombinant plasmid is based on SEQ ID NO.39, SEQ ID NO.9 is replaced by SEQ ID NO.12); based on Experimental Example 2, the recombinant plasmid pLKO.1shRNA-1 is replaced by the recombinant plasmid pLKO.1shRNA-4 to obtain the engineered polyclonal cell line 293T / pLKO.1shRNA-4 that secretes exosomes. The content of si-MMP-1 in the engineered exosomes secreted by the engineered polyclonal cell line 293T / pLKO.1shRNA-4 is shown in Fig.11 .Depend on Fig.11It can be seen that the absolute expression amount (expression level) of si-MMP-1 in the engineered exosomes loaded with si-MMP-1 is as high as 4.2 pmol / g total RNA. This result shows that positive cells can stably express si-MMP-1, and the engineered polyclonal cell line 293T / pLKO.1shRNA-4 that secretes exosomes is successfully constructed.

[0140] Experimental Example 6: Verification of the anti-aging properties of engineered exosomes

[0141] The specific process is as follows:

[0142] 7-8 week old female nude mice (purchased from Jicui Pharmaceutical Co., Ltd.) were irradiated with ultraviolet light (UVB, wavelength 311nm ultraviolet light) on the back skin (the lamp was placed about 30cm above the back) every other day for 10 weeks. Irradiation dose: 2MED (120mJ·cm) for the first 4 weeks. -2 ), 3 MED (180 mJ·cm ) from 5th to 10th week -2 ). The nude mice irradiated with ultraviolet light were randomly divided into 6 groups, with 3 mice in each group. The 6 groups of ultraviolet-irradiated photoaged nude mice were injected with 100 μL PBS buffer (PBS), 100 μg of unengineered exosomes (exo-WT, unengineered exosomes are secreted by unengineered 293T cells); 100 μg of engineered exosomes loaded with si-TNF-α of Experimental Example 2, 100 μg of engineered exosomes loaded with si-15-PGDH of Example 3, 100 μg of engineered exosomes loaded with si-HYAL2 of Example 4, and 100 μg of engineered exosomes loaded with si-MMP-1 of Example 5. Use a 1.5mL syringe for injection, dilute the sample to 200 μL with PBS buffer, and inject it into the midpoint of the back of the nude mouse. Take pictures before and 7 days after the injection, and the results of the pictures are shown in the figure. Fig.12 .

[0143] Depend on Fig.12It can be seen that before treatment, obvious wrinkles and folds appeared on the irradiated skin. After treatment with PBS buffer, there was no obvious improvement in wrinkles. After treatment with non-engineered exosomes, skin wrinkles were slightly reduced. After treatment with engineered exosomes loaded with si-TNF-α and engineered exosomes loaded with si-15-PGDH, wrinkles were slightly reduced, but the overall effect was not much different from that after treatment with non-engineered exosomes. After treatment with engineered exosomes loaded with si-HYAL2 and engineered exosomes loaded with si-MMP-1, wrinkles were significantly reduced and the skin surface was significantly smoother. Among them, engineered exosomes loaded with si-MMP-1 had the best effect on improving wrinkles and enhancing skin smoothness. It can be seen that engineered exosomes containing siRNA (especially HYAL2 and MMP-1) have better anti-skin aging effects than ordinary exosomes, and reduce skin wrinkles.

[0144] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A siRNA targeting a gene that promotes skin aging, characterized in that: The sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NOs. 1 to 4; the antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NOs. 5 to 8.

2. The siRNA according to claim 1, wherein The skin aging promoting genes include a gene encoding inflammatory factor α, a gene encoding 15-hydroxyprostaglandin dehydrogenase, a gene encoding hyaluronic acid glucosaminidase 2 and / or a gene encoding matrix metalloproteinase 1; When the skin aging-promoting gene is a gene encoding an inflammatory factor α, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.1, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.5; When the skin aging promoting gene is a gene encoding 15-hydroxyprostaglandin dehydrogenase, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.2, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.6; When the skin aging-promoting gene is a gene encoding hyaluronic acid glucosidase 2, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.3, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.7; When the skin aging promoting gene is a gene encoding matrix metalloproteinase 1, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.4, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.

8.

3. The siRNA according to claim 1 or 2, characterized in that The skin aging-promoting gene is a gene encoding hyaluronic acid glucosidase 2 or a gene encoding matrix metalloproteinase 1; When the skin aging promoting gene is a gene encoding hyaluronic acid glucosaminidase 2, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.7; When the gene promoting skin aging is a gene encoding matrix metalloproteinase 1, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO.

8.

4. A recombinant plasmid, characterized in that: The recombinant plasmid carries the siRNA according to any one of claims 1 to 3.

5. An engineered cell, characterized in that: The genome of the engineered cell is integrated with the siRNA according to any one of claims 1 to 3; or, the engineered cell carries the recombinant plasmid according to claim 4.

6. The engineered cell according to claim 5, characterized in that The method for preparing the engineered cells is: transfecting the recombinant plasmid described in claim 4 and the virus packaging plasmid into host cells simultaneously to obtain the engineered cells.

7. An exosome, characterized in that The exosomes are loaded with the siRNA according to any one of claims 1 to 3; or, the exosomes are loaded with the recombinant plasmid according to claim 4.

8. The exosome according to claim 7, characterized in that The exosomes are secreted by the engineered cells according to claim 5 or 6.

9. Use of the siRNA according to any one of claims 1 to 3, the recombinant plasmid according to claim 4, the engineered cells according to claim 5 or 6, or the exosomes according to claim 7 or 8 in the preparation of a product for resisting skin aging or treating diseases caused by genes that promote skin aging.

10. A product for resisting skin aging or treating diseases caused by genes promoting skin aging, characterized in that: The components of the product include the siRNA according to any one of claims 1 to 3, the recombinant plasmid according to claim 4, the engineered cells according to claim 5 or 6, or the exosomes according to claim 7 or 8.

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